CN1831010A - Regulatory factor for anti-reverse transcription of corn, and its coding gene and application thereof - Google Patents

Abstract

The invention discloses a maize anti-reverse transcription regulation factor, its coding gene and application. The corn anti-reverse transcription regulatory factor is a protein with one of the following amino acid residue sequences: 1) SEQ ID No.: 1 in the sequence listing; 2) the amino acid residue sequence of SEQ ID No.: 1 in the sequence listing through One to ten amino acid residues are substituted, deleted or added, and the protein interacts with the LTRE cis-element to improve plant stress resistance. This regulatory factor can act on the LTRE cis-acting elements in the promoter regions of multiple anti-abiotic stress-related genes, regulate the expression of multiple anti-abiotic stress-related genes, and improve the plant's resistance to abiotic stresses such as drought, low temperature, and salinity. Resistance. LBF is of great significance for cultivating stress-tolerant plant varieties, especially stress-tolerant maize varieties, and improving the yield of crops, especially maize.

Description

A maize anti-reverse transcription regulatory factor and its coding gene and application

technical field

The present invention relates to stress-related transcriptional regulatory factors in plants and their coding genes and their applications, in particular to a transcriptional regulatory factor and its coding genes for resistance to abiotic stresses such as low temperature, drought, and salinity in maize, and their use in cultivating stress resistance Improving applications in plants.

Background technique

Chilling injury is a natural disaster that often occurs in agricultural production, which greatly limits the planting of early spring crops and the harvest of late autumn crops.

Studies have shown that under low temperature conditions, plants will actively mobilize their internal defense systems to resist external adversity stress. At this time, great changes will occur in plants, such as the synthesis of heart protein, the transformation of metabolism, and the accumulation of anti-adversity substances etc. (Hans J, Adaptations to environmental stresses., Plant Cell, 1995, 7: 1009-1111). The resistance of plants to low temperature stress does not rely solely on one or two functional genes. There are many proteins involved in the response of plants to resist low temperature stress. At present, more than 100 genes related to low temperature stress have been found, and they synergistically regulate plant growth and development. Physiological, biochemical and metabolic changes can improve the resistance of plants to low temperature stress, and the existing research results have also proved this point of view. For example, people such as Steponkus transformed the COR15a gene into Arabidopsis (Steponkus P.L., Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis. PNAS, 1998, 1998: 14570-14575), Zhu B et al transformed the HVA1 gene into In rice (Zhu B, Overexpression of a 1-pyrroline-5-carboxylate synthetase gene and analysis of tolerance to water-and salt-stress in transgenic rice., Plant Sci, 1998, 139: 41-48), obtained by the above method Although the cold resistance of transformed plants has been improved, the actual effect is not ideal.

In order to improve the resistance of plants to abiotic stress, people have carried out a lot of research on the genes of plant resistance to abiotic stress, and began to turn to the research of transcriptional regulatory genes closely related to abiotic stress, because transcription factors can regulate the stress-related genes. The expression of a series of genes, not just one gene, so transcription factors play an important role in improving the ability of plants to resist stress, and have gradually become a research hotspot. In recent years, satisfactory research results have been obtained, such as DREB1A (DRE binding factor) factor, which can bind DRE (Droughtresponse element) cis-element to regulate the expression of RD29A, RD17, ERD10, KIN1, COR15a and other genes (Qiang Liu, Two transcription factors, DREB1 and DREB2, with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways indrought-and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 1998, 10: 13691- Improving plant drought, salt, and freezing tolerance by gene transfer of a single stess-inducible transciption factor., Nature Biotechnology, 1999, 17: 287-291); ABF (ABREbinding factor) factor can be combined with ABRE (ABA response element) cis Components that regulate the expression of RD28B, RAB18, ICK1 and other genes (Joung-youn Kang, Arabidopsis Basic Leucine Zipper Proteins That Mediate Stress-Responsive Abscisic Acid Signaling., Plant Cell, 2002, 14: 343-357). The results of transgenic experiments showed that the above-mentioned transcription factors can significantly improve the resistance of plants to abiotic stress.

The study of transcriptional regulatory genes related to cold resistance first started from the model plant Arabidopsis. Kisten R. et al. isolated the Arabidopsis CBF1 (C-repeat binding factor) factor, and CBF1 can interact with the C-repeat cis-element , regulate the expression of COR6.6, COR15a, COR47, COR78 and other genes (Kirsten R, Arabidopsis CBF1 Overexpression Induces COR Genes and Enhances Freezing Tolerance., Science, 1998, 280: 104-106), the results of transgenic experiments show that the CBF1 gene The cold resistance performance of Arabidopsis thaliana was significantly improved. In addition, it has been found that the promoter regions of multiple functional genes related to cold resistance contain low temperature response elements (LTRE), such as COR15a gene, COR78/LTI78/RD29A gene, KIN1, KIN2 gene and RAB18 gene (Liu Qiang, Zhang Guiyou, Chen Shouyi. The structure and regulation of plant transcription factors. Science Bulletin, 2000, 45(14): 1465-1474). Recently, Ming-Jun Gao et al. isolated BNCBFs (Brassica napus CBFs) factors interacting with LTRE cis-elements from rape, and BNCBFs were induced by low temperature (Ming-Jun Gao, Regulation and characterization of four CBF transcription factors from Brassica napus . Plant Molecular Biology, 2002, 49: 459-471). However, the trans factors that interact with LTRE cis elements have not been reported in monocots, especially in the important crop maize.

Contents of the invention

The purpose of the present invention is to provide a maize anti-reverse transcription regulator and its coding gene.

The maize anti-reverse transcription regulatory factor provided by the present invention is named LBF (LTRE Binding Protein), derived from Zea mays L., and is a protein with one of the following amino acid residue sequences:

1) SEQ ID №: 1 in the sequence listing;

2) The amino acid residue sequence of SEQ ID №: 1 in the sequence table is substituted, deleted or added with one to ten amino acid residues, and has a protein that interacts with the LTRE cis-element to improve plant stress resistance.

SEQ ID №1 in the sequence listing consists of 267 amino acid residues, and the DNA core sequence combined with LTRE is "CGAC".

The coding gene (LBF) of the above-mentioned maize anti-reverse transcription regulatory factor is one of the following nucleotide sequences:

1) The DNA sequence of SEQ ID №: 2 in the sequence listing;

2) A polynucleotide encoding the protein sequence of SEQ ID №: 1 in the sequence listing;

3) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID No. 2 in the sequence listing under high stringency conditions.

The high stringency conditions can be 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution, hybridization at 65°C and membrane washing.

SEQ ID №: 2 in the sequence listing consists of 1042 bases, and its open reading frame is from the 117th to the 920th base at the 5' end, encoding the amino acid residue sequence of SEQ ID №: 1 in the sequence listing protein.

The expression vector, transgenic cell line and host bacteria containing the gene of the present invention all belong to the protection scope of the present invention.

Primer pairs for amplifying any fragment of LBF are also within the protection scope of the present invention.

Using the plant expression vector, the LBF coding gene of the present invention is introduced into the plant cell, and the transgenic cell line and the transgenic plant with enhanced tolerance to adversity stress can be obtained.

When using LBF to construct a plant expression vector, any enhanced promoter or inducible promoter can be added before the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding selectable marker genes (GUS gene, luciferase gene, etc.) that can be expressed in plants or resistant Antibiotic markers (gentamicin markers, kanamycin markers, etc.). Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

The plant expression vector carrying the LBF of the present invention can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated, and transform plant tissues grown into plants. The transformed plant host can be either a monocotyledonous plant such as corn, rice, or wheat, or a dicotyledonous plant such as soybean, rapeseed, or cotton.

The present invention isolates and clones the LTRE-binding protein LBF from the important crop corn, which belongs to the AP1 class of transcription factors, is a transcriptional regulatory factor with binding specificity to LTRE, and can regulate the expression of more stress-related genes. The DNA core to which LBF binds The element sequence is "CGAC", which is obviously different from the reported transcription factors such as CBF1, DREB1A, BNCBF, osDREB, etc. The DNA core sequence they bind to is "CCGAC". Experiments have proved that this regulatory factor can act on multiple anti-abiotic The LTRE cis-acting element in the promoter region of stress-related genes regulates the expression of multiple anti-abiotic stress-related genes and improves the resistance of plants to abiotic stresses such as drought, low temperature, and salinity. LBF is of great significance for cultivating stress-tolerant plant varieties, especially stress-tolerant maize varieties, and improving the yield of crops, especially maize.

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Description of drawings

Figure 1 shows the results of the binding specificity analysis of LBF and LTRE in yeast cells

Figure 2 is the result of the functional analysis (EMSA experiment) of the combination of LBF and LTRE in vitro

Figure 3 is the DNA core sequence analysis results of LBF combined with LTRE

Figure 4 shows the verification of LTRE binding specificity and transcriptional activation function of LBF in yeast

Figure 5 shows the expression of LBF gene under stress conditions such as salt, drought, hydrogen peroxide, ABA, and low temperature

Figure 6 shows the specific expression of LBF during the development of maize immature embryos

Figure 7 shows the results of the cold resistance test of LBF transgenic Arabidopsis

Figure 8 shows the results of the salt tolerance test of LBF transgenic Arabidopsis

Figure 9 is the results of the drought resistance test of LBF transgenic Arabidopsis

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified. The primers and DNA fragments used were synthesized by Beijing Saibaisheng Bioengineering Company, and the sequencing work was completed by Shanghai Boya Biotechnology Co., Ltd.

Embodiment 1, the acquisition of LBF gene

1. Construction of maize immature embryo cDNA library

1. Extraction of total RNA and isolation of mRNA from immature maize embryos

Get 1g of immature embryos on the 17th day (17dpp) after pollination of corn variety Qi 319, use RNAgents Total RNAIsolation System kit (Promega Company) to extract the total RNA of the above-mentioned immature embryos of maize, then use PolyATtract mRNA Isolation System (Promega Company) from the above-mentioned mRNA was isolated from total RNA from immature maize embryos.

2. Construction of maize 17dpp immature embryo cDNA library

Using the mRNA of maize 17dpp immature embryos obtained in step 1 as a template, use GibcoBRL's SuperScriptTMPlasmid System for cDNA Synthesis and Plasmid Cloning kit and operate strictly according to the kit instructions to construct a cDNA library of maize 17dpp immature embryos, and finally the library capacity is 5.2×10 ⁶ cfu cDNA library of maize 17dpp immature embryos.

2. Screening LBF-encoding gene from maize 17dpp immature embryo cDNA library by yeast one-hybrid method

1. Construction of 4mer LTRE bait vector and 4mer mutant LTRE (mLTRE) rescue vector

1) Synthesize LTRE homeopathic elements (two complementary DNA fragments), the sequence is as follows:

LTRE(+): 5'-ATTTCATGGCCGACCTGCTTTTT-3'

LTRE(-): 5'-AAAAAGCAGGTCGGCCATGAAAT-3'

Take 20 μL of LTRE(+) and LTRE(-) at a concentration of 1 μg/μL each, mix well, add 4 μL of 3M NaOAc and 100 μL of absolute ethanol, and place at -20°C for 30 minutes to centrifuge to precipitate DNA. The precipitate was washed once with 70% ethanol, then dried to evaporate the ethanol, and then annealed by adding 6.5 μL sterile water and 1 μL 10×T4 _{polynucleotide} kinase (Promega Company) buffer, the annealing conditions were: 88°C for 2 min, 65 10min at ℃, 10min at 37℃, 5min at 25℃; add 1.5μL 20mM ATP and 1μL _T4 polynucleotide kinase, react at 37℃ for 2 hours, and extract with phenol-chloroform (mixing ratio: 1:1) and chloroform respectively Once, DNA was precipitated with absolute ethanol. Add 2 μL 10× ligase buffer, 1 μL ligase (5units/μL), 17 μL sterile water, connect at 16°C for 12-24 hours, perform 2% agarose gel electrophoresis for detection, and obtain a DNA fragment with a size of about 80 bp , using the DNA Fragment Rapid Purification/Recovery Kit (Dingguo Company) to recover the fragment, and clone it into the vector pBSK+ (Clontech Company) that has been digested by SpeI and filled in, and obtained by sequencing. The recombinant vector of the double-stranded DNA fragment of the element-bound DNA core element sequence is named pL4.

2) Synthesize LTRE homeopathic elements containing mutations, the synthetic sequences are as follows: mLTRE(+): 5'-ATTTCATGattagttTGCTTTTT-3' and mLTRE(-): 5'-AAAAAGCAaactaatCATGAAAT-3', use the same method to obtain the transcripts containing mutations The recombinant vector of the double-stranded DNA fragment of the DNA core element sequence combined with the factor and the LTRE homeopathic element is named pmL4.

3) Plasmid vectors pL4 and pmL4 were both digested with BamHI and Xba I and purified, and then respectively combined with the similarly treated vector pRS315His(Leu ⁺ ) (Wilson TE et al, Identification of the DNA binding site for NGFI-B by genetic selection in yeast. Science 252: 1296-300 (1991); ligated with T ₄ DNA ligase to obtain bait vector pRSL4(Leu ⁺ ) containing LTRE DNA fragment and rescue vector pRSL4(Leu ⁺ ) containing mLTRE DNA fragment.

2. Screening of maize 17dpp immature embryo cDNA library

Preparation of yeast yWAM2 (Leu ^- , His ^- , Trp ^- ) (Cheng X, Boyer JL, Juliano RL. Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library. Proc. Natl. Acad. Sci. USA 1997, 94: 14120-14125) competent cells, the bait vector pRSL4 (Leu ⁺ ) was transformed into the yeast strain yWAM2 (Leu ^- , His ^- , Trp ^- ), the transformation method was carried out according to the Two Hybrid System TRAFO protocol, and the integrated Yeast strain yL4 (His ^- , Trp ^- ) with pRSL4; then use yeast strain yL4 (His ^- , Trp ^- ) to screen the cDNA library of maize 17dpp immature embryos constructed in step 1, and the transformation method is the same as above, and the transformed cells are coated with His ^-Cultivate at 28°C for 3-5 days on the selection medium (Sigma Company). After the yeast grows, extract the yeast plasmid. The extraction method can refer to the literature (Christine Guthrie, Method I: Quick Plasmid DNA Preparations from Yeast, Methods in Enzymology, 1991, 194: 322); then the yeast plasmid was transformed into E.coli DH5α, and the plasmid of the positive clone was extracted and analyzed with Not I and Sal I, which showed that a DNA fragment of about 1.2Kb could be excised, and the obtained Sequence analysis of the positive clones showed that the gene encoding the library protein combined with the LTRE homeopathic element in the bait vector was obtained. This gene has the polynucleotide sequence of SEQ ID No. 2 in the sequence table, consisting of 1042 bases Composition, its open reading frame is from the 117th to the 920th base at the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID №: 1 in the sequence listing. Searching for the full sequence of the gene in GeneBank did not find the same sequence. The amino acid residue sequence analysis of the protein encoded by it found that it has the same conserved domain as that of AP1 transcription factors, and the 60th to 123rd amino acid residues from the N-terminal are the conserved domains of AP1 transcription factors, proving that The gene encoding the trans factor interacting with the LTRE cis-element in maize was obtained by yeast one-hybrid method, which was named LBF, and the protein encoded by the gene was named LBF.

Embodiment 2, the binding specificity analysis of LBF and LTRE

1. Binding specificity analysis of LBF and LTRE in yeast cells

The recombinant vectors pL4 and pmL4 were transformed into yeast yWAM2 (Leu ^- , His ^- , Trp ^- ) to obtain the yeast strain yWAM2/pL4 integrated with pL4 and the yeast strain yWAM2/pmL4 integrated with pmL4, and then the screening library in Example 1 The resulting plasmid transformation containing the LBF gene was transformed into recombinant yeast yWAM2/pL4 and yWAM2/pmL4 respectively, and then at 28°C, cultured for 3-5 days on the His ^- selection medium, the results as shown in Figure 1 (A in the figure is Recombinant yeast yWAM2/pL4, B in the figure is recombinant yeast yWAM2/pmL4), only recombinant yeast yWAM2/pL4 can grow, but recombinant yeast yWAM2/pmL4 cannot grow, indicating that LBF can specifically bind to the LTRE element in yeast, thereby activating the reporter The expression of the gene HIS3 gene, so it can grow on the His ^- selection medium. On the contrary, because LBF cannot combine with mLTRE, it cannot activate the expression of the reporter gene HIS3 gene, so it cannot grow on the His ^- selection medium. In vivo has binding specificity for LTRE.

2. Functional analysis of the combination of LBF and LTRE in vitro (EMSA experiment)

1. Obtain purified LBF protein

The full-length gene of LBF was cloned into the prokaryotic expression vector pGEX4T-1 (Amersham Pharmacia Biotech Company), then the recombinant expression vector was transformed into E.coli BL21, and at 37°C, the expression was induced with 0.3mM IPTG for 2-3 hours. The expression product was detected by SDS-PAGE electrophoresis, and a protein with a molecular weight of about 56KD was expressed, which was consistent with the size of the expected LBF (29.4KD) and fused 26KD GST protein. The expressed LBF protein was purified according to MicroSpin ^TM GST Purification ModuLe protocol of Amersham Pharmacia Biotech Company, and used for EMSA experiments.

2. Isotope-labeled LTRE and mLTRE probes

The LTRE probe sequence is: 5'-ATTTCATGGCCGACCTGCTTTTT-3'

The mLTRE probe sequence is: 5'-ATTTCATGattagttTGCTTTTT-3'

Promega’s DNA 5’End-Labeling System was used for isotope labeling of LTRE and mLTRE probes. The reaction system was: LTRE (or mLTRE) probe 1 μL, T ₄ PNK 10×buffer 5 μL, γ- ³² P-ATP 3 μL, T ₄ PNK (10U/μL) 2 μL, H ₂ O 39 μL; reaction conditions: 37°C for 20 minutes; add 2 μL of 0.5MEDTA, 68°C for 10 minutes to inactivate; 37°C for 10 minutes; store at 4°C for later use.

3. Binding reaction between LBF protein and DNA and non-denaturing SDS-PAGE detection

The reaction system for binding LBF protein to DNA is: 5× binding buffer (containing 125mM HEPES-KOH pH7.6; 50% glycerol; 250mM KCl) 4 μL, LBF (purified LBF protein prepared in step 1) about 4 μg (9 μL ), 1 μL of 1M DTT, 1 μL of LTRE (or mLTRE) probe labeled in step 2, 4 μL of H ₂ O. After mixing them, react in ice bath for 30 minutes, then add 3 μL of loading buffer (sterile water containing 0.025% bromophenol blue), and carry out non-denaturing polyacrylamide gel electrophoresis detection, formula of 5.4% polyacrylamide gel For: 30% acrylamide 9mL, 10X electrophoresis buffer 5mL (containing glycine 142.7g/L, EDTA 3.92g/L, Tris 30.28g/L), 50% glycerol 2.5mL, deionized water 33mL, 10% APS 400μL , TEMED 25 μL. After gelation, use 1× electrophoresis buffer for electrophoresis, pre-electrophoresis for 10 minutes (300V), then load the sample, and continue electrophoresis for 1 hour (300V); after electrophoresis, stick the gel with filter paper, and seal the comparison with plastic wrap. Afterwards, press the X-ray film for 1 hour; wash the film, develop it for 2 minutes, and fix it for 5 minutes. The results are shown in Figure 2 (swimming lane 1 is LBF protein, and swimming lane 2 is LBF protein). There is an obvious electrophoresis band in swimming lane 1, which is the result of LTRE being blocked by LBF protein. On the contrary, mLTRE is not blocked by LBF protein. stagnation, indicating that the expression product of the LBF gene also has the binding specificity of LTRE in vitro.

Example 3, DNA core sequence analysis of LBF combined with LTRE

Site-directed mutations were performed on the possible core sequence "GCCGACC" of LTRE, and the mutation sequences of m1-m7 were as follows (underlined bases are mutation sites):

LTRE: 5'-ATTTCATG GCCGACC TGCTTTTT-3'(CK)

m1: 5'-ATTTCATG a CCGACCTGCTTTTT-3'

m2: 5'- ATTTCATGGtCGACCTGCTTTTT -3'

m3: 5'- ATTTCATGGCtGACCTGCTTTTT -3'

m4: 5'-ATTTCATGGCC a ACCTGCTTTTT-3'

m5: 5'-ATTTCATGGCCG gCCTGCTTTTT -3'

m6: 5'- ATTTCATGGCCGAtCTGCTTTTT -3'

m7: 5'-ATTTCATGGCCGAC t TGCTTTTT-3' The same method as in Example 2 was used for EMSA experiments, and the results are shown in Figure 3. Lanes 1-7 are LBF and m1, m2, m3, m45, m5, m6, The combination product of the m7 probe sequence, and the lane CK is the control, which represents the combination product of the LBF and LTRE probe sequence, indicating that the DNA core sequence for the combination of LBF and LTRE is "CGAC".

In order to further confirm the specificity of LBF binding to the LTRE cis-element, the m1-m7 cis-element with single base mutation was constructed on pRS315His(Leu ⁺ ), and the yeast was transformed (the method was the same as in Examples 1 and 2). Cultured on His ^- selection medium, cultured at 28°C for 3 days, and counted separately. As a result, when the transformation efficiency was completely consistent, the number of colonies grown on m1 and m7 was basically the same as that of wild-type LTRE (CK), and the number of colonies grown on m2 About 30% of the wild type, m3, m4, m5, m6 have no colony growth. It shows that although the base mutation in m2 has an effect on the combination of LBF and LTRE, LBF can still combine with LTRE, which is consistent with the results of EMSA in vitro experiments, proving that the DNA core sequence bound by LBF is "CGAC".

Example 4, LBF transcriptional activation test in yeast cells

The full-length cDNA of LBF was cloned into the yeast expression vector YepGAP(Trp ⁺ ) (Qiang Liu, Twotranscription factors, DREB1 and DREB2, with an EREBP/AP2DNA binding domainseparate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression , respectively, in Arabidopsis.PlantCell, 1998, 10: 1391-1406), a recombinant yeast expression vector containing LBF full-length gene was obtained, named YepGAPLBF, and then YepGAPLBF was transformed into recombinant yeast containing LTRE elements in Example 2 yWAM2/pL4 and recombinant yeast yWAM2/pmL4 containing mLTRE elements were cultured on His ^- selection medium at 28°C for 3-5 days. The results are shown in Figure 4 (A is YepGAPLBF transformation of recombinant yeast yWAM2 containing mLTRE elements /pmL4, B is the recombinant yeast yWAM2/pL4 transformed with YepGAPLBF containing LTRE elements), indicating that the yeast containing LTRE elements transformed with YepGAPLBF can grow, but the yeast containing mLTRE elements cannot grow, indicating that LBF can specifically bind to LTRE elements in yeast , thereby activating the expression of the reporter gene HIS3, so it can grow on the His-selection medium. On the contrary, because LBF cannot combine with mLTRE, it cannot activate the expression of the reporter gene HIS3 gene, so it cannot grow on the His ^- selection medium, so , LBF not only has binding specificity to LTRE in yeast, but also has transcriptional activation function.

Example 5. Analysis of expression specificity of LBF under abiotic stress conditions and during the development of immature embryos

1. Analysis of expression specificity of LBF under abiotic stress conditions

1. Processing of corn materials

Take corn seeds, imbibition for 24 hours, plant them in flower pots, and cultivate them for about 20 days at 28°C and light time of 12 hours/day. After the corn seedlings grow three leaves and one core, perform the following treatments under different conditions:

1) Chilling damage treatment: Place corn seedlings in an incubator at 2°C, and cultivate them for 48 hours under 12hr/day light conditions, take out and wash off the soil on the roots, freeze them with liquid nitrogen, and store them at -80°C for later use.

2) Salt treatment: place corn seedlings in 0.6%, 0.8%, and 1% NaCl solution respectively, and cultivate them for 3 days under 12hr/day light conditions, take out and wash off the soil on the roots, and quickly freeze them with liquid nitrogen. Store at -80°C for later use.

3) Drought treatment: the corn seedlings were placed in soils with a water content of 8% (mixing 920g dry soil and 80mL water), 10%, and 13% respectively, under 12hr/day light conditions, cultivated for 3 days, took out and washed The soil on the roots was removed, quick-frozen with liquid nitrogen, and stored at -80°C for later use.

4) ABA treatment: put corn seedlings in 10 ^-4 M, 10 ^-5 M, 10 ^-6 M ABA solutions respectively (take 5mg of ABA and dissolve it in 0.1N KOH, and dilute it to 95mL water to get 10 ^-4 M ), under 12hr/day light conditions, cultivated for 24hr, took out and washed off the soil on the roots, quick-frozen with liquid nitrogen, and stored at -80°C for future use.

5) H ₂ O ₂ treatment: Put corn seedlings in 10mM H ₂ O ₂ (1.13mL 30%H ₂ O ₂ /L), 60mM H ₂ O ₂ (6.78mL 30%H ₂ O ₂ /L), 150mM In an aqueous solution of H ₂ O ₂ (14.95 mL 30% H ₂ O ₂ /L), under the condition of 12 hr/day light, cultivate for 24 hrs, take out and wash off the soil on the roots, freeze them with liquid nitrogen, and store them at -80°C for later use.

6) Water treatment (HCK): put the corn seedlings directly in water, cultivate them for 24 hours under the light condition of 12 hours/day, and store them at -80°C for later use.

7) Control treatment (CK): The corn seedlings without any treatment were directly frozen at -80°C as a control.

2. Extraction of RNA and removal of DNA in corn materials

1) Take about 200 mg of corn materials treated by the above different methods, grind with liquid nitrogen, and use RNAgents TotalRNA Isolation System Kit (Promega Company) to extract RNA according to the operation procedure of the kit manual.

2) Dissolve RNA in 85 μL of water, add 5 μL of RQ1 RNA Free DNase (1U/μL) (Promega) and 10 μL of 10× buffer, and incubate at 37°C for 5 min to remove DNA contamination.

3) Add 100 μL of phenol-chloroform for extraction once, take the supernatant, precipitate RNA with an equal volume of isopropanol, wash the RNA precipitate once with 70% ethanol, dissolve the RNA in 50 μL sterile water,

4) The concentration of quantitative RNA is 1 μg/μL.

3. Unified template DNA concentration by RT-PCR method

1) Use the RNA extracted in step 2 from _differently treated corn materials as a template, and synthesize its cDNA by reverse transcription. ), dNTP1μL (10mM) and water 27μL, after mixing, 65℃ for 5min, 0℃ for 2min; add 5×First-Strand buffer 10μL, DTT (100mM) 5μL and RNase Inhibitor 10U (40U/μL), after mixing for 42 ℃ 2-5min; add SuperScript II 1μL (200u/μL) (Gibcol BRL company), after mixing, 42℃ for 50min, 70℃ for 15min to inactivate, set aside.

2) Design the following primers according to the maize actin gene (Maize Actin1, Accession NO.J01238) registered on GenBank:

mAct1 F: 5'-CAC CTT CTA CAA CGA GCT CCG-3'

mAct1 R: 5'-TAA TCA AGG GCA ACG TAG GCA-3'

Using the cDNA of differently processed corn materials obtained in step 1) as a template, under the guidance of primers mAct1F and mAct1R, PCR amplification was carried out. The PCR reaction system was: 1 μL of template, 10 μL of 2×PCR buffer, 1 μL of 10 mM dNTP, 10 μM mAct1 F 1 μL, 10 μM mAct1 R 1 μL, Taq 1U, sterilized water 6 μL. The PCR reaction conditions were as follows: first at 94°C for 2 min; then at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec, a total of 30 cycles; finally, at 72°C for 5 min. After the reaction, agarose gel electrophoresis detection was carried out, showing that the amplified band had a size of 405bp. If the genomic DNA of the plant material was used as a template, a 512bp band (containing a length of 107bp) could be amplified. intron sequence). Then dilute the template DNA according to the electrophoresis results of the PCR product, adjust the amount of the template DNA until the amount of the DNA band amplified by the mAct1 F and mAct1 R primers is basically the same, so that the content of the template cDNA in each microliter solution is basically the same. unanimous.

4. PCR amplification of LBF gene

1) according to the LBF gene sequence design primer that embodiment 1 obtains, primer sequence is as follows:

Primer 1: 5'-CTT GAG CAC GTG CCT GTG AA-3'

Primer 2: 5’-TTA ACT TTG CTA GTA GTA GCT CC-3’

Using the cDNA of differently treated maize materials at a uniform concentration in step 3 as a template, PCR amplification was carried out under the guidance of primers 1 and 2. The PCR reaction system was: 1 μL of template, 10 μL of 2×PCR buffer, 1 μL of 10mMdNTP, 10uM mAct1 F 1μL, 10uM mAct1 R 1μL, Taq 1U, sterilized water 6μL. The PCR reaction conditions were as follows: first 94°C for 2min; then 94°C for 30sec, 55°C for 30sec, 72°C for 30sec, a total of 30 cycles; finally 72°C for 5min. After the reaction, 1% agarose gel electrophoresis was performed, and the results are shown in Figure 5 (lanes AP represent: A: CK1 (without any treatment), B: 2°C low temperature treatment, C: 8% H ₂ O drought Treatment, D: 10% H ₂ O drought treatment, E: 13% H ₂ O drought treatment, F: HCK (water treatment), G: 10 ^-6 M ABA, H: 10 ^-5 M ABA, I: 10 ^{- 4} M ABA, J: 10 mM H ₂ O ₂ , K: 60 mM H ₂ O ₂ , L: 150 mM H ₂ O ₂ , M: 0.6% NaCl, N: 0.8% NaCl, O: 1% NaCl, P: CK Control), electrophoresis results showed that the expression of LBF gene was induced by stress conditions such as low temperature, ABA and hydrogen peroxide.

2. Analysis of expression specificity of LBF in maize immature embryo development

Using the same method as step 1, extract RNA from maize immature embryos on days 15, 17, 21, 23, and 25 after pollination, and use RT-PCR to analyze the expression of LBF gene during the development of immature embryos. The results are shown in Figure 6 As shown, lanes 1-5 represent the expression of LBF gene on days 15, 17, 21, 23 and 25, respectively, indicating that the expression of LBF increases significantly with the development of immature embryos, which is consistent with the gradual dehydration of cells during seed development, The increase of osmotic stress and the increase of adversity showed a direct correspondence.

Example 6, Physiological Analysis Experiment of Arabidopsis Transformation of LBF Gene and Transgenic Plants

1. Arabidopsis transformation of LBF gene and detection of transgenic plants

1. Construction of plant expression vector and transformation of Agrobacterium

The LBF gene was cloned into the vector pBI121 to obtain a plant expression vector named pBI121-LBF; the pBI121-LBF was transformed into Escherichia coli JM109 by the CaCl ₂ method, the plasmid was extracted for enzyme digestion identification, and positive clones were selected for sequencing, indicating that the integration was obtained There is a recombinant of plant expression vector pBI121-LBF, which is transformed into Agrobacterium LBA4404.

2. Arabidopsis transformation

1) Culture of Arabidopsis

Arabidopsis thaliana seeds were first subjected to vernalization treatment for 2-3 days at 4°C, and then 7-10 seeds were sown in each pot (nutritional soil and vermiculite were mixed at 2:1); placed in a greenhouse for cultivation (22°C , light 16h/day), after Arabidopsis thaliana pulls out the primary moss, cut off the primary moss, and when it pulls out more secondary moss, and a few start pods, it can be used for the following transformation.

2) Cultivation of Agrobacterium

Pick a single colony of recombinant Agrobacterium transformed with pBI121-LBF and inoculate it in 3mL YEB (Kan 50mg/L, rifampicin 50mg/L) liquid medium, culture at 28°C 250rpm for 30 hours; transfer to 200mL at 1:400 In fresh YEB (Kan 50mg/L, rifampicin 50mg/L) liquid medium, culture at 28°C 250rpm for about 14 hours, measure OD ₆₀₀ ≈1.5; collect the bacteria by centrifugation at 4°C 7500rpm for 10min; resuspend the bacteria In the double volume (400mL) of permeate (1/2MS salt + 5% sucrose, pH5.7, sterilized at 121°C for 15min; before use, add the final concentration of 0.044uM 6-BA, VB6 1mg/L, VB1 10mg/L , SILWET 0.02%).

3) Transformation of Arabidopsis

Immerse the flower buds of Arabidopsis thaliana in step 2) containing the infiltrate of recombinant Agrobacterium, vacuumize (25 IN Hg, keep 5 minutes); After growing for 24-48 hours under the light intensity, it can be cultured normally.

4) Collect seeds for screening

Collect the seeds of the transformed plants, weigh 25-30mg seeds and put them into a 1.5mL centrifuge tube, add 1mL 75% ethanol (containing 0.05% Tween 20), shake on a shaker for 10 minutes (300rpm), centrifuge to remove the supernatant; add 1mL 95 Wash once with % ethanol, centrifuge to remove the supernatant; repeat once; add 0.3mL absolute ethanol to the ultra-clean bench, move to sterile filter paper, and dry; sprinkle the dried seeds on 1/2MS plate (Kan 50mg/L ) at 4°C for 2 days, then cultured at 22°C under light conditions of 16h/day; transplant positive plants (T ₀ generation) into pots for cultivation, and collect seeds for T ₁ generation screening.

3. PCR detection of transgenic Arabidopsis

The extraction of transgenic Arabidopsis genomic DNA comprises the following steps:

1) Take 0.1-0.2 g of leaves of transgenic Arabidopsis plants, grind them in liquid nitrogen, and transfer them to a 1.5 mL centrifuge tube.

2) Add 0.7mL CTAB (containing Tris 100mM, NaCl 1.4M, 20mM EDTA, CTAB 2%, mercaptoethanol 0.1%), bathe in water at 60°C for 30 minutes, and invert once every 10 minutes.

3) Add 0.7mL phenol chloroform (1:1), invert several times, centrifuge at 10000rpm for 5 minutes, transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform isoamyl alcohol (24:1), mix well, 10000rpm Centrifuge for 5 minutes. Transfer the supernatant to a new centrifuge tube.

4) Add an equal volume of isopropanol, mix evenly by inversion, centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, wash once with 70% ethanol, drain, and dissolve the DNA precipitate in 50 μL sterile water for PCR detection.

Primers were designed according to the 35S promoter sequence and LBF gene sequence in pBI121, and the primer sequences were as follows:

Primer 3: (forward primer) 5'-TCTGCCGACAGTGGTCCCAA-5'

Primer 4: (reverse primer) 5'-TTAACTTTGCTAGTAGTAGCTCC-5'

Using the genomic DNA of transgenic Arabidopsis as a template, under the guidance of primers 3 and 4, perform PCR reaction. The PCR reaction system is (20 μL): 1 μL (20ng-50ng) of transgenic plant DNA, 2 μL of 10×PCR buffer, MgCl ₂ (2.5mM) 2μL, Taq enzyme 0.2μL, dNTP (2.5mM) 2μL, primer 3 and primer 4 each 10μM, add sterile water to 20μL. The PCR reaction conditions are as follows: first 94°C for 5 minutes; then 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 60 seconds, a total of 35 cycles; finally 72°C for 5 minutes. After the reaction, 1% agarose gel electrophoresis was performed to obtain positive plants transformed with the LBF gene (the target band with a size of about 1000 bp was amplified).

2. Physiological analysis experiment of LBF transgenic Arabidopsis plants

1. Cold resistance experiment

The LBF transgenic plants and non-transgenic plants were kept at -6°C for 6 hours; then transferred to normal growth conditions to continue culturing. The results showed that the survival rate of transgenic plants was 95%, and that of non-transgenic plants was 5%. , LBF can significantly improve the cold resistance of plants, as shown in Figure 7 (A is the transgenic plant, B is the non-transgenic plant).

2. Salt resistance experiment

Soak LBF transgenic plants and non-transgenic plants in 600mM NaCl for 3 hours; culture at 22°C under light for 24 hours; transfer to Arabidopsis normal growth conditions and continue to cultivate. The results show that the survival rate of transgenic plants is 80%. , the survival rate of non-transgenic plants was 15%, and LBF could significantly improve the salt resistance of plants, as shown in Figure 8 (A is a transgenic plant, B is a non-transgenic plant).

3. Drought resistance experiment

The LBF transgenic plants and non-transgenic plants were placed under normal growth conditions of Arabidopsis thaliana without water and continuously cultured for 15-20 days. The results showed that the survival rate of transgenic plants was 80%, and the survival rate of non-transgenic plants was 10%. LBF It can significantly improve the drought resistance performance of the plants, as shown in Figure 9 (A is a transgenic plant, B is a non-transgenic plant).

Sequence Listing

<160>2

<210>1

<211>267

<212>PRT

<213> Zea mays L.

<400>1

Met Asp Thr Ala Gly Leu Val Gln His Ala Thr Ser Ser Ser Ser Ser Thr

1 5 10 15

Ser Thr Ser Ala Ser Ser Ser Ser Ser Glu Gln Gln Ser Arg Lys Ala

20 25 30

Ala Trp Pro Pro Ser Thr Ala Ser Ser Ser Pro Gln Gln Pro Pro Lys Lys

35 40 45

Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr Arg His Pro Val Phe

50 55 60

Arg Gly Val Arg Arg Arg Gly Ala Ala Gly Arg Trp Val Cys Glu Val

65 70 75 80

Arg Val Pro Gly Arg Arg Gly Ala Arg Leu Trp Leu Gly Thr Tyr Leu

85 90 95

Ala Ala Glu Ala Ala Ala Arg Ala His Asp Ala Ala Ile Leu Ala Leu

100 105 110

Gln Gly Arg Gly Ala Gly Arg Leu Asn Phe Pro Asp Ser Ala Arg Leu

115 120 125

Leu Ala Val Pro Pro Pro Ser Ala Leu Pro Gly Leu Asp Asp Ala Arg

130 135 140

Arg Ala Ala Leu Glu Ala Val Ala Glu Phe Gln Arg Arg Ser Gly Ser

145 150 155 160

Gly Ser Gly Ala Ala Asp Glu Ala Thr Ser Gly Ala Ser Pro Pro Ser

165 170 175

Ser Ser Pro Ser Leu Pro Asp Val Ser Ala Ala Gly Ser Pro Ala Ala

180 185 190

Ala Leu Glu His Val Pro Val Lys Ala Asp Glu Ala Val Ala Leu Asp

195 200 205

Leu Asp Gly Asp Val Phe Gly Pro Asp Trp Phe Gly Asp Met Gly Leu

210 215 220

Glu Leu Asp Ala Tyr Tyr Ala Ser Leu Ala Glu Gly Leu Leu Val Glu

225 230 235 240

Pro Pro Pro Pro Pro Ala Ala Trp Asp His Gly Asp Cys Cys Asp Ser

245 250 255

Gly Ala Ala Asp Val Ala Leu Trp Ser Tyr Tyr

260 265

<210>2

<211>1042

<212>DNA

<213> Zea mays L.

<400>2

caagctcgag acaagaaacc agaaccagct cactcctcac tccacttcca ctcccaacag 60

caagctcaag cagtcagtca ccggcagggg tcagggtcac agtcacagca gcagccatgg 120

acacggccgg cctcgtccag cacgcgacct cctcgtcttc cacctccacc tcggcgtcgt 180

cgtcctcgtc cgagcagcag agccgcaagg cggcgtggcc gccgtcgacc gcttcctcac 240

cacagcagcc gcccaagaag cgccccgcgg ggcgcacaaa gttccggggag acgcggcacc 300

cggtgttccg cggcgtgcgg cggcggggcg ccgcgggccg gtgggtgtgc gaggtgcgcg 360

tcccggggag gcgcggcgcg cggctgtggc tcggcaccta cctcgccgcc gaggcggcgg 420

cgcgcgcgca cgacgccgcg atactcgccc tgcagggccg cggcgcgggg cgcctcaact 480

tcccggactc cgcgcggctg ctcgccgtgc cgcccccgtc cgcgctcccg ggcctggacg 540

acgcccgccg cgcggcgctc gaggccgtcg cggagttcca gcgccgctct gggtccgggt 600

ccggggccgc cgacgaagcg acctcgggcg cgtctcctcc ctcctcgtcg ccgtcgctgc 660

cggacgtttc tgcggctggc tcgccggcgg cggcgcttga gcacgtgcct gtgaaggccg 720

acgaagcagt ggcgttggac ttggacggcg acgtgttcgg gcccgactgg ttcggggaca 780

tgggcctgga gttggatgcg tactacgcca gcctcgcgga agggttgctc gtggagccgc 840

cgccgccgcc ggcggcctgg gatcatggag actgctgtga ctccggagct gcggacgtcg 900

cgctctggag ctactactag caaagttaac aataataagc ttgacagcca accccaaaag 960

ccccccaact gattgtattc acctctgtaa caaaattcaa attgatttcc cagcaaatga 1020

acttcaaaaa aaaaaaaaaa aa 1042

Claims (10)

1, anti-reverse transcription of corn regulatory factor is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have to interact with the LTRE cis element and improve the protein of plant stress-resistance performance.

2, anti-reverse transcription regulatory factor according to claim 1 is characterized in that: described anti-reverse transcription regulatory factor and LTRE bonded DNA core sequence are CGAC.

3, the gene of the described anti-reverse transcription of corn regulatory factor of coding claim 1.

4, gene according to claim 3, it is characterized in that: it has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

5, gene according to claim 4 is characterized in that: it has SEQ ID № in the sequence table: 2 nucleotide sequence.

6, the plant expression vector that contains claim 3 or 4 or 5 described anti-reverse transcription of corn regulatory factor genes.

7, the transgenic cell line and the host bacterium that contain claim 3 or 5 or 5 described anti-reverse transcription of corn regulatory factor genes.

8, arbitrary segmental primer in amplification claim 3 or the 4 or 5 described genes.

9, a kind of method of cultivating resistance raising plant is to make up the plant expression vector that contains claim 3 or 4 or 5 described genes, again the plant expression vector that makes up is transformed the purpose plant.

10, method according to claim 9 is characterized in that: described purpose plant is corn, paddy rice, wheat, soybean, rape or cotton.

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