JP2020018310A - USE OF AT(n) INSERTIONS IN PROMOTER ELEMENTS FOR CONTROLLING EXPRESSION LEVELS OF CODING SEQUENCES IN PLANTS - Google Patents

Abstract

To provide: methods for controlling the expression levels of coding sequences in plants; gene expression cassettes containing promoter regions of a gene having different numbers of AT insertions fused to the GUS protein; and methods for obtaining genetically modified plants comprising AT-insertions.SOLUTION: The present invention relates to the use of ATinsertions in promoter elements for controlling the expression levels of coding sequences in plants. The invention also relates to gene expression cassettes containing promoter regions of a gene having different numbers of AT insertions fused to the GUS protein.SELECTED DRAWING: Figure 1

Description

The present invention provides a method for controlling the level of expression of a coding sequence in a plant, which may be artificially up-regulated (upstream) or down-regulated (downstream) within certain limitations, at a specific point in the promoter region sequence. The use of AT _(n) inserts.

  The promoter region of a gene is generally contained in a DNA sequence having 1 to 100 and 200 base pairs upstream from the gene transcription start site, and typically contains one or more transcription factor binding sites, and / or Adjacent. All genes have transcriptional regulation upstream (upper) and downstream (lower) of the transcription start site. Transcription factors recognize and bind these regulatory sequences and control the synthesis of transcribed messenger RNA. These sequences include promoters, enhancers and regulatory sequences. Promoters are used in genetic constructs and can be manipulated by genetic engineering means and induced by certain circumstances to overexpress, inhibit, or regulate the expression of a gene in a constitutive manner.

  Various techniques for overexpressing a gene are currently available in the field of biotechnology, the main one being to use the constitutive promoter 35S, isolated from cauliflower mosaic virus (CaMV). Expression cassette constructs. However, there are some exceptions to the use of this promoter in genetic constructs. Due to the use of short virus DNA and its insertion into the plant genome, there is a potential risk of genetic modification between the viral genomes infecting the plant or, more precisely, between the messenger RNA transcripts Was experimentally revealed. This risk leads to the creation of new virus strains, preferably of a more aggressive type.

  The promoter 35S CaMV is thought to be accidental and functions effectively in all plants and in green algae, yeast, E. coli. It has a modular structure with interchangeable pairs in common with other plant and animal virus promoters. It also has recombination hotspots, is located by multiple triggers involved in the recombination process similar to other recombination hotspots, and is frequently used in transgenic plants, of the Agrobacterium (Agrobacterium) species. Includes T-DNA vector boundaries. This hot spot tends to split and bind to other DNA, thereby increasing the likelihood of horizontal gene transfer and recombination processes. Thus, insertion of invasive foreign DNA into the genome, reactivation of dormant virus and renewal of new virus, which are only stable when contributing some selective advantages compared to wild-type virus Due to the occurrence, potential risks are mutagenesis and carcinogenesis.

  Partial or complete transcriptional events and / or inhibition of endogenous homologous expression were observed using the promoter 35S CaMV (Napoli et al (1990) The Plant Cell 2: 279-289; Van der Kroll et al (1990). ) Plant Cell 2 291-299). This inactivation occurs due to a complex mechanism involving methylation and epigenetic changes (Renckens et al (1992) Mol. Gen. Genet. 233: 53-64; Neuhuber et al (1994) Mol. Gen. Genet. 244: 230-241; Meyer & Heidmann (1994) Mol. Gen. Genet. 243: 390-399; Thierry & Vaucheret (1996) Plant Mol. Biol. 32: 1075-1083). The actual mechanism that directs certain sequences to methyltransferases or other enzymes involved in epigenetic changes remains unknown, but depends on the homologous interaction between DNA-DNA, DNA-RNA and RNA-RNA. There is a hypothesis suggesting an involvement of action (Grierson et al. (1991) Trends in Biotechnology 9: 122-123); Matzke & Matzke (1995) Plant Physiology 107: 679-685.

  There is data in the literature demonstrating that the CaMV gene was integrated into the canola chromosome recombined in the genome of the infecting virus, giving rise to a number of new malignant viruses. These recombination between CaMV viruses involves the promoter region (Vaden & Melcher (1992) Virology 177: 111) and can occur between DNA and DNA or between RNA and RNA, often with the parental It causes infections that are more severe than their counterparts (Mol. Plant-Microbe Interactions 5:48, 1992).

  In addition, the use of the promoter 35S CaMV usually results in the expression of the product of the endogenous gene at less than 1% of total protein. Some improvements in this promoter, such as duplication of certain sequences or the addition of enhancers, increased its expression, but for some applications the expression level of the endogenous gene product needs to be further increased .

  In general, experiments performed with the constitutive promoter 35S CaMV to obtain genetically engineered plants with respect to the characteristics of resistance to biological and abiotic stress show that in some cases this resistance is increased. However, one of the undesired effects derived from the use of this promoter is a reduction in plant size, where plant growth and development are usually affected and affect plantlets independently of the transformed plant species. (Kasuga et al. (1999) Nature America Inc. 287-291).

  Thus, while certain negative effects may occur, such as a delay in plant growth, strategies such as the use of stress-inducible promoters to regulate gene expression may prevent or reduce such effects. is there.

  In another work, Gelvin et al. (Plant Molecular Biology Manual. Norwell, MA: Kluer Acedicmic Publishers, 1995) reported that the bacteria Agrobacterium tumefaciens (Agrobacterium tumefaciens) and the genes of the Agrobacterium tumefaciens (P. The expression of GUS detected by several promoters, constructed using various combinations that regulate the sequence of the synthase (mas), was analyzed. Among various combinations, the hybrid promoter formed by three repeats of the ocs gene activation sequence fused using the mas activation element and the mas promoter region is called “(Aocs) 3AmasPmas”, and the promoter CaMV A 10-fold greater GUS marker gene was displayed at the expression level of the transformed tobacco cells when compared to cells containing 35S. This new promoter did not affect transformation efficiency, but high levels of marker gene expression facilitated the identification of a larger proportion of transformed cells. Expression levels were high in leaves, roots, and various cell types. This promoter was also very active in manioc and pea plants, and two cereals were difficult to transform with Agrobacterium species. This group further described success in studying the rapid transcription of genes introduced (18 hours later) into tobacco and corn plants using this promoter "(Aocs) 3AmasPmas" and expression regulated by the promoter CaMV 35S. Unlike, its activity is weak and does not allow any detection of gene expression within this period.

  There is a further option of using a stress-inducible promoter for overexpression of the gene in the gene construct, in which respect the promoter rd29 isolated from Arabidopsis thaliana is a good candidate for abiotic stress. Mostly used to search for plants that have tolerance. In general, these gene constructs are usually fused to genes of the family DREB (dehydration response element binding) that regulate gene expression in response to drought, salinity, and environmental stresses such as low and high temperatures. Some work with different plant species has reported plants that are more resistant to abiotic stress as a result of gene transformation using the induced stress promoter (Oono et al. (2003) The Plant Journal). Kasuga et al. (2004) Plant Cell Physiol 45 (3): 346-350).

  (2004) used a soybean genotype that was resistant and susceptible to cyst nematodes. This document contains initial data on molecular markers used for assisted selection of soybean genotypes resistant to cyst nematodes. However, no type of molecular difference between the tested individuals was detected with respect to the level of the DNA sequence or the expression of the mRNA encoded by this gene.

Fuganti (Master's Thesis, State University of Londona [Universidade Estadual de Londona], 2004) indicates the selection of resistant strains, wherein the resistance gene of the genomic region containing the gene of interest and the molecular features related to the resistant gene and molecular characteristics are shown. The used molecular markers were used. This study does not disclose qualitative and quantitative data showing a possible correlation between the size of the AT _(n) insert at the gene promoter and the resulting level of expression of its mRNA or protein. The present invention proposes the ability to regulate and / or up-regulate or down-regulate any expression level provided that a certain number of AT _(n) inserts have been inserted into the promoter region. Such proposals are not shown in this study and have not been tested.

Fuganti (PhD Thesis, State University of Maringa [Universidade Estadual de Maringa], 2007) is a gene Gmhsp17.6-L with different sized AT _(n) inserts within the promoter region of the promoter and various non-biological organisms. Quantitative and qualitative data showing a possible correlation between their expression levels in response to experimental stress (cold, heat, drought, salt, infection by nematode larvae) were presented. However, the coding region may be fused to promoter regions containing different AT _(n) sizes in the absence and / or presence of biological and abiotic stresses to up-regulate or up-regulate the expression level of any gene. The possibility of down-regulation is not shown or discussed.

Patent document US2005059623 discloses the use of controlled application of ultrasound to regulate the expression of therapeutic genes fused to a heat shock protein (HSP) promoter, which is expressed in selected cells. Provide a way to use fever. The proposed invention regulates, in the gene sequence, the expression level of any gene fused to the promoter region of a heat shock protein with various sizes of AT _(n) inserts expressed in all transformed cells. And up or down control. Biological or abiotic stress may be applied to the whole plant. Disclosed are any instructions as to the role of the AT insert in regulating gene expression, provided that the same is inserted into its promoter region, and its possible use as a tool to control any gene expression. Not.

Patent document US2003190706 relates to an endogenous gene fused to a DNA sequence exhibiting promoter activity, which promotes an increase in the total amount of gene product (protein). This region with promoter activity was generated from mutational alterations including substitutions and deletions of nitrogen-combined bases. The present invention requires the use of a complete gene promoter, including all the genetic elements required to drive mRNA synthesis. It is evident in the present invention that regulation of gene expression results in differentiated levels according to the size of the AT _(n) insert, rather than random mutations in the promoter region of the gene.

Patent document EP 1 930 423 discloses heat-shock protein promoter regions divided into different final sizes for use in systems for producing heterologous proteins whose expression levels are not regulated by other conventional promoters. Certain abiotic or chemical stresses are used to activate expression. The present invention uses a pre-existing variation in the tested genotypes, a fragment smaller than 1000 bp used in the document EP 193423, which is detected in the promoter region of the gene Gmhsp17.6-L. This variation is provided by a different number of AT (n) inserts in a particular region of the promoter, resulting in a size variation between 312 bp (AT ₉ ) and 358 bp (AT ₃₃ ) depending on the AT (n) insert.

US Pat. No. 5,929,302 uses a transient tissue-specific promoter that controls gene expression during maturation in tissues such as leaves and flower beds. The promoter was isolated from the dru gene itself in raspberry, or from its homolog, to express the chimeric gene. This promoter and specific genes, such as genes that control color and scent changes, genes of enzymes or catalyst products that modify plant cell processes, genes whose products affect ethylene synthesis, alternative fungal regulatory genes , As well as a combination with a sucrose storage gene. The present invention has alterations in the promoter region of any gene to alter the expression of any gene in all cells and throughout the life cycle of the plant. Variations in the expression levels of the gene products correlate with different numbers of AT _(n) inserts into the promoter and consequently with promoters of different sizes. The present invention proposes to regulate any gene expression involving these alterations.

  Thus, the present invention is innovative because it differs from all currently existing methods available in scientific papers overexpressing genes. To do so, the present invention uses the insertion of an AT sequence with a large and small number of repeats, allowing the modulation of the level of gene expression and increasing or decreasing it.

The present invention relates to the use of an AT _(n) insert in a promoter element to regulate the expression level of a coding sequence in a plant. The present invention also relates to a gene expression cassette comprising the promoter region of a gene having a different number of AT inserts fused to a GUS protein for transforming a soybean embryo.

The present invention provides a method for controlling the expression level of a coding sequence in a plant, the method comprising:
(I) stably transforming plant cells with an expression cassette carrying an AT _(n) insert in a promoter element operably linked to the gene of interest;
(Ii) culturing a plant cell that has been stably transformed under plant cell growth conditions;
(Iii) regenerating a transgenic plant in which the cassette of (i) is stably integrated into its genome, wherein the transgenic plant has a different level of gene expression when compared to a control plant. Show.

  Furthermore, the present invention provides an expression cassette comprising two promoter regions having different numbers of AT inserts linked to the promoter region of the Gmhsp17.6-L gene.

In another aspect of the invention, there is provided a method for obtaining a genetically modified plant comprising an AT _(n) -insert, said method comprising:
(I) stably transforming a plant cell with an expression cassette carrying an AT _(n) insert in a promoter element operably linked to the gene of interest;
(Ii) culturing a plant cell that has been stably transformed under plant cell growth conditions;
(Iii) The method comprising regenerating a stably genetically modified plant.

The result is that the expression level of certain genes can be artificially up-regulated (upstream) within certain limits by inserting AT _(n) repeated bases in the promoter region through genetic engineering tools. ) Or down-regulation (downstream).

Agarose gel using amplification of microsatellite marker SOYHSP176 in individuals of resistant parent sample PI595099, sensitive parent sample BRS133, and resistant population (sample 01 to sample 05) and sensitive sample (sample 06 to sample 10). Arrows indicate the bunt generated by marker amplification in susceptible parents and are also found in susceptible populations. The sequence of the gene Gmhsp17.6-L available from GenBank (accession number: M11317) is shown. Underline, primer pSoyHspPstI F and pSoyHsp-BamHI R defines the amplified fragment of the promoter region. The dot box is the AT _(n) insert, the gray box is the start codon (ATG) and stop codon (TAA) of the coding sequence. The sequence of the gene Gmhsp17.6-L (Accession number of GenBank: M11317) is shown. The gray box highlights the start codon (ATG) and stop codon (TAA) defining the coding sequence, the dot box highlights the AT (n) insert in the promoter region, and underlines are designed for complete gene sequencing. Primer pSoyHspPstI F and SoyHSP Cl R. Primer pSoyHsp designed based on the complete sequence of the gene available from GenBank (Accession number: M11317) AleI F and pSoyHsp NcoI 3 shows amplification of the promoter region of the gene Gmhsp17.6-L using R. The ladders are 100pb and 500pb. FIG. 7 shows amplification of the complete gene Gmhsp17.6-L using primers pSoyHSP PstIF and pSoyHSP Cl R designed based on the complete sequence of the gene available from GenBank (Accession number: M11317). The ladders are 100 pb and 500 bp. Primer pSoyHSPPstI defining promoter region F and pSoyHSPBamHI Using the set of R, the gene Gmhsp17.6-L generated by amplification of the resistant parent PI595099, the susceptible parent BRS133, the individuals of the susceptible population 256-S, 259-S, and the individuals of the resistant populations JF7002, JF7027 and JF7056. 2 shows an alignment of the sequence of the promoter region. Primers were defined based on the complete sequence of the gene Gmhsp17.6-L (GenBank accession number: M11317). In the dot box is the AT _(n) insert. The box is probably a TATA-box. The black box with white letters is one of the heat shock elements (HSE) of the consensus sequence 5′AGGAAnnTTCT3 ′. In the gray box with white letters, another consensus sequence 5'cTTCtaGAAgcTTCtaGAAg3 'with a core 5'CTnGAAnnTTCnAG3'. The gene sequence present between the promoter region of the gene Gmhsp17.6-L and the start of the coding sequence compared to the sequence of the gene available in GenBank (accession number: M11317) is shown, and the material of the study was sensitive (BRS133 ) And resistant (PI595099), susceptible populations 256-S, 259-S and 266-S, and individuals from the resistant populations JF7002, JF7027 and JF7056. The nucleotides in the gray box highlight the codon ATG at the transcription start site of the coding region. Sensitive parent BRS133 and resistant PI595099 of all materials under study, individuals of the susceptible populations 256-S, 259-S and 266-S, and resistance compared to the sequence of the gene available in GenBank (Accession number: M11317). Figure 2 shows an alignment of the coding sequence of the gene Gmhsp17.6-L from individuals in the populations JF7002, JF7027 and JF7056. The nucleotides in the gray boxes highlight the ATG start codon and the TAA thermal transcription codon that define the coding region. Sensitive parent BRS133 and resistant PI595099 of all materials under study, individuals of the susceptible populations 256-S, 259-S and 266-S, and resistance compared to the sequence of the gene available in GenBank (Accession number: M11317). Figure 2 shows an alignment of the coding sequence of the gene Gmhsp17.6-L from individuals in the populations JF7002, JF7027 and JF7056. The nucleotides in the gray boxes highlight the ATG start codon and the TAA thermal transcription codon that define the coding region. Sensitive parent BRS133 and resistant PI595099 of all materials under study, individuals of the susceptible populations 256-S, 259-S and 266-S, and resistance compared to the sequence of the gene available in GenBank (Accession number: M11317). 1 shows an alignment of the amino acid sequence encoded by the coding sequence of the gene Gmhsp17.6-L from individuals in the populations JF7002, JF7027 and JF7056. M. FIG. 4 shows ribonuclease protection performed using a probe (326 pb) constructed from a fragment of the gene Gmhsp17.6-L (GenBank accession number: M11317) using samples resistant and sensitive to Javanica. The numbers at the top are equivalent to the samples used: susceptible parent (BRS133) and resistant (PI5955099) and M.p. Treatments inoculated with Javanica eggs (2 and 4), positive control of kit (indicated by 5-arrow), RNase enzyme control, designed probe (6), and undigested RNase (7) is there. Cyst nematodes M. Figure 5 shows the relative quantification of the gene Gmhsp17.6-L in resistant (PI5955099) and susceptible (BRS133) parents in Javanica inoculated and uninoculated treatments. Using the complete sequence of the gene available in GenBank (accession number: M11317), data was obtained by real-time PCR with primers SOYHSP PSC F and SoyHSP PSC R designed using the program PrimerExpress® software v20. . Quantification was performed using the 2 ^-ΔΔCt method. Gene rRNA 18S was defined as a gauge and used as a non-inoculated sample. Samples were amplified in a three-point measurement in three independent runs using the SYBR method. Cyst nematodes M. Relative quantification (x) of gene Gmhsp17.6-L in susceptible populations 256-S, 259-S and 266-S in treatments with and without inoculation of Javanese; and in individuals of resistant populations JF7002, JF7027 and JF7056 Is shown. Using the complete sequence of the gene available in GenBank (accession number: M11317), data was obtained by real-time PCR with primers SOYHSP PSC F and SoyHSP PSC R designed using the program PrimerExpress® software v20. . Quantification was performed using the 2 ^-ΔΔCt method. Gene rRNA 18S was defined as a gauge and used as uninoculated sample 256-S. Samples were amplified in a three-point measurement in three independent runs using the SYBR method. 1 shows a schematic model of the plasmid pAG1, illustrating the Gus reporter gene encoding the enzyme β-glucuronidase, the gene Ahas and the promoter act2. Also, a restriction map is shown. Materials tested: BRS133, PI5955099, 256-S, 259-S and 266-S, JF7002, JF7027 and JF7056 All constructs of expression cassettes obtained, of the promoter region of the gene Gmhsp17.6-L. Figure 3 shows amplification via PCR. The reaction was performed using the primer pSoyHspPSTI which amplifies a 363 pb fragment. F and pSoyHSPBamHI R and then separated by agarose gel (1.0%) electrophoresis. The amplification reaction of samples BRS133 and JF7056 was concentrated during PCR. Material tested: BRS133, PI5955099, 256-S, 259-S and 266-S, JF7002, JF7027 and JF7056 All constructs of the expression cassette obtained using the part of the gene Ahas present in the plasmid pAG1. Figure 3 shows amplification via PCR. The reaction was performed using primer Ahas1 which amplifies a 654 bp fragment F and Ahas2 R and then separated by agarose gel (1.0%) electrophoresis. Materials tested: PCR of the terminal region Nos present in plasmid pAG1 of all constructs of the expression cassettes obtained using BRS133, PI5955099, 256-S, 259-S and 266-S, JF7002, JF7027 and JF7056. FIG. The reaction was performed using primer Nos1 which amplifies a 200 bp fragment. F and Nos3 R and then separated by agarose gel (1.0%) electrophoresis. Figure 2 shows a schematic model illustrating the structure of test plaques for abiotic and biological stress, and is susceptible to cyst nematodes, BRS133 and Pintado (spots) transformed with different expression cassettes. Varieties were given soybean embryos. CN—negative control, untransformed embryo; CP—positive control, embryo transformed with plasmid pAG1; embryo transformed with an expression cassette constructed using the promoter region of S-sensitive parental origin , PAG1 / promoter GmHSP BRS133; embryo transformed with an expression cassette constructed using a promoter region of an individual origin of the R-resistant population, pAG2 / promoter region GmHSP JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Tissues performed on soybean varieties BRS133 and pintado embryos transformed with bioballistics using JF7027 and subjected to heat stress at 25 ° C. for 2 hours, 4 hours and 24 hours 3 shows a chemical test. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Histochemical studies performed on soybean varieties BRS133 and pintado embryos transformed by biovaritics using JF7027 and subjected to heat stress at 35 ° C. for 2 hours, 4 hours and 24 hours Is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Histochemical studies performed on soybean varieties BRS133 and pintado embryos transformed by biovaritics using JF7027 and subjected to heat stress at 45 ° C. for 2 hours, 4 hours and 24 hours Is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter Gmhsp BRS133 and pAG1 / promoter Gmhsp Histochemical studies performed on embryos of soybean varieties BRS133 and pintado (spots) transformed by biovaritics using JF7027 and subjected to heat stress at 4 ° C for 2 hours, 4 hours and 24 hours Is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter Gmhsp BRS133 and pAG1 / promoter Gmhsp Histochemical studies performed on soybean varieties BRS133 and pintado embryos transformed by biovaritics using JF7027 and subjected to heat stress at 15 ° C. for 2 hours, 4 hours and 24 hours Is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp JF7027 transformed by biotechnology burr probiotic using shows 24 hours, 200 mM, the histochemical tests carried out in embryos varieties BRS133 and Pintado soybeans were subjected to a salt of NaCl ₂ of 400mM concentration (spots). Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Soybean varieties BRS133 and pintado transformed by biovaritics using JF7027 and subjected to drought stress while maintaining embryos on paper filters at 37 ° C. in a greenhouse for 2, 4 and 6 hours 3 shows histochemical tests performed on embryos of the present invention. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp ₂ shows histochemical studies performed on embryos of soybean variety BRS133, transformed by biovaritics with JF7027 and subjected to biological stress by inoculation of 2000-3000 J ₂ / mL. The samples remained in contact with the pathogen for 24, 48 and 72 hours. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to the cyst nematode Javanica, the expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp JF7027 transformed by biotechnology burr probiotics with shows histochemical tests were carried out in embryos 2000~3000J ₂ / mL varieties of soybeans that have been subjected to biotic stress by the inoculation Pintado (spots). The samples remained in contact with the pathogen for 24, 48 and 72 hours. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027.

The present invention relates to the use of an AT _(n) insert in a promoter element to regulate the expression level of a coding sequence in a plant. Among the resistant and susceptible individuals in the population, the expression levels of heat shock protein (Gmhsp17.6-L) were compared, and the maximum expression level per quantitative PCR was found in the individual containing the largest AT insert in the promoter region. Was. To transform soybean embryos, a gene expression cassette containing the promoter region of the gene was constructed, along with different numbers of AT inserts fused to the GUS protein. According to the experimental results of the present invention, the expression level of certain genes can be artificially controlled within certain limits by inserting AT _(n) repeating bases into the promoter region through genetic engineering tools. It is suggested that control (upstream) or down-regulation (downstream) could have been performed. Thus, the technology developed in the present invention allows for the regulation of gene expression levels, provided that the AT _(n) repeat insert is inserted at a specific point within the sequence of the promoter region.

  Based on previous work trying to identify molecular markers for use in assisted selection of soybean genotypes resistant to the nematode meloidogine javanica, the F-linked microsatellite marker SOYHSP176 was identified for the population tested. It showed high significance and explained the characteristic number of cysts in the roots, with 43% phenotypic variation and 70% of the variation on entry scale. It is important to point out that fragments amplified by this marker are only present in susceptible individuals and not in resistant individuals.

  This marker was sequenced and subsequently searched for similarities to other sequences of the nucleic acid deposited in the gene bank, which sequence is associated with the promoter region of the low molecular weight heat shock protein (Gmhsp17.6-L). It showed homology and was found in soybeans and was deposited at GenBank (accession number: M11317). Using the complete sequence of this gene available in GenBank, a new primer pair was designed to amplify fragments in both genotypes.

This strategy was successful and fragments were obtained in both susceptible and resistant individuals. All of the resulting fragments were cloned and sequenced and showed homology to the same promoter region of the gene Gmhsp17.6-L. However, alignment of the sequences showed that when compared to susceptible plants, resistant plants had larger inserts of microsatellite in the promoter region of the gene, characterized by AT _(n) repeats. Thus, the fragment from the resistant parent PI595099 showed 15 and 13 additional AT repeats when compared to the sequence of the gene Gmhsp17.6-L available in GenBank. On the other hand, the fragment from the susceptible parent BRS133 has 6 AT repeats less than the sequence available in GenBank. These results were repeated for population resistant and susceptible individuals resulting from this intercross that had also been sequenced.

  Alignment also indicated that all of the amplified fragments had a sequence complementary to the primer for microsatellite marker SOYHSP176. If this is achieved, the following question then arises: Did amplification not occur initially in resistant plants, even if the annealing region of the microsatellite primer was present at a high probability? One possibility is a higher number of AT repeats in resistant individuals, which gives rise to the formation of certain tertiary structures, such as hairpins, thus making it difficult for the DNA polymerase to anneal to the primers during the amplification process, In conclusion, it may have inhibited amplicon formation in these plants.

  Insertion of the microsatellite within the promoter region of this gene was considered to inactivate it in susceptible plants, since the marker SOYHSP176 was amplified only in these individuals. However, studies indicate that all samples showed microsatellite insertion, presumably at AT repeat numbers within the promoter region that may be interfering in the response of soybean to nematodes. The presence of more or less insertions of a repeat sequence in the promoter region of a gene may inactivate, activate, or alter the level of expression of a protein regulated by this promoter.

  According to the literature, one of the main functions of these AT-elements that is repeated in regulating the gene expression of heat shock proteins is to facilitate access of RNA polymerase to the transcription start site, Elimination of histones to promote binding of heat shock proteins involved in induction. According to the literature, heat shock proteins act as molecular sheperons in cells, establish nascent polypeptide chains, assist in their correct folding, re-establish protein and / or denatured membranes, and further signal transduction Plays an important role in

  Therefore, the length of insertion of the gene Gmhsp17.6-L into the promoter region was M. A ribonuclease protection assay was used to test the hypothesis of altering the expression level of the protein Gmhsp17.6-L in resistant and susceptible individuals affected by Javanica. The results showed that all individuals expressed the protein, whether resistant or susceptible, and regardless of treatment, whether or not inoculated with nematodes. Due to differences in the biological function of the AT sequence in gene regulation and the number of repeats within the promoter between individuals, the ribonuclease protection assay was thought to show an expressed strand of the gene Gmhsp17.6-L in different individuals. Still, the results indicate that, in theory, there was no difference in expression of this gene in response to infection by nematodes, at least not at levels detectable by the technique used.

  In an attempt to confirm these results, a new study was devised using real-time PCR (RT-PCR), an accurate and precise technique for quantifying gene expression levels. Parental BRS133 (susceptible) and PI595099 (resistant), as well as lines from susceptible populations (256-S, 259-S and 266-S) and resistant populations (JF7002, JF7027 and JF7056) derived from this cross. Insects were inoculated. The roots of the treatments inoculated and uninoculated with Meloidogyne jawanica were recovered for RNA extraction and total cDNA synthesis was subsequently used in RT-PCR reactions for relative quantification.

The results showed that all resistant parent individuals PI595099 and their populations JF7002, JF7027 and JF7056 exhibited higher expression of the gene Gmhsp17.6-L inoculated with cyst nematodes. These individuals in this population are identical in sequencing to the higher AT _(n) repeats within the promoter region of the gene. Our hypothesis is that, when infected by a nematode, certain genes interacting with the AT-rich region of the promoter of the resistant plant gene Gmhsp17.6-L, which activates its transcription with the function of an intracellular chaperone. That is, to generate factors. In plants susceptible to the interaction of factors such as the AT region, it does not occur in such a vigorous manner, which indicates that the expression level of the protein Gmhsp17.6-L is lower in plants susceptible to nematodes. means.

  Through the techniques of the present invention, it is possible to control the expression levels of different genes, including different sized AT inserts at specific points in the promoter region. In addition, it becomes possible to develop a GM plant that overexpresses or underexpresses a gene present in the promoter region in proportion to the size of the AT insertion.

Biomaterial constructs were generated using parental PI595099 and BRS133, soy genotypes, which are each resistant and susceptible to the root-knot nematode, Meloidogyne javanica. From this high distribution, in the F4 generation, individuals 256-S, 259-S and 266-S of the susceptible population and individuals JF7002, JF7027 and JF7056 which were the resistant population were selected and used in the present test.

In addition, the cultivar Pintado susceptible to nematodes was constructed from different samples and using an expression cassette containing different AT _(n) in the promoter region of the gene Gmhsp17.6-L (GenBank accession number: M11317). Used during particle transformation in soy embryo transformation.

Example 1 Isolation and Characterization of Promoter and Complete Gene Using the complete sequence of the gene Gmhsp17.6-L obtained from the initial sequencing of amplified fragments of sensitive material using the microsatellite marker SOYHSP176 primer, Taking into account that only the susceptible genotype amplified the microsatellite marker SOYHSP176, a new primer set was designed to obtain bands for all genotypes under analysis (FIG. 1).

Primer (pSoyHsp AleIF 5 'CAC CGC GGT G GAA TTC TGA AAT TGG GTC TTT TTG 3'; pSoyHsp NcoI R 5 ′ CCA TGG AAT GGG GAC ACT CGA GGT ATT3 ′) was synthesized at the start of the promoter region of the gene Gmhsp17.6-L, with a restriction site for subsequent cloning. FIG. 2 shows the primer annealing sites in the sequence of the gene Gmhsp17.6-L available in GenBank.

Newer primer set (pSoyHSP PstI F 5′GGG CTG CAG GAA TTC TGA AAT TGG GTC TTT TTG3 ′; SoyHSPCL R 5 ′ CCC CCC GGG TTA ACC AGA GAT TTC TAT AGC CT 3 ′), which amplifies all genes from the start of the coding region promoter to the stop codon, and is a root-knot nematode, M. cerevisiae. This was to confirm the presence of other differences in the gene sequence besides the promoter's common sense AT _(n) insert between the genotypes sensitive to and resistant to javanica. FIG. 3 shows the primer annealing sites in the sequence of the gene Gmhsp17.6-L, available in GenBank, designed to amplify and sequence the complete gene.

Genomic DNA from seeds was extracted from all analyzed samples. Approximately 50 mg of seed from each analyzed sample was sliced into slices with a razor blade, collected in a 1.5 mL microtube, and added with 300 μL of extraction buffer. The material was ground and an additional 700 μL of extraction buffer was added. The extraction buffer was prepared according to the protocol shown in the table below:
Table 1: Reagents used to prepare genomic DNA extraction buffer:

  The solution was homogenized using a vortex for 30 seconds to 1 minute and centrifuged at 16,000 xg for 4 minutes at room temperature. The suspension was transferred to a new 1.5 mL tube, provided to a new centrifugation step under the same conditions as the previous time, and transferred to a new 1.5 mL tube.

To exclude proteins, 0.1 mg of proteinase K and 0.01 mM CaCl ₂ were added to the samples and incubated in a water bath at 37 ° C. for 90 minutes. 900 μL of cold isopropanol was added, incubated for 2 minutes, and centrifuged at 16,000 × g for 7 minutes. The supernatant was discarded and the pellet was dried for 90 minutes. The pellet was resuspended in 300 μL of TE buffer (Tris-HCl 1 M, pH 8.0, EDTA 0.5 M, pH 8.0) containing 40 μg / μL RNase A, and again in a water bath to eliminate RNA. The samples were incubated for 90 minutes at 37 ° C in water. Next, a fresh precipitation step was performed, and finally the DNA pellet was resuspended in 300 μL of TE buffer and quantified using a spectropolarimeter, and its quality and integrity were determined on a 0.8% agarose gel. confirmed.

  To amplify the promoter region and complete gene sequence from all resistant and sensitive samples, a PCR reaction was performed using a Perkin Elmer 9600 thermocycler, 30 ng of template DNA, 10 × reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, and 400 μL ultrapure water), 1.5 mM MgCl 2, 1.3 mM dNTP, 1 U Taq DNA polymerase, and 5 μM of each F and R primers. To a final volume of 10 μL to complete.

  The thermal cycling program used to amplify the DNA sample was configured as follows: an initial denaturation step at 94 ° C. for 7 minutes, followed by denaturation at 94 ° C. for 1 minute, annealing at 60 ° C. for 1 minute, And 30 cycles of extension at 72 ° C for 2 minutes. A 7 minute cycle at 72 ° C. was finally performed. The amplification products are separated on a 1% agarose gel and used with 1 × TBE buffer (108 g Tris base, 55 g boronic acid, 40 mL EDTA 0.5 M pH 8.0, and distilled water, complete to a final volume of 1 L). And stained with ethidium bromide (10 mg / mL). Images were obtained using a Kodak Digital DC290 system.

  The amplified fragments (FIGS. 4 and 5) were extracted using the PureLink ™ Quick Gel Extraction (Invitrogen) kit according to the manufacturer's instructions. After the DNA was purified, it was stored at -20 ° C. Next, the DNA was quantified, and a ligation reaction between the fragment and the plasmid vector was performed using a TOPO (registered trademark) TA cloning kit (Invitrogen). The ligation reaction consisted of the following: 2 μL ultrapure water; 1 μL salt solution (dilute 7 μL salt solution: 21 μL ultrapure water); 1 μL TOPO® vector and 2 μL (about 10 ng) purified band . The sample was incubated at room temperature for 5 minutes.

  The resulting vector was used for E. coli (DH10B strain) cell transformation via electroporation. Electrocompetent cells were prepared and colonies isolated from the glycerol stock through plate streaking were obtained. The five single colonies are then cultured overnight in 10 mL of LB medium-Luria Bertani (20 g mixture of tryptophan, yeast extract and NaOH for 1 L of distilled water) at 37 ° C. at 200 rpm. A 5 mL pre-inoculation was added to a 500 mL LB medium rotated at 300 rpm at 37 ° C. until the OD500 reached 0.5-0.7. After this period, the cells were incubated on ice for 20 minutes and transferred aseptically to a 250 mL centrifuge tube pre-cooled on ice. A centrifugation step was performed at 4,278 × g for 15 minutes at 4 ° C., the supernatant discarded and the pellet resuspended in 500 mL of cold 10% glycerol. A new centrifugation was performed under the same conditions, the supernatant was discarded, and the pellet was resuspended in 250 mL of cooled 10% glycerol. Another centrifugation step was performed and the pellet was resuspended in 20 mL of cold 10% glycerol. The solution was transferred to a 50 mL sterile tube, and centrifuged at 3997 × g for 15 minutes at 4 ° C. As a final step, the pellet was resuspended in 1-2 mL of chilled 10% glycerol. The electrocompetent cells were dispensed into 500 μL microtubes and stored in a −80 ° C. freezer.

Electroporation was performed with a MicroPulser (BioRad) electroporator set at 2.5 kV, 25 μF and a pulse controller set at 200 or 400 ohms. 2 μL of the ligation reaction and 100 μL of electrocompetent cells were used. Cell rescue was performed in a 15 mL sterile Falcon tube in 1 mL SOC medium (0.5 g yeast extract, 2 g tryptophan, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose, and 96 mM Of distilled water) was obtained by culturing and incubating at 37 ° C. for 1 hour at 200 rpm. After this period, 300 μL of electroporated cells were supplemented with ampicillin (100 μg / 30 mL), IPTG / X-Gal (50 μL of IPTG-isopropyl-β-thiogalactopyranoside 0.1 M, and 10 μL of X -Gal- -bromo-4-chloro-3-indolyl-β-D-galactoside (50 mg / mL) / plate) was seeded on a solid LB medium. Plates were incubated at 37 ° C. overnight for colony growth. Selection of recombinant colonies was performed using the lacZ system, with recombinant colonies showing white colonies and non-recombinant colonies showing blue colonies. This is due to the resulting product of the reaction of the lacZ gene product with the substrate (X-Gal).

  Next, to confirm the cloning and size of the fragments, the plasmid DNA extract was combined with 1 mL of CG medium-cycle glow (4 g of a commercial mixture in 100 mL of water) and 100 μL / mL of ampicillin (50 mL of CG medium). Was performed by inoculation of a single white colony in a 96-well microplate containing 50 μL of ampicillin (100 mg / mL). The plate was sealed and the material was incubated at 37 ° C. at 320 rpm for 22 hours.

  Before proceeding with plasmid extraction, a permanent culture of growing bacteria is obtained and 75 ml of overnight grown CG medium and 50% sterile glycerol are contained in a sterile plate. This culture was stored at -80 ° C in an ultra-freezer and used as a stock.

  The micropreps were centrifuged at 1,310 × g, 10 ° C. for 6 minutes to continue the miniprep and obtain cell deposits. The supernatant was discarded and the plate was quickly inverted on absorbent paper. Next, 200 μL of GET (20% glucose, 0.5 M EDTA pH 8.0, 1 M Tris-HCl pH 7.4 and water to fill 500 mL) was added to each plant, the plate was sealed and vortexed for 2 minutes. Shake vigorously using to resuspend the cells. A new centrifugation at 1,310 × g and 10 ° C. was performed for 9 minutes, the supernatant was discarded, and the plate was quickly inverted on absorbent paper and dried.

  65 μL of GET containing RNase A (10 mg / mL) was added to each well of the microplate. All material was resuspended using a vortex and transferred to a “U” bottom plate. Next, 65 μL of NaOH 0.2 N / SDS 1% (0.5 mL NaOH 4 M + 1 mL SDS 10% + ultra pure water to make a volume of 10 mL) was added to each well. This solution was prepared just before the steps used. The plate was sealed; the materials were mixed by inversion for 5-10 minutes and incubated for 10 minutes at room temperature. A few seconds of rapid centrifugation was performed until no solution remained on the adhesive seal.

  To each well, 60 mL of 3M potassium acetate (KOAc) stored at 4 ° C. was added, the plate was sealed, the material was mixed by inverting 10 times, and a 10 minute incubation was performed on ice. Perform a centrifugation step at 1,310 × g, 4 ° C. for 15 minutes, transfer 80 μL of the supernatant to a Millipore® (MAGV N22) plate, and place it on top of a 250 μL “V” bottom polypropylene microplate. Fixed. Plates were centrifuged for 5 minutes at 1,310 × g, 4 ° C., or performed until the entire volume was transferred to the bottom plate (V-bottom). The Millipore® plate was removed, discarded, and 80 μL of isopropanol was added to the filtered solution transferred to the “V” bottom plate.

  The "V" bottom plate was sealed with an alcohol resistant adhesive and the material was inverted and mixed. A centrifugation step at 1,310 × g, 4 ° C. was performed for 45 minutes, the supernatant was discarded by inverting the plate, and 150 μL of cold 70% ethanol was added to each sample. The plate was again centrifuged for 5 minutes at 1,310 × g at 4 ° C., and after discarding the supernatant, the plate was inverted on absorbent paper and quickly spun down at 82 × g at 4 ° C. The plate was covered with a paper towel, dried for 60 minutes at room temperature, and finally the DNA was resuspended in 30 μL of ultrapure water. Plates were sealed and incubated overnight at room temperature for complete elution of plasmid DNA. After this period, the plate containing the plasmid DNA was stored in a -20 ° C freezer.

  Samples were subjected to a digestion reaction using restriction enzymes to confirm the presence of the insert and its size before being prepared for sequencing. The digestion reaction consisted of 1.5 μL of reagent 3 enzyme buffer; 0.5 μL of EcoRI (10 U / μL); 5 μL of plasmid DNA (about 10 ng) and 8 μL of ultrapure water and incubated at 37 ° C. for 2 hours did. After this period, an aliquot of the reaction was subjected to 1% agarose gel electrophoresis.

  Fragments are sequenced on an ABI Prism 3100 (Applied Biosystems) capillary sequencer, where the ABI Prism BigDye Terminator Cycle Sequencing (Applied Biosystem) kit is used based on both orientations of the DNA duplex. A different sequencing experiment, M13R and F primers were used. The sequencing reaction used 1.5 μL of DNA from each sample on a plate and used 8.5 μL of a reaction mixture prepared according to the protocol described in the table below.

Table 2: Reagents used in preparing the reaction mixture used for the sequencing reaction

  The sequencing reactions were performed on an Applied Biosystem thermocycler under the following conditions: 96 ° C for 2 minutes; 30 cycles of 96 ° C for 15 seconds, 50 ° C for 15 seconds, and 60 ° C for 4 minutes. Finally, the reaction for DNA precipitation was performed by adding 80 μL of 75% isopropanol to each sample, then incubating for 15 minutes at room temperature, protected from light and 45 minutes at 1,310 × g Centrifugation was performed.

  The supernatant was discarded and the pellet was washed with 100 μL of 70% ethanol, followed by a centrifugation step at 1,310 × g for 15 minutes. The supernatant was discarded, light was shielded at room temperature, and the pellet was dried. For sample application on a sequencing machine, the pellet was resuspended in 10 μL hi-formamide and then incubated for 5 minutes at 95 ° C. in a thermocycler to cause DNA denaturation. The plate was immediately transferred to ice to avoid re-annealing of the DNA and then placed on a sequencing machine.

  After sequencing of the promoter region and the complete gene region, a sequence database search of biological sequences was performed to verify the similarity between the resulting fragments and known DNA and protein sequences (FIG. 6). To achieve this, the programs BLASTn and BLSATx (Altschul et al. (1997), Nucleic Acids Res. 25: 3389-3402) were used. Alignment of nucleotide sequences (FIGS. 7 and 8) and amino acid sequences (FIG. 9) can also be performed using appropriate software, such as Vector NTI Advanced 10.0.1 (Invitrogen Corp.), BioEdit Sequence Alignment Editor and ClusterW. I went.

Example 2: Gene expression regulation study-RPA
Parental genotypes PI595099 (resistant) and BRS133 (sensitive) were selected for RPA experiments. From the complete sequence of the heat shock protein Gmhsp17.6-L (GenBank accession number: M11317) present in soybean, a primer set was designed for the coding sequence (RPA2 F 5 ′ GAC ATC ATC AAA CAA GAG AA3 ′ and RPA2 R 5 'TCT CTC CGC TAA TCT GAA 3').

  Seed DNA extract from the parent sample analyzed, amplification of the fragment through PCR, cloning and sequencing of the product generated by PCR were performed according to the protocol detailed previously in the preceding section.

  Total RNA from the parent PI595099 (resistant) and BRS133 (sensitive) was extracted from soybean roots that had been subjected to two different treatments (inoculation and non-inoculation of M. javanica nematode eggs). Extraction was performed using a Trizol reagent (Life Technology) kit. Grind the roots in liquid nitrogen using a mortar and pestle, transfer to an autoclaved Falcon tube and store in a fume hood, which contains 20 mL of Trizol. After homogenization, the samples were incubated for 5 minutes at room temperature (15-30 ° C). 0.2 mL of chloroform was added to each 1 mL of Trizol, and after 15 seconds of vigorous stirring, the solution was incubated for an additional 3 minutes at room temperature (15-30 ° C.). Next, a centrifugation step at 12.240 × g for 15 minutes at 4 ° C. was performed. The liquid phase containing the RNA was transferred to a new Falcon tube and precipitated with 0.5 mL of isopropanol for each 1 mL of Trizol used initially. The solution was incubated at room temperature (15-30 ° C) for 10 minutes. A new centrifugation step at 12.240 × g for 10 minutes at 4 ° C. was performed. The pellet formed at the bottom of the tube contains the precipitated RNA. All supernatant was removed and the RNA pellet was washed with 75% ethanol (1 mL ethanol for each 1 mL of Trizol used). The solution was centrifuged at 4.285 × g for 5 minutes at 4 ° C., and the entire supernatant (ethanol) was removed again. To dissolve the pellet, 400 μL of DEPC water was added (if necessary, the temperature was increased to 50-60 ° C. to dissolve the pellet). After extraction, total RNA was quantified on a spectropolarimeter; its integrity was verified on a 2% agarose gel and finally stored at -80 ° C in an ultrafreezer.

Initially, the plasmid containing the fragment of interest was linearized. Reactions containing 30.6 μL DEPC water, 4 μL buffer, 1 μg purified plasmid, 0.4 μL BSA, and 1 μL restriction enzyme ApaI were incubated at 37 ° C. for 2 hours in a thermocycler. After this period, the plasmid was purified using one volume of phenol, mixed using a vortex for 2 minutes, and subjected to a centrifugation step at 11.750 × g for 10 minutes at 4 ° C. Next, the aqueous phase was collected and ammonium acetate (NH ₄ OAc) was added to a final concentration of 0.5M. Three volumes of cold 95% ethanol were added and the solution was incubated at -20 C for 1 hour. A new centrifugation step at 11.750 × g, 4 ° C., 15 minutes was performed and the supernatant was carefully discarded. The tube was opened; the pellet was dried for 5 minutes, then resuspended in 30 μL of DEPC water and stored in a freezer at −20 ° C.

Obtaining the Probe The transcription reaction for obtaining the probe was performed using MAXISScript ™ in vitro transcription (Ambion Inc.) kit. RNA polymerase T7 and P ³² - using labeled phosphate closed deoxynucleotides (CTP). First, all kit reagents were thawed and kept on ice. The exception is Transcription Buffer (registered trademark), which must be stored at room temperature. The transcription reaction using a total final volume of 20 μL was performed in a 1.5 mL microtube at room temperature according to the protocol described in the following table.

Table 3: Reagents used for the transcription reaction of radioactive probes

  Next, all reagents were mixed gently in a microtube and the reaction was transferred one at 37 ° C. and incubated using a thermocycler. 2 U of DNase I RNase free was added and the reaction was incubated for 15 minutes at 37 ° C. One volume of gel loading buffer (95% formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 0.5 mM EDTA, 0.025 SDS) is added to the reaction and the tube is left for 3-5 minutes , Heated at 85-95 ° C. and all samples were applied on a 5% polyacrylamide gel, 8M urea, 1 × TBE buffer.

  A final volume of 25 mL was prepared using 12.01 g urea, 3.12 mL 40% acrylamide: bis-acrylamide (19: 1) and 2.5 mL 10 × TBE. A small amount of water was further added to avoid exceeding the final volume of the gel. The solution was maintained in a magnetic stirring system equipped with a heater to dissolve the urea, then cooled; transferred to a cylinder and the final volume of the gel was filled with DEPC water. Next, 12.5 μL of TEMED was added, and finally 156.2 μL of 10% ammonium persulfate was added.

  This solution was immediately applied to a glass plate, the well comb was placed, and the polymerization was allowed to occur without moving the plate. The glass plate and the electrophoresis chamber can be washed and pretreated with RNase AWAY (registered trademark) (Invitrogen-Life Technology). On a smaller plate, 1-3 mL of Repel can be provided in the fume hood and the plate can be dried for 5-10 minutes. This treatment promotes gel removal after electrophoresis.

  Prior to electrophoresis, the plates were washed to eliminate excess urea, and after removal of the well comb, the wells were washed with running buffer with the aid of a syringe. The gel was pre-driven for 20-30 minutes using a predetermined voltage to the chamber, after which use was applied. Electrophoresis for about 2 hours was performed at 200-300 volts, depending on the size of the probe.

  Only the full length probe was gel purified. To accomplish this, after electrophoresis, plates were opened, the gel was covered with plastic film, and the autoradiographic film marked to guide band position identification was exposed for about 1 hour. The film was then developed by immersion in a developer for 5 minutes in a dark room, removing excess, immersing in water for about 3 minutes for washing, and finally immersing in fixative solution for 5 minutes. Next, the film was immersed in water and air-dried. The film and gel were aligned to locate the full-length transcript. The area of interest of the gel was removed and transferred to a microtube containing 350 mL of elution buffer (0.5 M ammonium acetate, 1 mM EDTA, 2% SDS). Tubes containing the probe were incubated at 4 ° C. overnight to maximize probe regeneration and stored at 20 ° C. until the time of hybridization. The kit for labeling the probe provided a positive control to monitor its quality.

  Probes were synthesized from control DNA containing a 250 bp rat β-actin gene insert, the linearized pTRIPLEscript plasmid, and hybridized to rat liver total RNA. They were also given in the kit. Two control tubes contained only yeast total RNA and were used to verify the activity of the RNase enzyme. In this way, the enzyme positive control tube contained RNase and buffer, while the negative control tube contained only buffer without enzyme.

Sample Hybridization and Digestion The ribonuclease protection assay (RPA) was performed using the HySpeed ™ RPA-Hyg-Speed Hybridization Ribonuclease Protection Assay (Ambion Inc.) kit. For each microtube, a labeled probe was added to total RNA (20 mg) (about 100-800 pg of 250 nt or 1-10 fmol of 2-8 × 104 cpm with high specific activity). Also, 30 mg of yeast total RNA was added to the sample to obtain a final 50 mg / sample. Two other enzyme control tubes containing 10 mL of yeast total RNA (50 mg) were obtained, one containing rat liver total RNA as a probe positive control. For co-precipitation of the probe + sample, the addition of NH ₄ OAc and three volumes of cold 95% ethanol 0.5M, after homogenization, was incubated tubes at 15 min -20 ° C.. The centrifugation step was performed at 4 ° C. for 5 minutes at maximum speed (minimum 8.160 × g). The supernatant was removed and the pellet was dried. 10 mL of HySpeed Hybridization Buffer® (preheated to 95 ° C.) was added to each sample and the tubes were immediately incubated at 95 ° C. (water bath or thermocycler). The sample was vortexed for several seconds and returned to 95 ° C. to dissolve the pellet. After complete dissolution of the pellet, the tubes were incubated at 95 ° C for 3 minutes and 68 ° C for 10 minutes. The temperature must be maintained at 68 ° C. and the transfer process must be less than 30 seconds. First, the HySpeed RNase Digestion Buffer® is thawed and a mixture of RNase A / T1 and buffer (1: 100 dilution-1 mL RNase in 99 mL buffer) is added to each sample for 100 mL of appropriate It was prepared in various volumes and stored at room temperature. 100 mL of mixed RNase A / T1 + buffer was added to each sample and to a control tube containing only yeast total RNA, and 100 mL of HySpeed RNase Digest® buffer without enzyme was added to one tube and the enzyme was added. The complete mixture containing was added to the other control tubes. Samples were vortexed and incubated at 37 ° C. for 30 minutes to digest. 150 mL of HySpeed Incubation / Precipitation Buffer® was added to the sample, vortexed quickly, and the tubes were incubated for 15 minutes in a -20 ° C freezer. After digestion, samples were centrifuged for 15 minutes at maximum speed at 4 ° C., the supernatant was removed and resuspended in gel loading buffer (usually 9-10 mL volume). After pipetting homogenization, the tubes were heated to 90-95 ° C. for 3-4 minutes and immediately transferred to ice to avoid regeneration. The sample was applied to a 5% polyacrylamide gel, 8M urea, diluted in 1 × TBE buffer, and subjected to electrophoresis at 200 to 300 volts for about 2 hours to separate protected fragments. After electrophoresis, standard procedures were followed for film exposure and development as detailed in the aforementioned protocol (FIG. 10).

Example 3: Gene expression-RT-qPCR-Experiment set in greenhouse For the expression test of the promoter of the gene Gmhsp17.6-L, the materials PI5995099, BRS133, 256-S, 259- Seeds derived from S, 266-S, JF7002, JF7027 and JF7056 were sown on germination paper in a growth chamber, and after 8 days the seedlings were transferred to plastic containers. In the greenhouse, these seedlings were infected with the larva meloidogine jawanica at the infection stage of development J2. The nematode population used was obtained from soybean plants from the variety Doko. A nematode egg extract from the root was obtained by grinding the material with a blender in a 0.5% hypochlorite solution for 30 seconds. The roots were washed in water and eggs were collected using a miniscreen with an exclusion size of 500. Thereafter, the free egg suspension was transferred to a hatching chamber set at 26 ° C., and every 24 hours, Javanica (J2) was collected and stored in a refrigerator. Javanica J2 was quantified in a Peters chamber. With the help of a pipette, inoculate 664 J2 / ml (per plant), bulk the roots from three soybean plants of each of the eight materials under test on days 1, 3 and 6 after inoculation. And transferred to liquid nitrogen for those inoculated with Javanica and those not inoculated (control treatment). Roots were stored in an ultra-freezer (-80 ° C) until the start of the RNA extraction experiment.

RT-qPCR primer design Primers used for real-time PCR (SoyHspPSC F 5 'GCT GTG TGT CAT TGT CAT CGA A3'; SoyHspPSC R5 'CAC GGT CTA TTT CTT GCC TAC ATC 3') was constructed using the Primer Express 2.0 (Appied) using the sequence pos stop codon (TAA-nucleotide position 884) of the Gmhsp17.6-L gene (GenBank accession number: M11317). Biosystems) program. These primers were used to amplify an approximately 80 bp fragment. The selected parameters applied to the Primer Express program to design the primers were: amplicon length of 50 bp to 150 bp (120 bp is recommended for RT-PCR), CG content of 40% to 60%, max. There are four G bases, primer Tm (melting temperature) between 58 ° C and 60 ° C, the maximum difference in Tm between F and R primers is 1 ° C, and up to four identical bases in a row should be avoided. Thereafter, to analyze primer dimer formation, the program OMIGA was used to observe the minimal presence of 6 free bases at the 3 'end.

Obtaining Total RNA for cDNA Synthesis To perform real-time PCR experiments, total RNA was extracted using Trizol reagent (Invitrogen-Life Technology). First, 1 mL of Trizol / sample was dispensed into a Falcon tube and heated to 55 ° C. Sample plant tissue was homogenized in liquid nitrogen and 0.1 g was dispensed into a tube maintained in nitrogen. Thereafter, 1 mL of heated Trizol was added to the sample, vortexed for 1 minute, spun down quickly, and incubated at 55 ° C. for 2 minutes. The sample was centrifuged at 16.000 × g at 4 ° C. for 20 minutes, the supernatant was transferred to a tube containing 200 μL of chloroform, and the residue was discarded. Samples were shaken and incubated at room temperature (at 22-25 ° C) for 2 minutes, then centrifuged at 16.000 xg for 30 minutes at 4 ° C. The supernatant was transferred to a new tube and 1/3 volume of 8M LiCl was added. After shaking, the tubes were incubated in a freezer at -80 ° C for 1 hour. To thaw the solution, the tubes were kept in a 40 ° C. water bath for 1-3 minutes and centrifuged at 16.000 × g at 4 ° C. for 30 minutes. The supernatant was removed, discarded, and 400 μL of 75% ethanol was added to each sample to avoid disturbing the pellet containing RNA, and the tubes gently inverted. A centrifugation step of 16.000 × g was performed at 4 ° C. for 5 minutes, and the supernatant was removed and completely discarded. Add 100 μL of ultrapure water to the pellet and gently tap the tube to dissolve the pellet (if the pellet does not dissolve quickly, another 100 μL of ultrapure water can be added, but in the next step The volume of sodium acetate and isopropanol should be doubled). Next, 10 μL of sodium acetate (3M) and 100 μL of isopropanol were added and the tube was gently inverted. The samples were incubated at -80 ° C for 30 minutes, transferred to a water bath at 37 ° C for 1-3 minutes, and centrifuged at 16.000 xg at 4 ° C for 15 minutes. The supernatant was removed, 400 μL of 70% ethanol was added, and the tube was again centrifuged at 16.000 × g at 4 ° C. for 15 minutes. The supernatant was removed, discarded and the pellet was dried on the bench for 10 minutes. Finally, 50 μL of ultrapure water (or 100 μL if the pellet does not dissolve immediately) was added and the pellet and solution were heated at 37 ° C. for 10 minutes to facilitate dissolution of the pellet. RNA was quantified and stored in a -80 ° C freezer. For the reverse transcription reaction, the RNA was diluted and cDNA synthesis was performed using reverse transcriptase (Moloney murine leukemia virus: M-MLV). In this way, 1.5 mg of total RNA was dispensed into microtubes, DEPC water was added to a final volume of 9 mL, 6 mM random primer was added to the reaction, and then incubated at 80 ° C. for 3 minutes. After this period, the tubes were cooled on ice and 14 mL of the mixture was added to the sample. This mixture was prepared according to the protocol described in the table below.

Table 4: Reagents used in the preparation of the mixture for cDNA synthesis Reagent Volume per reaction 5 x 6 mL of first strand buffer dNTP (2.5 mM) 4 mL
DTT (0.1M) 2mL
Reverse transcriptase 2mL

  The reaction was incubated for 1 hour at 37 ° C. using a thermocycler, followed by a 10 minute step at 65 ° C. The cDNA was stored at -20C.

RT-qPCR
The PCR reaction was performed using a Platinum® SYBER® Green qPCR SuperMix UDG (Invitrogen-Life Technology row) kit using the Thermocycler 7300 real-time system (Applied Biosystem) according to the manufacturer's instructions. Was done. As recommended by Applied Biosystem, amplification efficiency curves were performed for the target gene Gmhsp17.6-L and the endogenous control gene rRNA 18S (GenBank accession number: X02623.1) (used for sample normalization). Was. Experimental plates were set up with samples in triplicate measurements for both genes. The curve gives the slope, which is used to calculate the amplification efficiency of the primer and should be similar for both genes and close to 100% (value 1). Amplification reactions for relative quantification were performed using bulk cDNA from day 3 of collection for each of the samples analyzed. In separate experiments, two real-time PCR experiments for parental use and two for population samples were performed. Parental strains BRS133 and PI5955099, and the six resulting individuals from susceptible populations, 259-S, 259-S and 266-S, and resistant JF7002, JF7027 and JF7056 were analyzed for nematode and non-inoculation treatments. Was done. The reaction was performed in triplicate and consisted of 8.0 μL of ultrapure water, 0.5 μL of ROX, 12.5 μL of SYBER® Green qPCR SuperMix UDG, and 2 μL of bulk DNA (about 1.5 μg). . The cycling parameters for the amplification reaction were as follows: 50 ° C. for 2 minutes; 95 ° C. for 2 minutes; then 95 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 minutes. 45 cycles of seconds. Data was collected in the extension step (72 ° C.). After concluding the relative quantification, dissociation curves were performed to verify primer dimer formation, inspecific amplification, possible errors and contamination. Interpretation of the data generated by RT-PCR- was performed using SDS-Sequence Detection System (Applied Biosystem) software.

  In these analyses, the relative gene expression (RQ) calculations were performed separately and the samples were compared separately for the nematode inoculation and non-inoculation treatments using the uninoculated sample as the calibration (value 1). The RQ value was calculated using the ΔCt method. In this way, the level of gene expression (RQ) is calculated by subtracting the Ct of the target sample for each treatment by the endogenous control Ct, yielding ΔCt. This value is subtracted from the control sample's ΔCt (calibration-value 1) to give a value of ΔΔCt. RQ is obtained through Equation 2-ΔCt. Here, 2 corresponds to the sum of the target gene efficiency (100% = 1) and the synthesis of the endogenous control (100% = 1) obtained from the 100% efficiency curve (Livak and Schmittgen (2001), Methods 25). : 402-408). The efficiency of the target gene and the endogenous gene must be close to 100%, but not necessarily, they may be lower, however, they must be close. Then, in this case, the value 2 of Equation 2-ΔΔCt is replaced by the sum of the efficiencies of the target gene and the endogenous gene. Root-knot nematode M. For the analysis of parent strains BRS133 and PI5955099 in the treatment of inoculation and non-inoculation with Javanica, analysis of the susceptibility groups (256-S, 259-S and 266-S) using the uninoculated sample PI5955099 as a calibration (value 1X) For the resistant populations (JF7002, JF2027 and JF7056), the uninoculated susceptible sample 256-S was used as a calibration sample. This material was chosen due to the fact that sequencing of the promoter region of the Gmhsp17.6-L gene showed a low number of AT (n) inserts.

Example 4 Expression Cassette Constructs Containing Different Sizes of AT (n) Inserts in the Promoter Region of the Gmhsp17.6-L Gene Resistant individuals have a high number of AT (n) insertions in the promoter region of the Gmhsp17.6-L gene , And these individuals, when subjected to RT-PCR, had a clubroot nematode M. When confirmed to show high levels of expression of this gene when inoculated with Javanica, the expression cassette may be constructed to contain different numbers of AT inserts in the promoter region of the Gmhsp17.6-L gene. It was decided. The purpose of utilizing these constructs is that these promoters, along with the AT (n) insert, drive the expression of other genes and also respond to those genes, their activation or deactivation by different stresses. Can be evaluated. The cassette was used to transform soybean embryos from the nematode susceptible varieties BRS133 and Pintado using particle bombardment. In the resulting construct, the promoter of the Gmhsp17.6-L gene is located after the embryo's exposure to different stresses, before the Gus gene, which has expression via histochemical assays.

Expression cassettes containing the promoter regions of eight analyzed samples, BRS133, PI5955099, 256-S, 266-S, JF7002, JF2027 and JF7056, were obtained, for a total of 96 cassettes (12 for each material). Was. However, only two constructs were used in the next step of this test, which is AT (9), a cassette pAG1 / promoter containing an amplified promoter region from the susceptible parental BRS133 with fewer inserts. Gmhsp Cassette pAG1 / promoter Gmhsp containing BRS133 and amplified promoter region from individuals from resistant population JF7027 having AT (32) JF7027. Plasmid pAG1 was chosen as a template for constructing the expression cassette. The strategy designed was the removal of the act2 promoter from the actin gene present in the plasmid and the insertion of the amplified parental promoter BRS133 and the amplified promoter from the material to be analyzed, individuals of the resistant population JF7027. . This plasmid also shows the reporter gene Gus encoding the β-glucuronidase enzyme, which is permitted by visualization in transgenic embryos through histochemical assays if the transformation process with these cassettes is successful. Plasmid pAG1 also contains the herbicide Imazapyr (2- [4,5-dihydro-4-methyl-4- (1-methylethyl) -5-oxo-1H-imidazol-2-yl) due to the mutation at position 653. -3-Pyridinecarboxylic acid) (Tu et al. (2004), Weed control methods handbook; London: Academic) belongs to a class to which a herbicide imidazolinone, a herbicide imidazolinone, belongs. (AHAS) -Contains the promoter of the als / ahas gene encoding acetolactate synthase (ALS) (ahas 5), the coding sequence (ahas), and the terminator (ahast). In this way, this gene allows the selection of positive plants after transformation in a herbicide-containing medium. FIG. 13 shows a plasmid map. In order to develop this strategy, plasmid DNA extraction was performed using the Wizard Plus Maxipreps DNA Purification System (Promega) kit. In this way, a single colony was inoculated into 2-5 mL of LB medium containing the antibiotic ampicillin (0.5 mg) and incubated at 37 ° C. for 8 hours with rotation at 200 rpm (pre-inoculum). . One mL of the pre-inoculum was transferred to 500 mL of LB / ampicillin medium and incubated at 37 ° C. for 12-20 hours with rotation at 200 rpm. The cells are dispensed into a 250 mL tube, centrifuged at 5.000 × g for 10 minutes at room temperature, the supernatant is discarded, and a 15 mL cell resuspension (50 mM Tris-HCl, pH 7.5, 19 mM EDTA The pellet was completely resuspended in 100 μg / mL RNase A). 15 mL of cell lysis solution (0.2 M NaOH, 1% SDS) was added, mixed by inversion and the solution was incubated for 20 minutes at room temperature. Next, 15 mL of neutralization solution (1.32 M potassium acetate, pH 4.8) was added, the solution was inverted and mixed, resulting in the formation of white flakes, and the lysate was less viscous. The precipitated material contains genomic DNA, proteins, cell debris and SDS. A centrifugation step was performed at 14.000 × g for 15 minutes at room temperature, and the supernatant was filtered through a cylinder containing Whatman 1 filter paper. The volume was measured, transferred to a 50 mL centrifuge tube, 0.5 volume of isopropanol at room temperature was added, inverted and mixed. A new centrifugation step at 14.000 × g, 15 minutes at room temperature was performed, the supernatant discarded and the pellet resuspended in 2 mL of TE buffer. To the solution containing this DNA was added 10 mL of Wizard Maxipreps DNA Purification Resin®, and the resin + DNA mixture was transferred to Maxicolumn, which was connected to a vacuum pump. Vacuum was applied, 25 mL of column wash solution (80 mM potassium acetate, 8.3 mM Tris-HCl, pH 7.5, 40 μM EDTA, 55% ethanol) was added, and the vacuum was further applied. 5 mL of 80% ethanol was applied to the Maxicolumn and a vacuum was applied, after which the whole cough ethanol was passed through the column and the vacuum was maintained for another minute. Next, Maxicolumn was transferred to a 50 mL Falcon tube and centrifuged at 1.300 × g for 5 minutes. Vacuum was applied for an additional 5 minutes to dry the resin. Finally, the Maxicolum was transferred to the tube provided by the kit and 1.5 mL of preheated water (65-70 ° C.) was added to the column. After 1 minute, a centrifugation step (1.300 × g, 5 minutes, room temperature) was performed to elute the DNA. After extraction, plasmid DNA was quantified and its integrity was verified on a 1% agarose gel. The excision of the act2 promoter (-1000 bp) was digested with restriction enzymes. In the first step, pAG1 was subjected to double digestion with enzymes Pst1 and Not1. This digestion resulted in approximately 5.500 bp (corresponding to the promoter-ahas 5, coding sequence-ahas and terminator-ahast of the als / ahas gene) and 6.500 bp (Gus gene, act2 promoter and plasmid backbone, Ori and Nos). Corresponding) were obtained. The digestion reaction consisted of 10 μL of plasmid DNA (about 1.5 μg), 2 μL of React 2 buffer, 5.0 μL of Pst1 enzyme (10 U / μL), and 3 μL of ultrapure water. The tubes were incubated at 37 ° C for 2 hours using a thermocycler. After this period, another 2 μL of Pst1 enzyme (10 U / μL) was added to the solution and incubated at 37 ° C. for 2 hours using a thermocycler. In the same microtube, a reaction using Not1 was performed, and 3.5 μL of Not1 enzyme (10 U / μL), 1.5 μL of Reat 2 buffer, and 2 μL of NaCl (1 M) were added. After another 2 hour incubation at 37 ° C., after this period, an additional 2 μL Not1 enzyme was added. The reaction was maintained overnight using a thermocycler at 37 ° C. The pAG1-digested fragments were separated on a 0.8% agarose gel and the gel was extracted using the PureLink ™ Quick Gel Extraction (Invitrogen) kit according to the protocol described above. In the second step of the digestion, the larger fragment (6.500 pb) was digested with the restriction enzyme BamHI for excision of the act2 promoter. The reaction consisted of 0.5 μL BamHI enzyme, 15 μL React 3 buffer, 12 μL DNA digested fragment, and 1 μL ultrapure water. The microtubes were incubated for 2 hours at 37 ° C. using a thermocycler. After this period, an additional 0.5 μL of BamHI enzyme was added and the reaction was further incubated at 37 ° C. overnight. Fragments were separated on a 0.8% agarose gel and the larger fragments (corresponding to Gus gene, plasmid DNA, Ori and Nos) were purified according to the manufacturer's instructions PureLink ™ Quick Gel Extraction (Invitrogen) kit. The gel was purified using. To obtain an expression cassette construct containing a different amplified promoter from the parent strain BRS133 and the resistant individual JF7027, the ligation reaction was performed with 11 μL of ultrapure water, 2 μL (about 10 ng) of Ahas fragment from each tested sample, 1 μL ( About 10 ng) of the Gus fragment, 1 μL (about 10 ng) of the purified band derived from the Gmhsp17.6-L promoter, 4 μL of ligase buffer and 1 μL of T4 ligase enzyme were mixed. The reaction was incubated overnight at 14 ° C. The promoter region fragments from the two selected samples were prepared using the primers pSoyHspPstI F (5′GGG CTG CAG GAA TTC TGA AAT TGG GTC TTT TTG3 ′) and pSoyHspBamHI Amplified from genomic DNA using R (5 'CCC GGA TCC AAT GGG GAC ACT CGA GGT ATT3'). The restriction enzyme sites designed and synthesized in the primers allowed the cloning of the correct orientation of the promoter of the Gmhsp17.6-L gene at the same position where the act2 promoter was previously located. According to the protocol described above, E. coli. E. coli cells, strain DH10B, were electroporated using cassette ligation and grown overnight at 37 ° C. in LB medium. From the obtained colonies, a series of PCR amplification using specific primers and a digestion reaction using restriction enzymes were performed to verify that the cloning of the construct was successful. After this step, demonstrating the success of obtaining the expression cassette, a primer specific to the promoter region of the Gmhsp17.6-L gene, the Ahas gene (654 bp) of the pAG1 plasmid (Ahas1 Ahas2 F5'ACT AGA GAT TCC AGC GTC AC3 '; R5'GTG GCT ATA CAG ATA CCT GG3 ') and Nos terminator (Nos1 Nos3 F5′GAA TCC TGT TGC CGG TCT TG3 ′; PCR reactions using R5 'TTA TCC TAG TTT GCG CGC TA 3') were performed using all the obtained samples. FIGS. 14, 15 and 16 show the amplification of the promoter region of Gmhsp17.6-L, part of the Ahas and part of the Nos region, respectively, of 12 samples of each of the obtained expression cassettes.

Example 5: Study of transient expression or GUS (β-glucuronidase) activity Acquisition of transgenic plants via particle bombardment Expression cassette pAG1 / promoter GmHSP BRS133 and pAG1 / promoter GmHSP To obtain transgenic plants transformed with JF7027, plasmid DNA extraction was first performed according to the protocol described above. This technique resulted in a large amount of plasmid DNA sufficient for use in biovaritics, gene gun or particle bombardment transformation techniques. In this approach, the introduction of genetic material into plants is performed using microparticles and is usually made of gold or tungsten with a diameter of 0.4-0.2 mm, coated with endogenous DNA. The particles are coated with the DNA molecule of interest and accelerated and fired at high speed by a shock wave caused by a helium gas discharge under high pressure. When colliding with a target cell or tissue, it penetrates the cell wall and plasma membrane without destruction (Klein et al. (1987), Nature 327: 70-73), and endogenous DNA is microparticled by the action of cell fluid. And must be integrated into the plant genome. After being cultured and regenerated in vitro, these transgenic tissues or cells give rise to genetically modified plants (Brasileiro and Aragao (2001), Plant Biotechnol J. 3 (3): 113-121; Rech et al. (2008), Nat Protocol 3: 410-418).

Seed decontamination Soybean seeds derived from varieties BRS133 and Pintado which are both susceptible to root-knot nematodes were used. After weighing, the seeds were decontaminated by immersion in 70% ethanol for 10 minutes followed by 1% sodium (V / V) for 20 minutes. The seeds were washed three times with autoclaved distilled water in a laminar flow hood, embedded in twice as much water as the seeds, and kept soaked for hydration for about 16-18 hours.

Embryo Isolation and Preparation of Support Plates for Impact Embryos were isolated from hydrated seeds using a dehumidifier and an air conditioner with low room humidity. With the aid of sterile forceps and a surgical blade, the seeds were dissected, transferred to a Petri dish containing distilled water to remove the hypocotyl and avoid drying. Next, with the help of a stereomicroscope, the leaf primordium was excised, exposing the apical meristem area. Transfer the embryos to filter paper in a laminar flow hood to remove excess water, which can reduce the transformation efficiency, which acts as a barrier to refracting particles. For the collisions, the 16 mm diameter central salt (dead zone) was placed in a small petri dish containing MS medium (Murashige and Skoog Salt), 3% sucrose and 0.8% phytagel (pH 5.7) using sterile forceps. Designed. With the aid of a stereomicroscope, the embryo was placed in the groove in the plate, with the apical meristem area facing up. The carrier membrane support and support cylinder were sterilized by fire and the four fracture membrane sets were separated and kept immersed in isopropanol until use. Carrier membranes were collected in their supports.

Sterilization and Washing of Tungsten Particles In a centrifuge tube, weigh out 60 mg of M10 tungsten particles, which is sufficient material for about 100 shots. To this centrifuge tube, 10 mL of 70% ethanol was added; the solution was homogenized vigorously and maintained on a stirrer for 15 minutes at a speed sufficient to maintain movement. A centrifugation step at 15.000 g for 5 minutes was performed and the supernatant was removed and discarded with the aid of a micropipette to avoid inhibition of microparticle sedimentation. 1 mL of sterile distilled water was added to the tube, the mixture was homogenized vigorously using a stirrer and centrifuged again at 15.000 g for 5 minutes. The supernatant was discarded and the washing step was repeated twice. After a final wash and discarding the supernatant, the microparticles were resuspended in 1 mL of 50% glycerol (V / V).

DNA coating of tungsten microparticles Dispense 50 μL of the microparticle suspension into a 1.5 mL centrifuge tube and subject it to a sonicator for homogenization for 15 minutes, thus avoiding microparticle aggregation and uniform precipitation. Enabled. DNA equivalent to a minimum of 6 μg was added to the supernatant and gently homogenized with the aid of a micropipette. 50 μL of CaCl ₂ (2.5 M) was added to the solution and after gentle homogenization, 20 μL of spermidine was added. After further homogenization, the solution was incubated for 10 minutes at room temperature with gentle rotation on a suitable tube shaker. The tubes were spun down at maximum speed for 10 seconds and the supernatant was carefully removed and discarded to avoid resuspension of the microparticles. 150 μL of 100% ethanol was added; the solution was homogenized vigorously and centrifuged again at maximum speed (spin) for 10 seconds. The supernatant was removed and the washing step was repeated once more. After discarding the supernatant, in the final wash, 24 μL of 100% ethanol was added to the vigorously homogenized sample. Finally, a 3.2 μL aliquot of the supernatant was applied to the central region of each carrier membrane previously placed on the membrane support. Each precipitate is sufficient to prepare six carrier membranes containing microparticles coated with DNA. After application of the microparticles, the membrane was immediately stored on a plate containing silica gel and kept in a dissection chamber. That is because exposure of DNA-coated microparticles to conditions of air relative humidity above 50% allows aggregation, and consequently reduces the frequency of foreign gene expression.

Gene Gun Use and Collision Experiments With some modifications, the varieties BRS133 and B. susceptible to clubroot nematodes, according to the protocol developed by Aragao et al. (Crop. Sci. 42: 1298-1302 (2002)). The meristem site of the soybean seed embryo from Pintado was targeted for firing. Prior to impact, the laminar flow hood was cleaned with 70% ethanol and sterilized by UV irradiation (15 minutes). A retaining screen, a petri dish containing embryos, a set of four, ruptured membranes immersed in isopropanol, and a carrier membrane containing foreign DNA were maintained near the operator. The valve on the helium gas cylinder was opened and the pressure was set at 1.200 psi. Thereafter, a rupture membrane set (300 psi / membrane) was placed at the far end of the high pressure helium gas chamber, the sealing screw was tightly closed, the high pressure chamber was inverted and fitted to the vacuum chamber. The plate containing the material to be bombarded was placed, as well as a holding screen and carrier membrane support containing DNA coated microparticles on the support cylinder. Carefully positioned the support cylinder and closed the vacuum chamber. The value allowing vacuum in the chamber was slowly opened until the pressure reached 27 pol / Hg, at which time the valve was closed. The valve for pushing helium gas into the high-pressure chamber was opened, and when the rupture film showed bending due to the presence of helium gas, it was fired by pushing the trigger. After firing, the valve was closed, while the valve for releasing helium gas from the high pressure chamber was opened. Thereafter, the valve for releasing the vacuum was slowly opened, and the plate containing the embryo to be bombarded was removed from the apparatus. At each collision, these steps were repeated. After each collision, most embryos were transferred to different plates and subjected to abiotic and biological stress. However, some embryos were transferred to plates containing MS medium supplemented with BAP 5 mg / mL (benzylaminopurine) 3% sucrose, 0.6% agar (pH), in which, for induction of regeneration, Maintained for 18 hours and protected from light at 28 ° C.

Selection of Transgenic Embryos The distinction between transgenic cells and non-transgenic cells can be performed individually or linked to the gene of interest in the same transformation vector to introduce a selection gene that expresses a protein with enzymatic activity into the plant genome. It can be done by doing. According to the mode of action, marker genes are classified as genes that confer resistance to antibiotics, genes that confer resistance to herbicides, and marker genes with positive selection (Brasileiro and Dusi (1999), In: TORRES). Caldas, L.S .; BUSO, JA Ed. Cultura de tecidos. And transformamagao genetica de Plantas. Brasilia: Embrapa-SPI / Embrapa-CNPH. 2). The gene ahas is classified into genes that confer resistance to herbicides. This gene encodes a modified form of the acetohydroxylate synthase (AHAS) enzyme, also known as acetolactate synthase (ALS). Mutation of this sequence to position 653 results in replacement of serine by asparagine, resulting in a modified enzyme, which is not recognized by the herbicide class of imidazolinones. The meristem cells transformed with this gene are selected in the presence of the herbicide Imazapyr in a process based on total transfer of the selected molecule to the meristem site and inactivation of the endogenous AHAS enzyme. Since the selection agent is localized in the apical meristem, non-transgenic cells die and support the survival of transgenic cells growing in plants (Aragao and Brasileiro (2002), JP Physio, 14 (I). )). Thus, after regeneration, some embryos are transferred to plastic cups containing the selection medium MS, 3 & sucrose, 0.15 μM Imazapyr herbicide, 0.8% agar and B5 vitamins (pH 5.7), They were grown for about 45 days in a growth chamber at 28 ° C. with a 16 hour photoperiod. The light intensity was 50 μmol m−2s−1 and the relative humidity exceeded 80%. Next, the regenerated plants were transferred to plastic cups containing autoclaved sand: vermiculite (1: 1) and retouched with a nutrient solution (pH 6.6). Later, for acclimation, the cups were covered with plastic bags, sprinkled with nutrient solution as needed, and grown in growth chambers for an additional 28-30 days. After the passage of this organ of acclimation, the plastic bag can be removed and allowed to grow normally, via PCR technology with specific primers, as far as sample collection is possible for molecular analysis and identification of positive plants. Was.

Experiments using transgenic embryos subjected to various stresses It is susceptible to clubroot nematodes and various expression cassettes (parent-sensitive strain BRS133 (S-pAG1 / promoter GmHSP) BRS133) and embryos derived from the soybean varieties BRS133 and Pintado transformed with the resistant population JF7027 (including the promoter region of the Gmhsp17.6-L gene amplified from the R-pAG1 / promoter GmHSP JF7027). Stress, in this case, M. Javanica was inoculated with Javanica J2 and subjected to abiotic stresses, such as heat, cooling, salinity and dehydration (drought). Non-transgenic embryos (CN-negative control) and embryos transformed with the pAG1 plasmid alone (CP-positive control) were subjected to the same treatment. Heat stress was applied and embryos were maintained at 25 ° C (room temperature), 35 ° C and 45 ° C (in a greenhouse). Treatments at 4 ° C. (refrigerator) and 15 ° C. (greenhouse) were used for cooling stress. These stresses were applied to the embryos in solution at 2, 4, and 24 hours. Dehydration (drought) was performed in a greenhouse at 37 ° C. and maintained on filter paper at 2, 4, and 6 hours. For salinity stress, embryos were maintained at 200 mM and 400 mM NaCl concentrations for 24 hours. Biological stress was performed by maintaining embryos in a solution containing nematodes at the J2 developmental stage (2.00 / 3000 J2 / mL) for 24, 48 and 72 hours. FIG. 17 shows how the stress was applied.

Histochemical assays in embryos transformed with various expression cassettes and subjected to various stresses The expression product of the Gus gene is detected by detecting its enzymatic activity in plant tissues through histochemical assays. Can be used as a marker reporter of choice. This quantitative method uses 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X), which produces a dimer by the b-glucuronidase enzyme in the presence of oxygen to give an insoluble blue precipitate. -Gluc) Based on cleavage of the substrate. This methodology assists in gene expression control studies on tissue specificity, promoter isolation, transgenic plant identification, and migration conditions (Brasileiro (1998), In: Brasileiro, ACM; Carneiro, V. T.C. (ed.) Manual de transforma genetica de plantas.Brasia: Embrapa-SPI / Embrapa-Cenargen, 1998, p.143-154; Aragao et al. (2000), Arago et al. (2000). 101: 1-6)). Thus, embryos of the two genotypes, BRS133 and Pintado, transformed with different cassettes and then subjected to different stresses, are transferred to a plate and X-expressed in sufficient volume to cover the sample. Gluc reaction buffer was added. This buffer was prepared by diluting 8.5 mg of X-Gluc in 85 mL of dimethylformamide. Phosphate buffer (NaH2PO4-50 mM pH 7.0) was added to a volume of 17 mL and finally 17 mL of Triton X-100. After preparing this solution, it was stored in a refrigerator. Plates containing embryos and buffer were sealed and incubated in the dark at 37 ° C. for about 16 hours in the dark. Thereafter, the buffer was removed and the reaction was stopped by adding 1 mL of 70% ethanol. Samples were analyzed under a stereomicroscope (SQZ-DS4-BI (Tecnival)), images were acquired and the results were written. Histochemical assays showed that cultivar BRS133 showed a stronger response when subjected to the stress and then the reagent X-Gluc, indicating that the blue spots were clear and that the transformed cells This is because it is visually recognized in the restricted area shown. This insoluble blue precipitate is the product of the Gus gene reaction present in the construction cassette encoding β-glucuronidase, which reacts with the X-Gluc substrate to form a dimer that precipitates in the presence of O2. . When analyzing embryo assay results from the cultivar Pintado, no positive blue spots were detected for most of the stress. In this breed, endogenous GUS expression is higher, resulting in intact blue embryos, the results of which interfere with the analysis. Background endogenous GUS activity in soybean embryos has been reported in previous studies. Hu and co-workers (Plant Cell Reports, 9: 1-5, 1990) analyzed 53 different plant species and tested for intrinsic GUS activity in leaves, fruit parts, seeds and embryos. In this study, soybeans showed strong positive staining in mature embryos from fresh fruits and mature embryos from dehydrated seeds, indicating the result of endogenous GUS activity. In this way, heat stress was applied by subjecting the transgenic embryos to temperatures of 25 ° C., 35 ° C., and 45 ° C. for 2 hours, 4 hours, and 24 hours in a greenhouse. Figures 18, 19 and 20 respectively show these treatments for both varieties tested. Cooling stress was applied by subjecting the transgenic embryos to temperatures of 4 ° C. and 15 ° C. for 2 hours, 4 hours, and 24 hours in a refrigerator and greenhouse (FIGS. 21 and 22). For this stress, the differences between the varieties with respect to the positive blue spots of histochemical reactions are much more pronounced in Pintado embryos, they are structurally blue and are due to endogenous GUS activity. This is described in a previous document (Hu et al. (1990), Plant Cell Reports, 9: 1-5). Once again, embryos from breed BRS133 show a better response to histochemical assays, indicating that this breed is the one that should be selected and must be used in the next step of this study . Salinity stress experiments were performed by maintaining transgenic embryos in water as a control, NaCl concentrations of 200 mM, 400 mM for 24 hours. Embryos from cultivar BRS133 continued to respond positively to this assay, showing a blue spot in a restricted area, unlike previous stress, cultivar Pintado showed no apparent and detectable response. FIG. 23 shows the results. Drought stress was applied by placing embryos at 37 ° C. on filter paper and maintaining in a greenhouse for 2, 4, and 6 hours. Once again, cultivar BRS133 showed a positive response in the histochemical assay, while cultivar Pintado again showed a negative response. The latter showed non-specific blue staining of the embryo as a result of endogenous GUS expression, as shown in FIG. Root-knot nematode M. Using Javanica J2, an infection stage of Javanica, infection of embryos from varieties BRS133 and Pintado built up biological stress. Approximately 2000-3000 J2 / mL were incubated with the transgenic samples and maintained for 24, 48 and 72 hours. The results show that cultivar BRS133 (FIG. 25) showed a positive blue spot in the histochemical assay, indicating that cassette expression occurred satisfactorily. At the end of this time, the embryos have been modified in their aspect, exhibiting a reddish brown color, indicating that they contain control samples. This change is more visible after 72 hours and can be caused by infection at the time of infection by attacking the embryo with an infectious form of staining that injects hormones and substances, just as occurs in the roots of the host plant, Enables cell modification. However, the embryo, designated R, has the resistance cassette pAG1 / promoter Gmhsp Samples that had been transformed with JF7027 and had a low-impact aspect, and were inoculated at 72 hours, retained the original aspect and were white, which apparently indicates a higher number of AT ( n) Repeatedly, Gmhsp17. from individuals from resistant population JF7027. Cassettes containing the promoter region of the β-L gene suggest a better response to pathogen challenge, probably due to higher expression levels of the chaperone. Pintado-derived embryos also show a constitutive blue staining histochemical assay, as well as a negative color response, when exposed to biological stress by inoculation of J2 nematodes. And was more pronounced from 48 hours, especially from 72 hours. FIG. 26 shows the results.

  After being subjected to biotic and abiotic stress, histochemical assays detected the presence of the marker gene Gus, which can be concluded from the results shown for varieties BRS133 and Pintado, but high endogenous. Sexual activity was also observed, especially in the cultivar Pintado, suggesting that the different materials showed a clear response to the transformation process.

  While it is worth mentioning that Gus expression is just considered to be evidence of transformation, it is insufficient as definitive evidence of Gus gene integration into the plant genome (Potrykus (1990), Physiological Plantarum 79: 129). -134). Definitive evidence of integration of the endogenous DNA sequence is provided by genomic DNA hybridization with specific probes via molecular biology techniques.

Claims (18)

A method for controlling the expression level of a coding sequence in a plant,
(I) stably transforming plant cells with an expression cassette carrying an AT _(n) insert in a promoter element operably linked to the gene of interest;
(Ii) culturing a plant cell that has been stably transformed under plant cell growth conditions;
(Iii) regenerating a transgenic plant in which the cassette of (i) is stably integrated into its genome, wherein said transgenic plant has a different level of gene expression when compared to a control plant. The method shown.   The method of claim 1, wherein the AT inserts are different sizes.   2. The method of claim 1, wherein the AT insert is localized at a specific site within a promoter region.   The method according to claim 1, wherein the promoter region is related to a Gmhsp17.6-L gene.   The method according to any one of claims 1 to 4, which is used for controlling plant nematodes.   6. The method of claim 5, wherein the nematode is Meloidogyne javanica, which is preferentially a gall nematode.   An expression cassette comprising two promoter regions with different numbers of AT-inserts linked to the promoter region of the Gmhsp17.6-L gene.   The expression cassette of claim 7, comprising the ability to transform soy embryos.   The expression cassette according to claim 7 or 8, wherein the cassette is used for controlling plant nematodes.   The expression cassette according to claim 9, wherein the nematode is preferentially a root-knot nematode, meloidogine javanica. A method for obtaining a genetically modified plant comprising an AT _(n) -insert, comprising:
(I) stably transforming a plant cell with an expression cassette carrying an AT _(n) insert in a promoter element operably linked to the gene of interest;
(Ii) culturing a plant cell that has been stably transformed under plant cell growth conditions;
(Iii) The method comprising regenerating a stably genetically modified plant.   The method of claim 11, wherein the AT-inserts are different sizes.   12. The method of claim 11, wherein said AT-insert occurs at a specific site within a promoter region.   14. The method of claim 13, wherein said promoter region is associated with a Gmhsp17.6-L gene.   15. The method according to any one of claims 11 to 14, wherein the plant is present in the promoter region at the specific size of the AT-insert and has an overexpressed gene.   15. The method of any one of claims 11 to 14, wherein the plant has an under-expressed gene present in the promoter region at the AT-insert specific size.   17. The method according to any one of claims 11 to 16, wherein the plant is resistant to nematodes.   18. The method of claim 17, wherein the nematode is preferentially a root-knot nematode, meloidogine javanica.