CN106011157A - Capsella bursa-pastoris peroxidase gene and application of capsella bursa-pastoris peroxidase gene to improvement of cold resistance of economic plants - Google Patents

Abstract

The invention belongs to the technical field of plant molecular biology, in particular to a shepherd's purse peroxidase gene and its application in improving the cold resistance of economic plants. The nucleotide sequence of the shepherd's purse peroxidase functional gene of the present invention is shown in SEQ ID NO: 1. The nucleotide sequence encodes a gene that is induced by low temperature and is expressed at a low level at normal temperatures. After expression, it can regulate the active oxygen in plant cells. , to further promote the production of low-temperature induction-related genes in cells, and ultimately improve the cold resistance of plants. The overexpression vector constructed by using the gene fragment can be overexpressed in plants, thereby regulating the expression of cold resistance genes and protecting plant cells from low temperature damage. The invention also provides an application method for cultivating cold-resistant plants based on the above-mentioned gene, which is used for the improvement of economically valuable crop varieties.

Description

Shepherd's purse peroxidase gene and its application in improving cold resistance of economical plants

technical field

The invention belongs to the technical field of plant molecular biology, and specifically relates to a shepherd's purse peroxidase gene, a preparation method thereof, a recombinant plasmid of the gene, and its application to plant adversity regulation, especially to the inheritance of economic plant cold resistance genes Transformation to increase plant cold resistance.

Background technique

Temperature is an indispensable environmental condition in the process of plant growth. Plants, as a kind of fixed growth organisms, cannot avoid adverse environments by walking and moving, and are more restricted by environmental factors. Low temperature freezing damage is a serious natural disaster in agricultural production. It will not only limit the planting range of crops, but also cause crop yield reduction, causing huge losses of crops every year. Therefore, the research on plant cold resistance should transform the genetic characteristics of crops to make them Getting rid of freezing damage has always been one of the hot spots in the field of agricultural research, and it is also the ideal and pursuit of scientists. With the development and wide application of biotechnology, the understanding of the molecular mechanism of cold resistance has been deepened, and the use of modern biotechnology to cultivate new cold-resistant varieties has become one of the main means.

Using modern molecular biology techniques, people have cloned many genes involved in the formation of low temperature tolerance of plants from various plants. These genes are all specific genes induced by plants in low temperature environments, called cold-regulated genes or cold Domestication genes, the proteins they produce can protect plants from low temperature damage, so it is of great significance to study the role of low temperature-induced genes and apply them to crop improvement. In recent years, a class of low-abundance cold-inducible genes, called RCI (Rare Cold Inducible) genes, have been found in the study of the model plant Arabidopsis thaliana. The RCI genes of Arabidopsis thaliana are divided into four categories, the third category is the RCI3 gene, which encodes a cationic peroxidase (Llorente F, López-Cobollo RM, Catalá R, Martínez-Zapater JM, Salinas J. A novel cold-inducible gene from Arabidopsis, RCI3, encodes a peroxidase that constitutes a component for stress tolerance. Plant J.2002,32(1):13-24), the role in plants is to enhance The detoxification function of reactive oxygen species (Reactive Oxygen Species, ROS) in plant cells to improve plant cold resistance (Kim MJ, Ciani S, Schachtman DP. A peroxidase contributes to ROS production during Arabidopsis root response to potassium deficiency. Mol. Plant. 2010 , 3(2):420-427). A large number of studies have shown that when the external conditions of plants change sharply, such as in the process of low temperature stress, the ability to use _O2 in the body decreases, and the excess _O2 is converted into ROS in the metabolic process, and excessive ROS accumulates Can cause damage to plant cells, an antioxidant defense mechanism exists in plants to counteract the adverse effects of ROS. At the same time, ROS is also an important signal regulator molecule, which participates in the regulation of plant growth and development and various stress responses. Current studies have found that the production of ROS in plants can affect the expression of various genes, indicating that plant cells have acquired the mechanism of using ROS as a signaling molecule during the evolution process. As a signaling molecule, ROS can selectively act on a target protein that can sense ROS, and then transmit this signal to regulate the expression of certain genes. In plant cells, the results of ROS regulation are both positive and negative. The double-faced regulation of ROS has been confirmed in various abiotic stresses. Functional studies will help to better control the concentration of ROS in cells. The regulation of the level of ROS in plant cells has a good application prospect for improving the stress resistance of crops.

Shepherd's purse ( Capsella bursa -pastoris) is a 1 or 2-year wild herbaceous plant. It is spread on the ground and loves shade. It is an edible vegetable that can be seen everywhere in the south. It belongs to Cruciferae (Cruciferae), Capsella bursa (Capsella ), under low temperature conditions such as early spring and autumn and winter can normally grow and develop and flower and set. The shepherd's purse peroxidase gene involved in the present invention is cloned from the genomic DNA of the shepherd's purse leaf. There is no report about the use of shepherd's purse peroxidase gene in cultivating cold-resistant plants.

Contents of the invention

The first object of the present invention is to provide a new shepherd's purse peroxidase gene, which is cloned from the genome of shepherd's purse and is a highly expressed protein produced by plants under cold induction and cold acclimation.

The second object of the present invention is to provide a new shepherd's purse peroxidase.

The third object of the present invention is to provide the application of the shepherd's purse peroxidase and its gene sequence in improving plant stress resistance (low temperature resistance) by transgenic technology.

In one aspect of the present invention, an isolated DNA molecule is provided, that is, a new shepherd's purse peroxidase gene, which is cloned from the genome of shepherd's purse. The shepherd's purse peroxidase gene is selected from:

(1) Its nucleotide sequence is shown in bases 20-1532 in SEQ ID No.1;

(2) Its nucleotide sequence has at least 70% homology with the nucleotide sequence of the DNA molecule from nucleotides 20-232; 337-525; 615-777; 1116-1532 in SEQ ID No. 1 , and encode the same functional protein; or

(3) Its nucleotide sequence hybridizes with the nucleotide sequence from 20th to 1532nd nucleotides in SEQ ID No.1 under moderately stringent conditions, and encodes the same functional protein.

In another aspect of the present invention, a recombinant plasmid comprising the above-mentioned shepherd's purse peroxidase gene is also provided.

In another aspect of the present invention, there is also provided a shepherd's purse peroxidase, which comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the shepherd's purse peroxidase is the polypeptide of SEQ ID No.2 sequence.

In another aspect of the present invention, also provide the method for preparing above-mentioned shepherd's purse peroxidase gene, described method comprises the following steps:

(1) After the shepherd's purse seeds are sterilized by 70% alcohol, they are sown on MS medium;

(2) After the shepherd's purse seeds grow leaves, extract the genomic DNA of the leaves, and analyze its quality by 1% agarose gel electrophoresis; and

(3) The full-length sequence of the shepherd's purse peroxidase gene genome was cloned by genome walking technology, wherein the upstream primer used was CbRCIF1:5-ATGAACTGCTTGAGAGCTATTGCCC-3 (denoted as SEQ ID No.3), and the downstream primer was CbRCIR: 5-TTAACTATTTGCAACGGAACATTGCCT-3 (denoted as SEQ ID No. 4).

In another aspect of the present invention, also provide the method for preparing above-mentioned recombinant plasmid, described method comprises the following steps:

(1) Design a pair of primers to amplify the complete DNA fragment of the above-mentioned shepherd's purse peroxidase gene, the upstream primer of the primer pair is CbRCIF1:5-ATGAACTGCTTGAGAGCTATTGCCC-3 (SEQ ID No.3), and the downstream primer is CbRCIR: 5-TTAACTATTTGCAACGGAACATTGCCT-3 (SEQ ID No. 4); and

(2) Cloning the complete DNA fragment into the intermediate vector pMD18-T, and further cloning it into the plant expression vector p1304 to obtain the above-mentioned recombinant plasmid.

In step (1), a restriction enzyme cutting site Nco I is introduced into the upstream primer, and a restriction enzyme cutting site BstE II is introduced into the downstream primer.

In another aspect of the present invention, application of the above-mentioned shepherd's purse peroxidase gene or the above-mentioned recombinant plasmid in plant cold resistance and cold resistance is also provided. Preferably, the plants are economically valuable crops, more preferably including rice, wheat, corn, cotton, rapeseed, tomato and cucumber.

In another aspect of the present invention, a host cell transformed with the above vector is also provided. In examples the host cell is Arabidopsis, tobacco and other plant cells and the like.

In another aspect of the present invention, there is also provided a method for producing a polypeptide having shepherd's purse peroxidase activity, the steps are as follows:

(1) The purified nucleotide sequence encoding the polypeptide having shepherd's purse peroxidase activity is operably connected to the expression control sequence to form a shepherd's purse peroxidase gene expression vector, and the nucleotide sequence is identical to SEQ ID No. .1 have at least 70% homology to the nucleotide sequence from nucleotide position 20 to 1532;

(2) transferring the expression vector in step (1) into a prokaryotic host cell to form a recombinant cell of shepherd's purse peroxidase;

(3) culturing the recombinant cells in step (2) under conditions suitable for expressing the shepherd's purse peroxidase polypeptide;

(4) Isolating a substantially pure polypeptide having shepherd's purse peroxidase activity.

Preferably, the nucleic acid sequence used in the method is the sequence at position 20-1532 in SEQ ID No.1.

In another aspect of the present invention, there is also provided a method for utilizing transgenic technology to transform the nucleotide sequence encoding a polypeptide with shepherd's purse peroxidase activity into plants to improve the tolerance of plants to low temperature, the steps are as follows:

(1) A purified nucleotide sequence encoding a polypeptide having shepherd's purse peroxidase activity is operably linked to a plant expression control sequence to form a plant expression vector containing a shepherd's purse peroxidase gene, and the nucleotide The sequence has at least 70% homology with the nucleotide sequence from nucleotide 20-1532 in SEQ ID No.1;

(2) Transform the expression vector in step (1) into Agrobacterium, co-cultivate the Agrobacterium containing the expression vector with eukaryotic host cells, and culture in the dark at 22-28°C for 1-2 days, and then pass screening such as Antibiotic screening, obtaining transformed cells containing shepherd's purse peroxidase gene, and finally regenerating transgenic plants and their progeny, including plant seeds and plant tissues. The transgenic plant containing the shepherd's purse peroxidase gene has an enhanced effect on the drought tolerance of the plant.

Preferably, the nucleotide sequence used in the method is the 20th-1532nd sequence in SEQ ID No.1.

In another aspect of the present invention, antibodies specifically binding to the shepherd's purse peroxidase polypeptide are also provided, including polyclonal antibodies and monoclonal antibodies.

In the present invention, "isolated", "purified" or "substantially pure" DNA means that the DNA or fragments have been separated from the sequences on both sides of it in the natural state, and also refers to the DNA. Or the fragment has been separated from the components that naturally accompany the nucleotide, and has been separated from the protein that accompany it in the cell.

In the present invention, the term "shepherd's purse peroxidase (or polypeptide) gene sequence" refers to a nucleotide sequence comprising a polypeptide encoding shepherd's purse peroxidase activity, such as No. 20-232; 337- in SEQ ID No.1 525; 615-777; 1116-1532 nucleotide sequence and its degenerate sequence. The degenerate sequence refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the nucleotides 20-1532 of the coding frame of the sequence of SEQ ID No.1 . Due to the degeneracy of codons, the nucleotide sequence homology with the 20-232; 337-525; 615-777; 1116-1532 coding region in SEQ ID No.1 is as low as about 70% The degenerate sequence can also encode the sequence described in SEQ ID No.2. The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotides 20 to 1532 of SEQ ID No. 1 under moderately stringent conditions, more preferably under high stringent conditions. The term also includes at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 70% homology with the nucleotide sequence from nucleotide 20-1532 in SEQ ID No.1 At least 95% nucleotide sequence.

The term also includes variants of the coding region sequence of SEQ ID No. 1 that can encode a protein having the same function as natural shepherd's purse peroxidase. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion and/or substitution, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) at the 5' and/or 3' end ) nucleotides.

In the present invention, "substantially pure" protein or polypeptide means that it accounts for at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight) of the total sample substance. or wet weight meter). Purity can be measured by any suitable method, such as measuring the purity of the polypeptide by column chromatography, PAGE or HPLC. A substantially pure polypeptide is substantially free of components that accompany it in its natural state.

In the present invention, the term "shepherd's purse peroxidase or polypeptide" refers to the polypeptide of SEQ ID No. 1 sequence having shepherd's purse peroxidase activity. The term also includes variants of the sequence of SEQ ID No. 2 that have the same function as natural shepherd's purse peroxidase. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of shepherd's purse peroxidase.

The variant forms of the shepherd's purse peroxidase polypeptide of the present invention include: homologous sequence, conservative variant, allelic variant, natural mutant, induced mutant, can be combined with shepherd's purse peroxidase under high or low stringent conditions The protein encoded by the DNA hybridized with enzyme DNA, and the polypeptide or protein obtained by using the serum of shepherd's purse peroxidase polypeptide.

In the present invention, "shepherd's purse peroxidase conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID No.1, there are at most 10, preferably at most 8, and more preferably at most 5 amino acid properties are similar or similar amino acids to form polypeptides. These conservative variant polypeptides are preferably produced by substitutions according to Table 1.

The invention also includes analogs of shepherd's purse peroxidase or polypeptide. The difference between these analogues and natural cold-inducible protein polypeptides may be the difference in amino acid sequence, or the difference in modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides listed above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their proteolytic properties or to optimize their solubility properties.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids and the like. When producing the shepherd's purse peroxidase polypeptide of the present invention, the shepherd's purse peroxidase coding sequence can be operably linked to the expression control sequence, thereby forming a shepherd's purse peroxidase expression vector.

Table 1

As used herein, "operably linked" refers to the condition that certain parts of a linear DNA sequence are capable of affecting the activity of other parts of the same linear DNA sequence. For example, the signal peptide (secretion leader) DNA is operably linked to the polypeptide DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position to enable its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention, the term "host cell" is a eukaryotic cell. Commonly used eukaryotic host cells include yeast cells, Arabidopsis cells, tobacco cells, and other plant cells.

Northern blot technique or fluorescent quantitative PCR technique can also be used to analyze the expression of the shepherd's purse peroxidase gene product, that is, to analyze the existence and quantity of the RNA transcript of shepherd's purse peroxidase in the cells.

Northern blot analysis or fluorescent quantitative PCR analysis of shepherd's purse peroxidase gene and Western blot analysis of shepherd's purse peroxidase-specific antibody can be used in combination to confirm the expression of shepherd's purse peroxidase in biological samples.

In addition, the nucleic acid molecules that can be used as probes in the present invention usually have 8-100 continuous nucleotides of the shepherd's purse peroxidase nucleotide coding sequence, preferably have 15-50 continuous nucleotides . The probe can be used to detect whether there is a nucleic acid molecule encoding shepherd's purse peroxidase in a sample.

The invention relates to a method for detecting whether there is shepherd's purse peroxidase nucleotide sequence in a sample, which comprises the steps of using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence of shepherd's purse peroxidase, and can be located on both sides or in the middle of the coding sequence. Primers are generally 15-50 nucleotides in length.

In addition, according to the shepherd's purse peroxidase nucleotide sequence and amino acid sequence of the present invention, the shepherd's purse peroxidase homologous gene or homologous protein can be screened on the basis of nucleic acid homology or expressed protein homology.

In order to obtain the dot array of the shepherd's purse cDNAs related to the shepherd's purse peroxidase gene, the shepherd's purse genomic library can be screened with DNA probes, which are all or part of the shepherd's purse peroxidase with ³² P under low stringency conditions. obtained by radioactive labeling. The most suitable DNA library for screening is the genomic library from shepherd's purse leaves. Methods for constructing genomic libraries from cells or tissues of interest are well known in the art of molecular biology. In addition, many such cDNA libraries are commercially available, eg, from Clontech, Stratagene, Palo Alto, Cal. This screening method can identify nucleotide sequences homologous to the shepherd's purse peroxidase gene family.

The full-length sequence of the shepherd's purse peroxidase gene or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially some conserved sequences, and can be prepared with commercially available genomic libraries or by conventional methods known to those skilled in the art. As a template, related sequences are amplified. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial chemical synthesis. Before this application, in the prior art, it was possible to obtain the nucleic acid sequence encoding the shepherd's purse peroxidase of the present invention by first synthesizing a plurality of small polynucleotide fragments and then connecting them. This nucleic acid sequence can then be introduced into various DNA molecules (such as vectors) and cells available in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to recombinant production, fragments of the protein of the invention can also be produced by direct peptide synthesis using solid-phase technology (Guan X, Chaffey PK, Zeng C, Tan Z. New methods for chemical proteins synthesis. Top Curr Chem. 2015 , 363:155-92). Protein synthesis in vitro can be performed manually or automatically. For example, an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA) can be used to automatically synthesize peptides. Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

The shepherd's purse peroxidase of the invention can improve the cold resistance ability of economic crops through the transgenic technology, and the invention has great application prospects under the situation of global land shortage and population explosion.

Description of drawings

Fig. 1 is the phylogenetic tree analysis result of shepherd's purse peroxidase and peroxidase protein family members in other species.

Figure 2 shows the expression analysis of shepherd's purse peroxidase gene in shepherd's purse leaves, roots and stems under cold acclimation (A), cold induction (B), various hormones (C) and various ions (D) treatment conditions.

Fig. 3 is the result of PCR identification of hygromycin resistance gene in transgenic tobacco with shepherd's purse peroxidase gene. Lane 1. DNA marker; 2. Wild-type tobacco; 3, 4 Blank control; 5. Empty vector-transferred tobacco; 6. Agrobacterium-positive bacteria; 7-13. Transgenic tobacco-resistant seedlings.

Fig. 4 is the PCR identification result of the target gene in the transgenic tobacco positive seedlings of shepherd's purse peroxidase. Lane 1. DNAmarker; 2-5 transgenic tobacco positive seedlings.

Figure 5 is the determination of shepherd's purse peroxidase gene expression in transgenic tobacco positive plants (A), the phenotype observation of transgenic tobacco positive plants under cold stress (B) and the results of the determination of cold-resistant physiological indicators of transgenic tobacco seedling plants (C) .

Fig. 6 is the identification result of active oxygen in transgenic tobacco positive plants.

Fig. 7 is the result of measuring the expression level of endogenous cold-induced genes in transgenic tobacco positive plants.

detailed description

Below in conjunction with laboratory specific test data and in conjunction with specific examples, further elaborate the present invention. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, generally according to the condition described in conventional condition (such as Sam Brook (author), Huang Peitang (main translation). Molecular Cloning Test Guideline (Refine Edition), Chemical Industry Publishing Society, 2008) or as recommended by the manufacturer.

Example 1 Isolation and identification of shepherd's purse peroxidase gene

1. Cultivation of shepherd's purse seedlings

The shepherd’s purse seeds come from Shanghai Seed Company. After being sterilized with 75% ethanol, the shepherd’s purse seeds are sown on medium containing MS ₀ , cultured at 26°C for 2 weeks, and then transplanted to soil (vermiculite: black soil: perlite =9:3:1) to continue growing, the photoperiod during the growth process was 16h light, 8h dark, and the humidity was kept constant and moderate.

2. Extraction of Genomic DNA and RNA from Shepherd's Purse Leaf

The leaves of shepherd's purse of appropriate size were ground into powder in liquid nitrogen, and the total RNA and whole genome DNA of shepherd's purse were extracted by plant RNA extraction kit (CW0588) (Beijing Kangwei Century Biotechnology Co., Ltd.) and CTAB method, respectively. Agarose gel electrophoresis was used to detect the quality of RNA and DNA, respectively.

3. Cloning of the core sequence of the coding region of shepherd's purse peroxidase gene

A pair of primers CbRCIF2 (5 -CTGAAGGACCTTGTCTTACTCTCC-3 (denoted as SEQ ID NO.5) and CbRCIR (5-TTAACTATTTGCAACGGAACATTGCCT-3 (SEQ ID No.4)), using RT-PCR technology to amplify to obtain the coding region sequence, the amplified PCR product was subjected to 1.0% agarose gel electrophoresis detection, a highly specific fragment was obtained with a length of about 250bp. The fragment was recovered and connected to the pMD-19T vector, and the connection product was transformed into E. coil DH5a competent cells and sequenced to obtain The sequence length is 267bp. Blast analysis found that the nucleotide sequence of this fragment is 94% homologous to the mRNA sequence of RCI3A (ATU97648) of Arabidopsis ( A. thaliana ), and XM_013728218 of cabbage ( Brassica oleracea ). The homology of the mRNA sequence of 1 is 92%, confirming the correctness of the sequence.

4. Cloning of Shepherd's Purse Peroxidase Gene Sequence

It is divided into three stages:

(1) Cloning of the intermediate sequence Use step 3 to synthesize primers, and use the genomic DNA sequence as a template for PCR amplification. The PCR reaction system is: 10×PCR Buffer 5 μL; MgCl ₂ (25mM) 10 μL; dNTP Mix (10mM) 5 μL; TaqDNA polymerase (5U/μL) 1 μL; Primer1 (10 μM) 1 μL; Primer2 (10 μM) 1 μL; DNA1 μL; dH ₂ O 26 μL; total volume 50 μL. The program used is: 94°C for 5 min, 30 cycles (94°C for 30 sec, 56°C for 30 sec, 72°C for 50 sec). The amplified PCR product was detected by 1.0% agarose gel electrophoresis, and a highly specific fragment was obtained with a length of about 450bp. The fragment was recovered and ligated to the pMD-18T vector, the ligated product was transformed into E. coil DH5a competent cells, and the sequence length was 447bp obtained by sequencing. Sequence comparison analysis with step 3, found that except for an extra intron sequence, the rest of the sequence is the same as step 3, indicating that the sequence we obtained is the middle sequence of the shepherd's purse peroxidase genome;

(2) According to the sequence obtained in step (1), design primers for amplifying the upstream of the intermediate sequence, and the two upstream primers are respectively marked as CbRCI5-1 (5-GAGATACACCGATAGTGTGAGCCCCGGA-3 (recorded as SEQ ID No.6)) and CbRCI5 -2 (5-TGTTTTGCGGCTCCCTGGATCCATCTCA-3 (denoted as SEQ ID No. 7)). Using the shepherd's purse genomic DNA extracted in step 2 as a template, the adapter primers AP1 and AP2 in the Universal GenomeWalker Kit (Clontech) kit provided by CIONTECH were combined with the upstream gene-specific primers for nested amplification, and the obtained target band contained There are sequences shifted a certain distance to the 5' end of the intermediate sequence. The reaction system and procedures of PCR were carried out according to the instructions of the kit. The amplification of the upstream sequence was detected by 1.0% agarose gel electrophoresis, and a highly specific fragment was obtained with a length of about 1200bp. Recovery The two fragments were recovered and connected to the pMD-19T vector, and the ligated product was transformed into E. coil DH5a competent cells, and the length of the upstream sequence obtained by sequencing was 1140bp;

(3) Perform sequence splicing of the amplified fragments according to steps (1) and (2), and then design and amplify the full-length primers according to the spliced sequences. The primer sequences are CbRCIF (5-AACCCCCACAACTTTAAAGATGAAC-3 (denoted as SEQ ID No.8) ), the downstream primer is CbRCIR (5-TTAACTATTTGCAACGGAACATTGCCT-3 (SEQ ID No.4)). The full-length sequence was obtained by PCR amplification using the shepherd's purse genomic DNA extracted in step 2 as a template. The PCR reaction system and procedure were the same as step (1). The amplified PCR product was detected by 1.0% agarose gel electrophoresis, and a highly specific fragment was obtained with a length of about 1500 bp. The fragment was recovered and ligated to the pMD-19T vector, and the ligated product was transformed into E. coil DH5a competent cells, and the sequence length obtained by sequencing was 1532bp. It is the same as the spliced sequence of step (1) and step (2), indicating that the sequence we obtained is the complete sequence of shepherd's purse peroxidase genome.

Example 2 Bioinformatics analysis of shepherd's purse peroxidase gene

1. Gene structure analysis

Comparing the obtained sequence with the CDS sequence of shepherd's purse peroxidase gene (AY566573), it was found that the genome sequence of shepherd's purse contains 3 introns, and all 3 introns contain the gt-ag intron conserved sequence. Intron, the CDS sequence of shepherd's purse peroxidase gene contains 9811bp.

2. Homology analysis

Use http://www.ncbi.nlm.nih.gov/blast to search by Protein-Protein BLAST and other peroxidase protein family members including peroxidase in Arabidopsis ( A. thaliana ) (NM100405and NP188814), peroxidase in soybean ( Glycine max ) (AAD11481), peroxidase in Eucommia ulmoides ( AAU04879 ), peroxidase in spinach ( Spinaciaoleracea ) (CAA76374), rice ( Oryza sativa ), Peroxidase (CAH69331) in Ziping ( Spirodelapolyrrhiza ) (CAA80502), Peroxidase in tobacco ( Nicotianatabacum ) (BAA07664), Peroxidase in corn ( Zea mays ) Enzyme (AAS75418), the amino acid sequence of BrCOR (ABF60663) in rapeseed ( B.rapa ) was analyzed by Protein-Protein BLAST (http://www.ncbi.nlm.nih.gov/blast) and Vector NTI v10.0 software Analysis showed high homology, among which the homology with Arabidopsis peroxidase AtRCI3 was the highest (identity 95%, similarity 94%).

3. Phylogenetic tree analysis

Using the Clustal X software, the phylogenetic tree of CbRCI3 and other peroxidase family members was constructed by the neighbor joining method (Fig. 1). From the results of protein sequence alignment and evolutionary relationship, CbRCI3 is the closest to AtRCI3.

The expression specificity analysis of embodiment 3 shepherd's purse peroxidase

1. Various treatment conditions of shepherd's purse plants

(1) Cold induction treatment: move the shepherd's purse seedlings grown for 4w under long-day conditions at 22°C to 4°C for 4h, 8h, and 24h respectively;

(2) Cold acclimatization treatment: put shepherd's purse seedlings grown under long-day conditions at 22°C for 4w, and put them in 12°C for 4 days, 4°C for 4 days, and 0°C for 2 hours;

(3) Hormone treatment: The shepherd's purse seedlings grown for 4w under long-day conditions at 22°C were soaked in 5 μM GA, 300 μM MeJA, 20 μM IAA, and 100 μM ABA aqueous solutions for 1 hour, 6 hours, and 24 hours, respectively;

(4) Ionic solution treatment: The shepherd's purse grown for 4w under long-day conditions at 22°C was soaked in 80mM KCl, 50mMMgCl ₂ , 5mM ZnCl ₂ , 30mM LiCl, 0.1mM CuCl ₂ , and 80mM CaCl ₂ aqueous solutions for 4h, 8h, respectively. 24h.

2. Extraction of total RNA from three tissues of shepherd's purse leaf, root and stem

The plant RNA extraction kit (CW0588) (Beijing Kangwei Century Biotechnology Co., Ltd.) was used to extract total RNA from the three tissues of shepherd's purse root, stem, and leaf, and the steps were the same as in Example 1 (2).

3. Real-time quantitative PCR analysis

Fluorescent quantitative PCR technology was used to detect the changes in the expression of shepherd's purse peroxidase gene under various treatment conditions. The experiment was repeated in three groups, and shepherd's purse 18s rRNA gene (GenBank Accession No.: AY662285) was used as an internal reference. The results showed that the basal expression of shepherd's purse peroxidase gene was only lower in the root, and there was almost no expression in the stem and leaf. Both cold induction (Fig. 2A) and cold acclimation (Fig. 2B) can significantly increase the expression of shepherd's purse peroxidase gene in various tissues. Different hormone treatments have different effects on the regulation of shepherd's purse peroxidase expression. ABA can significantly enhance the expression of shepherd's purse peroxidase gene, and with the increase of time, the expression level will continue to increase; MeJA can instantaneously increase the expression level of shepherd's purse peroxidase gene, showing feedback inhibition; GA ₃ can inhibit the peroxidation of shepherd's purse expression of the enzyme gene; IAA had no effect on the expression of its gene (Fig. 2C). Metal ion treatment found that calcium ions, copper ions and lithium ions could all induce the expression of shepherd's purse peroxidase gene, while magnesium ions, zinc ions and potassium ions could inhibit the expression of shepherd's purse peroxidase gene (Figure 2D).

Example 4 Agrobacterium-mediated leaf disk method plant transformation

1. Construction of plant expression vectors

In this embodiment, the shepherd's purse peroxidase gene is placed behind the 35S promoter to construct a plant expression vector for plant transformation.

2. Agrobacterium-mediated transformation of tobacco

(1) Cultivation of Agrobacterium. Pick a single colony, add it to 2mL Agrobacterium liquid medium, and cultivate overnight at 28°C; take more than 2mL of the culture, add it to 50mL Agrobacterium liquid medium, and cultivate it to OD ₆₀₀ =0.6-1.0 at 28°C; centrifuge at 8000rpm for 10min, and collect Cells, resuspended with MS ₀ to make OD ₆₀₀ =1.0;

(2) Co-culture. Take young and strong leaves of sterile tobacco seedlings, remove the main veins, cut the leaves into leaf discs of about 0.8cm×0.8cm, put them on MS ₁ medium to prevent dehydration of the leaf discs; put the leaf discs into Agrobacterium In culture medium, 190rpm, 10min on a shaker at 28°C; take out the leaf disk with a medicine spoon, put it on sterilized absorbent paper, and absorb the excess bacterial liquid on the leaf disk; put the dried leaf disk on a flat layer with a cover On MS ₁ medium on filter paper, co-cultivate in the dark for about 2 days at about 25°C;

(3) Induce clustered buds. After co-cultivation, wash off the Agrobacterium on the surface of the leaf disk according to the following steps, and shake it from time to time to make the leaf disk fully contact with the following solutions: sterile water, 15 minutes; sterile water + carbenicillin (350mg/L), 15 minutes; MS ₀ + carbenicillin (350mg/L), 20min. Remove the leaf disk with a medicine spoon, put it on absorbent paper, and absorb excess water; put the leaf disk on MS ₂ medium, and gently press the edge of the leaf disk into the medium; culture at about 26°C for 16 hours under light;

(4) Induce rooting. After being induced on MS ₂ medium for about 4w, the young shoots growing from the leaf edge were cut from the base and the leaf disc, and the young shoots were inserted into MS ₃ medium to induce rooting, and cultured at about 26°C for 16 hours under light.

Example 5 Molecular detection of transgenic tobacco

1. Extraction of DNA from transgenic tobacco leaves and PCR detection of resistance genes

The steps for extracting DNA from transgenic tobacco leaves can refer to the method in Example 1 (2). Using the extracted genomic DNA of transgenic tobacco seedling leaves as a template, hyg-F (5-GTCGAGAAGTTTCTGATCG-3 (denoted as SEQ ID No. 9)) and Hyg-R (5-GTTTCCACTATCGGCGAGTACT-3 ( Denoted as SEQ ID No.10)) for PCR amplification, negative control of wild-type tobacco and tobacco samples transformed into empty vector, 1% agarose gel electrophoresis to detect whether the transgenic tobacco genome contains hygromycin resistance gene (700bp), the results are shown in Figure 3, and the test results showed that a total of 35 transgenic resistant seedlings included the hygromycin resistance gene.

2. PCR detection of target genes in transgenic tobacco

Using the extracted transgenic tobacco genomic DNA as a template, PCR amplification was performed using specific primers for the shepherd’s purse peroxidase gene. -TTAACTATTTGCAACGGAACATTGCCT-3 (SEQ ID No. 4)). Using wild-type and empty vector-transferred tobacco samples as negative controls, 1% agarose gel was used to detect whether the transgenic tobacco genome contains the gene of shepherd’s purse peroxidase (1200bp). The results are shown in Figure 4. The detection results found that 35 tobacco plants Among the resistant seedlings, 30 seedlings contained the shepherd's purse peroxidase gene.

3. RNA extraction of transgenic tobacco and identification of target gene expression

The extraction of transgenic tobacco RNA can refer to the method in Example 1 (2). Fluorescent quantitative PCR was used to detect the expression of shepherd's purse peroxide gene in 30 transgenic tobacco plants. The primer sequences used were: Realtime-CbRCI35-F (5-CATTAGCCAACATTCCTCCTCCGACCA-3 (denoted as SEQ ID No.12)) and Realtime-CbRCI35- R(5-CTGACTGAAACAGACCTCTACGCTTG-3 (denoted as SEQ ID No.13)), the GAPD gene in tobacco (GenBankAccession No.: AJ133422) was used as an internal reference. The results found that compared with wild-type tobacco, the expression levels of different transgenic lines were different, and the gene expression levels in the transgenic tobacco were all up-regulated. Figure 5A shows the expression levels of two transgenic tobacco positive plants.

Example 6 Cold tolerance test of transgenic tobacco positive plants

Two lines with higher expression levels were selected, and pure lines were obtained after continuous planting. The two transgenic pure-line plants were cultured under light at 12°C, cold-adapted for 4 days, then placed at 4°C for 24 hours, -4°C for 1 hour, and recovered at 22°C for 3 days. Phenotypic differences between transgenic wild-type tobacco seedlings and tobacco seedlings transformed with empty vector (Fig. 5B). The results showed that the transgenic tobacco had no growth delay and dwarfing phenomenon under the normal temperature of 22°C; under the cold treatment of 4°C, the non-transgenic and empty vector control plants suffered more severe chilling damage, with wilting leaves and lodging stems, and the positive seedlings of transgenic tobacco suffered slightly chilling damage , only a few leaves shrunk; at -4°C, non-transgenic control plants suffered severe chilling damage, and leaf wilting and stem lodging aggravated. Transgenic tobacco also suffered chilling damage, and leaf wilting occurred, but the phenomenon was milder. The positive transgenic tobacco seedlings and the control were put back at 22°C to resume growth, and the positive transgenic tobacco seedlings could return to normal growth state after 3 days, while the tobacco seedlings of the control group would shrink and die.

Example 7 Determination of Cold-resistant Physiological Indexes of Transgenic Positive Tobacco

Take the tobacco leaves treated in Example 6 to measure the leaf electrical conductivity, relative water content and glucose content. The experiment was repeated in three groups, and the significant difference between the transgenic group and the control group was evaluated by t-test.

1. Determination of blade electrical conductivity

Take 8 leaf discs on both sides of the main vein of the leaves of the same part of each tobacco plant using an 8mm caliber puncher, soak them in 10ml Milli-Q water for 2 hours, and use a conductivity meter DDS-11A (Shanghai Suo Shen) to detect the apparent conductivity . After boiling at 100°C, cool to room temperature and measure the absolute conductivity. Relative conductivity (%)=apparent conductivity/absolute conductivity×100%. The results showed that under cold stress, the electrical conductivity and plasma membrane permeability of the tobacco leaves in the control group increased significantly under cold stress; while the plasma membrane permeability of the leaves of transgenic tobacco positive plants increased significantly slowly, and the amplitude was also small, indicating that the cytoplasmic The membrane was slightly damaged. Statistics showed a significant difference between the two (Fig. 5C).

2. Determination of relative water content

Using a 6 mm caliber puncher, 8 leaf disks were taken from both sides of the main vein of the leaves of the same part of the various treated tobacco plants, and the fresh weight (FW) was weighed; the leaf disks were placed in Milli-Q water and vacuumed for 4 h , weigh the wet weight (TW); blot the leaf disk with absorbent paper, dry at 85°C for 24 h, and weigh the dry weight (DW); relative water content (%=(FW-DW)/(TW-DW)× 100%. The results show that under cold stress, the relative water content of cold-sensitive plant tobacco significantly decreases, indicating that water and cell active components are lost under cold stress. Compared with wild type and empty vector control, the two high-expression lines of transgenic tobacco are relatively The water content was significantly higher (Fig. 5C). At the same time, there was no significant difference in the relative water content values among different transgenic lines, indicating that the overexpression of shepherd's purse peroxidase in plants can complete the low temperature protection of cells.

3. Glucose Assay

The glucose content (HK method) assay kit (Sigma-Aldrich, Inc., USA) was used to detect the relative glucose content of tobacco leaves. Under each treatment method and each treatment gradient, the leaves of 4 plants were taken from each strain, and the leaves of about 1.5 cm square in the same position were taken from each plant, and the mass of the leaves was weighed. Vacuumize for 2 hours to obtain the glucose extract of the sample. The absorbance of NADH products at 340 nm was measured with a spectrophotometer (Eppendorf, Germany), and deionized water was used as a control. The glucose content is proportional to the absorbance value, and the calculation formula is: glucose content=0.319×[Atest-(Asample blank +Areagent blank)]. A _Test , A _{Sample Blank} and A _{Reagent Blank} are sample tube, sample blank tube (replace the Glucose Assay Reagent in the sample tube with an equal volume of deionized water), and reagent blank tube (replace the sample glucose extract in the sample tube with into an equal volume of deionized water). Glucose plays a role in regulating the osmotic potential of cells. From the results of the determination of glucose content in the leaves of wild-type plants and transgenic positive plants, it can be seen that the intracellular glucose content of transgenic tobacco-positive plants is higher than that of wild-type plants (Fig. 5C) , indicating that the intracellular glucose content increased under low temperature stress in response to water shortage and cell damage caused by low temperature.

Example 8 Peroxide Analysis of Transgenic Tobacco Plants

The peroxide analysis of transgenic tobacco uses DCFH-DA fluorescent probe, which can pass through the cell membrane and be converted into DCFH by peroxidase and stay in the cell. Then DCFH in the cells will be oxidized by reactive oxygen species to emit fluorescence. At the end of the experiment, the leaf tissue was cut off first, then immersed in a DCFH-DA fluorescent probe solution with a concentration of 10 μM, incubated at 37°C for 20 min, and then the leaf surface was washed with deionized water to make the DCFH-DA attached to the leaf cell surface DA is washed away so as not to affect subsequent observations and cause interference. The results showed that transgenic tobacco positive plants significantly increased peroxides compared with wild-type tobacco control plants (Figure 6), indicating that shepherd's purse peroxidase is a peroxidase capable of producing ROS.

Example 9 Molecular Mechanism Analysis of the Formation of Cold Resistance in Transgenic Tobacco Plants

From the above cold acclimation phenotypes, it can be seen that the cold tolerance ability of transgenic tobacco positive plants has been greatly improved, and this improvement must be related to substances such as plant cell membrane protection proteins, that is to say, among the transgenic tobacco positive plants The shepherd's purse peroxidase may improve the cold resistance of tobacco by activating the key genes of the cold-responsive signaling pathway. There are cold response pathway-related genes similar to those in Arabidopsis in tobacco, among which NtDREB1 and NtDREB3 encode CBF/DREB transcription factors, which play an important role in the cold signal pathway; NtERD10a and NtERD10b encode some downstream COR genes; NtTSI1 is A transcription factor related to diseases, etc. The extraction of transgenic tobacco RNA can refer to the method in Example 1 (2). Fluorescent quantitative PCR was used to detect the expression changes of five endogenous cold-responsive genes in transgenic tobacco plants. The results showed that under the culture condition of 26°C, relative to the control group tobacco, the expression levels of genes related to the cold-induced pathway of the transgenic tobacco positive plants themselves had been up-regulated to a certain extent; after being treated at 4°C, the transgenic tobacco positive plants The expression levels of NtDREB1 , NtDREB3 , NtERD10a , and NtERD10b were also significantly up-regulated compared with the control plants ( FIG. 7 ). There was no significant difference in the expression of NtTSI1 between transgenic tobacco and control plants. This shows that the shepherd's purse peroxidase gene can participate in the signal transmission pathway in the cold tolerance pathway, and through signal transmission, the expression level of the endogenous downstream cold-induced genes in tobacco can be greatly increased, thereby improving the plant's cold resistance.

<110> Fudan University

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Claims (10)

1. a shepherd's purse peroxidase gene isolated from shepherd's purse with low-temperature induction properties, characterized in that, said shepherd's purse peroxidase gene is selected from one of the following: (1) Its nucleotide sequence is shown in bases 20-1532 in SEQ ID No.1; (2) Its nucleotide sequence has at least 70% homology with the nucleotide sequence of the DNA molecule from nucleotides 20-232; 337-525; 615-777; 1116-1532 in SEQ ID No. 1 , and encode the same functional protein; or (3) Its nucleotide sequence hybridizes with the nucleotide sequence from 20th to 1532nd nucleotides in SEQ ID No.1 under moderately stringent conditions, and encodes the same functional protein. 2. A recombinant plasmid, characterized in that it contains the shepherd's purse peroxidase gene according to claim 1. 3. a shepherd's purse peroxidase, is characterized in that, described shepherd's purse peroxidase is selected from one of following: (1) Its amino acid sequence is shown in SEQ ID No.2; or (2) A conservative variant polypeptide of the polypeptide having the amino acid sequence of SEQ ID No. 2, or an active fragment thereof, or an active derivative thereof. 4. a method for preparing the shepherd's purse peroxidase gene according to claim 1, is characterized in that comprising the following steps: (1) After the shepherd's purse seeds are sterilized by 70% alcohol, they are sown on MS medium; (2) After the shepherd's purse seeds grow leaves, extract the genomic DNA of the leaves, and use 1% agarose gel electrophoresis to analyze its quality; and (3) The full-length sequence of the shepherd's purse peroxidase gene genome was cloned by genome walking technology, wherein the upstream primer used was: SEQ ID No.3, and the downstream primer was SEQ ID No.4. 5. A method for preparing the recombinant plasmid according to claim 2, characterized in that it may further comprise the steps: (1) Design primer pairs to amplify the complete DNA fragment of the shepherd's purse peroxidase gene described in claim 1, the primer pair used for amplification is: the upstream primer is SEQ ID No.3, and the downstream primer is SEQ ID No. 4; (2) Cloning the complete DNA fragment into the intermediate vector pMD18-T, and further cloning it into the plant expression vector p1304 to obtain the recombinant plasmid. 6. The preparation method according to claim 5, further comprising introducing a restriction enzyme site Nco I into the upstream primer, and introducing a restriction enzyme site BstE II into the downstream primer. 7. application of the shepherd's purse peroxidase gene according to claim 1 or the recombinant plasmid according to claim 2 in plant cold resistance and cold resistance. 8. The application according to claim 7, wherein the plants are crops with economic value, including rice, wheat, corn, cotton, rapeseed, tomato and cucumber. 9. The application according to claim 7, characterized in that the recombinant plasmid is transformed into Agrobacterium tumefaciens EHA105, and then used to transform the target plant to improve the tolerance of the plant to cold. 10. application according to claim 7, is characterized in that, utilizes transgenic technology to transform the nucleotide sequence encoding has shepherd's purse peroxidase activity polypeptide into plant, to improve the tolerance of plant to cold, its steps are as follows : (1) A purified nucleotide sequence encoding a polypeptide having shepherd's purse peroxidase activity is operably linked to a plant expression control sequence to form a plant expression vector containing a shepherd's purse peroxidase gene, and the nucleotide The sequence has at least 70% homology with the nucleotide sequence from nucleotide 20-1532 in SEQ ID No. 1; (2) Transform the expression vector in step (1) into Agrobacterium, co-cultivate the Agrobacterium containing the expression vector with eukaryotic host cells, culture in dark at 22-28°C for 1-2 days, and pass the screening, The transformed cells containing the shepherd's purse peroxidase gene are obtained to finally regenerate transgenic plants and their progeny, including plant seeds and plant tissues.