CN103060350A - Sclerotia oxalic acid decarboxylase gene SsOXDC2 and application thereof in improvement in soybeans for disease resistance - Google Patents

Abstract

The invention belongs to the technical field of plant pathology and molecular biology thereof, and relates to the plant transgene technology. A sclerotinia oxalic acid decarboxylase gene SsOXDC2 capable of resisting sclerotiniose is obtained by utilizing the plant gene cloning technology, wherein the nucleotide sequence of the gene SsOXDC2 is shown in SEQ ID NO: 1 in a sequence table; and the coded protein sequence of the gene SsOXDC2 is shown inSEQ ID NO: 3 in the sequence table. The invention further discloses a method for obtaining a transgenic plant by the clone, the vector construction and the screening of the sclerotinia oxalic acid decarboxylase gene SsOXDC2. The gene SsOXDC2 provided by the invention is suitable for the genetic improvement in enhancement of the capability of the soybeans for resisting the sclerotiniose.

Description

Sclerotinia oxalate decarboxylase gene SsOXDC2 and its application in improvement of soybean disease resistance

technical field

The invention belongs to the technical field of plant genetic engineering. It specifically relates to the isolation, cloning and functional verification of an oxalate decarboxylase gene SsOXDC2 derived from Sclerotinia sclerotiorum. The oxalate decarboxylase gene SsOXDC2 can endow soybean with the ability to resist soybean sclerotinia caused by Sclerotinia sclerotiorum. the

Background technique

Sclerotinia sclerotiorum is an important plant pathogenic fungus with a wide range of hosts, which can infect 75 families and more than 450 kinds of plants, mainly herbaceous and succulent woody plants, causing plant sclerotinia (Purdy , 1979; Boland et al, 1994; Bolton et al, 2006), causing huge losses to the production of a variety of commercial crops. In my country, Sclerotinia has become a huge hazard to soybean production. the

Sclerotinia can secrete a large amount of oxalic acid in the process of pathogenicity. Most of the researches in recent years believe that oxalic acid is inseparable from the pathogenicity of Sclerotinia, and oxalic acid is the determinant of its pathogenicity (Maxwell et al., 1970; Godoy et al ., 1990; Wu Chunren et al., 1991). Oxalic acid provides an acidic microenvironment for the infection of S. sclerotiorum, where oxalic acid accumulates at the site of infection, and the pH value drops to about 4.5. The acidic environment can increase the activity of cell degradation enzymes, such as polygalacturonase (polygactu-ronase , PG), cellulase and hemicellulase (Riou et al., 1991) and so on. At the same time, oxalic acid can inhibit the activity of substances related to plant resistance (Noyes and Hancock, 1981; Magro et al., 1984). the

In organisms, oxalic acid can be decomposed by oxidation and decarboxylation. There are three main types of enzymes involved in the hydrolysis of oxalate. Oxalate oxidase (EC 1.2.3.4), which oxidizes oxalic acid to produce 2 molecules of CO ₂ and 1 molecule of H ₂ O ₂ , can be cloned from higher plants and a few microorganisms. The acetyl-CoA decarboxylase (EC 4.1.1.8) system is mainly associated with microorganisms. Oxalate is converted to oxalyl-CoA and _CO2 by acetyl-CoA decarboxylase. Oxalate Decarboxylase (OXDC) (EC 4.1.1.2) decarboxylates oxalate to produce 1 molecule of CO ₂ and 1 molecule of formic acid. Oxalate decarboxylase, which was first discovered in the hyphae of the wood-rot basidiomycetes Collybia velutipes and Coriolus hirsutus, is a manganese-containing enzyme. Oxalate decarboxylase, recently obtained from Bacillus subtilis, is induced by acidic media, but not necessarily by oxalate. Compared with other oxalate degrading enzymes, oxalate decarboxylase is specific for oxalate, the process is simple, and it is active at low pH.

With the establishment and development of tissue culture and Agrobacterium tumefaciens mediated transformation (ATMT), plant genetic engineering has achieved fruitful results. At present, research on soybean transgenics mainly focuses on its insect resistance, stress resistance, herbicide resistance, and quality improvement. The genes used mainly include Bt insect resistance gene Cry1A, atrazine resistance gene, phytase gene, r-cystathionine synthase gene, etc., but there are few studies on the resistance of sclerotinia, which is the most important soybean production. Table 1 lists the main achievements of plant oxalate metabolizing enzymes in recent years. Only a few genes have been tried to transform soybean (Donaldson et al., 2001; Cunhaab et al., 2010). Since the enzyme-catalyzed oxalate degradation reaction occurs in the acidic environment created by S. sclerotiorum infection, and the efficiency of the enzyme is greatly affected by the environment, the activity of plant-derived oxalate oxidase is likely to be affected by the environment And lower. The oxalate decarboxylase derived from S. sclerotiorum has very good adaptability to the acidic environment created by S. sclerotiorum infection, and will not be affected by substances related to the inhibition of enzyme activity secreted by S. sclerotiorum, so it may It has better performance in the interaction between Sclerotinia and soybean. the

Table 1 Application of genes related to oxalate metabolism in transgenic plants

Contents of the invention

The purpose of the present invention is to isolate and clone the oxalate decarboxylase gene in Sclerotinia sclerotiorum, and adopt virus-mediated expression strategy to overexpress in soybean, and efficiently use this gene to improve the anti-sclerotinia ability of soybean varieties. the

The present invention relates to the separation and application of a DNA fragment containing the SsOXDC2 gene, its nucleotide sequence is shown in the sequence table SEQ ID NO: 1, and the sequence of its encoded protein is shown in the sequence table SEQ ID NO: 3. The gene (fragment) endows soybean with specific disease-resistant response to Sclerotinia sclerotiorum caused by Sclerotinia sclerotiorum. This invention is applicable to all plants sensitive to the pathogenic bacteria. These plants include major oil crops such as soybeans and canola and vegetable crops. In addition to the above-mentioned DNA fragment shown in SEQ ID NO: 1, the gene defined in the present invention also includes a DNA sequence substantially equivalent to that shown in SEQ ID NO: 1, or its function is equivalent to SEQ ID NO: 1 Subfragments of the indicated sequences. the

The cloned SsOXDC2 gene can be used as a probe to screen the gene or homologous gene of the present invention from cDNA and genome libraries. Similarly, using PCR (polymerase chain reaction) technology, the SsOXDC2 gene of the present invention and any piece of DNA of interest or a piece of DNA homologous thereto can also be amplified from the genome, mRNA and cDNA. Using the above techniques, a sequence containing the SsOXDC2 gene or a sequence containing a segment of the SsOXDC2 gene can be isolated, and this sequence can be connected with a suitable vector to be transferred into plant cells to improve the ability of plants to resist sclerotinia. the

In the example part of the present invention, we described the isolation, functional verification and application process of the gene SsOXDC2 and the characteristics of the gene. the

Description of drawings

Sequence Listing SEQ ID NO: 1. The present invention clones the Sclerotinia oxalate decarboxylase gene SsOXDC2 nucleotide sequence, which is derived from the genome of Sclerotinia bacterial strain Ep-1PNAa367. the

Sequence Listing SEQ ID NO: 3. is the sequence of the protein encoded by the oxalate decarboxylase gene SsOXDC2. the

Fig. 1. The flow chart of the separation and cloning of the oxalate decarboxylase gene SsOXDC2 and functional verification of the present invention. the

Figure 2. Cloning of the oxalate decarboxylase gene SsOXDC2 from the genome of Sclerotinia strain Ep-1 PNAa367. In the figure: lane 2 is a blank control, and lane 2 and lane 3 templates are genomic DNA samples of Ep-1PNAa367. the

Figure 3. Maps of gene cloning and transformation vectors pMD18T, T-AON, pGR106 and pGR106O+. Figure 3A is the transformation vector pMD18T and T-AON; Figure 3B is the transformation vector pGR106; Figure 3C is the transformation vector pGR106O+. the

Figure 4. Expression of oxalate decarboxylase gene SsOXDC2 in soybean Zhonghuang 18 transgenic plants. In the figure: Swimming lane 1 is a blank control, and swimming lanes 2-5 templates are RNA samples of transgenic soybean plants. the

Fig. 5. Analysis results of antibacterial sclerotinia lesion expansion on leaves of transgenic plants (quantitative test). the

Figure 6. The expansion of antibacterial sclerotinia lesions on the leaves of transgenic plants (visual diagram of lesions). In the figure: the first row is the soybean variety Zhonghuang 18; the second row is the transformation of blank pGR106; the third row is the transformation of pGR106O+. the

Figure 7. Anti-sclerotinia expansion of petioles of transgenic plants. In the picture: the left side of the picture is transformed into pGR106O+; the right side of the picture is transformed into blank pGR106. the

Detailed ways

The present invention is based on the analysis and prediction of the genome sequence of Sclerotinia sclerotiorum, using the Sclerotinia sclerotiorum Ep-1PNAa367 bacterial strain as research material, isolating and cloning the full-length coding region sequence of the SsOXDC2 gene by RT-PCR technology, and merging it with the viral PVX vector pGR106 for agricultural production. Bacillus-mediated expression in soybean significantly improves the anti-sclerotinia ability of soybean (as shown in the flow chart of Figure 1). In order to better understand the present invention, the following examples are used to further illustrate, but not to limit the present invention. the

According to the following examples, those skilled in the art can ascertain the basic characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention so that it is applicable to various uses and conditions . the

In the following specific examples, unless otherwise specified, all culture media and sterile test supplies were sterilized by conventional high-pressure steam method (121° C., high-pressure sterilization for 30 minutes), and stored at room temperature. Unless otherwise specified, all restriction endonucleases and polymerases were purchased from Bao Biological Engineering (Dalian) Co., Ltd. the

Embodiment 1: the isolated clone of sclerotinia oxalate decarboxylase gene SsOXDC2

Obtain some information of the sclerotinia oxalate decarboxylase gene from the fungal genome database (http://www.broadinstitute.org/scientific-community/data), containing two oxalate decarboxylase gene sites in the sclerotinia genome, respectively are SS1G_08814 and SS1G_10796, located on different chromosome segments. The SS1G_08814 transcribed region contains 4 exons, 3 introns, the full length is 1575bp, the cDNA coding region contains 1368bp, encoding 455 amino acids; the SS1G_10796 transcribed region contains 3 exons, 2 introns, the full length is 1665bp , the cDNA coding region contains 1515bp, encoding 504 amino acids. Analysis of the two amino acid sequences by TargetP 1.1Server found that both amino-terminals contained signal peptide sequences. the

The present invention shows that the Solexa expression profile analysis of Sclerotinia strain Ep-1PNAa367 in different growth stages shows that the expression levels of these two genes have great changes in the different growth stages of Sclerotinia, and the expression of the two genes in the sclerotinia formation stage The levels reached the highest, and the expression levels of the two genes also had obvious differences in different growth stages (see Table 2). In view of the differences in expression levels, the present invention selects the SS1G_10796 gene as the research object. The oxalate decarboxylase gene SsOXDC2 mentioned below refers to this gene. the

Table 2 Expression levels of oxalate decarboxylase gene in different growth stages of Sclerotinia

1. Sclerotinia total RNA extraction

Sclerotinia strain Ep-1PNAa367 (collected by Jiang Daohong et al. from Jiamusi City, Heilongjiang, Liu et al., 2009) was inoculated on a PDA plate (90cm petri dish) covered with cellophane, cultured at 20°C for 3-4d, and mycelium was collected . Take 100 mg of mycelium, grind it fully in liquid nitrogen, quickly transfer it to 1 ml of RNArose (purchased from Bao Biological Engineering Dalian Co., Ltd.), and place it on ice for 15 minutes to completely separate the nucleic acid-protein complex. Centrifuge at 12,000r/min at 4°C for 10min, and carefully transfer the supernatant to a new centrifuge tube. Add 0.2ml chloroform to every 1ml RNArose used, shake vigorously for 15s, and place at room temperature for 3min. Centrifuge at 12,000r/min at 4°C for 10-15min, discard the precipitate, add an equal volume of isopropanol to the aqueous phase solution, mix well, and place at -20°C for 20-30min. Centrifuge at 12,000r/min at 4°C for 10min, and discard the supernatant. Then add 1ml 75% ethanol to wash the precipitate. Centrifuge at 5,000r/min for 3min at 4°C. Discard the supernatant, dry it at room temperature, add 30 μl RNase-free water to fully dissolve the RNA, and store the obtained RNA sample in a -80°C refrigerator for later use. the

Wherein: PDA medium (i.e., the formula of potato dextrose agar medium is: fresh potato (peeled) 200g, add water and boil to get juice, then add glucose 20g, agar 17-20g, replenish water to 1000ml, kill according to the conventional method described above Bacteria.

2. Cloning of the full-length coding region of the sclerotinia oxalate decarboxylase gene SsOXDC2

Use the RevertAid ^™ First Strand cDNA Synthesis Kit (reverse transcription kit; purchased from Fermentas) to synthesize the first strand of cDNA: prepare the following system on ice with sterile, diethylpyrocarbonate (DEPC)-treated Eppendorf tubes : RNA 10 ng-0.5 μg, oligo(dT) ₁₈ primer 1 μl, DEPC-treated ddH ₂ O to make up to 12 μl. Gently mix, centrifuge, 65 ° C water bath for 5 minutes to fully dissociate the secondary structure in the RNA, and quench on ice. Then add in the following order: 5× buffer 4μl, RiboLock ^TM RNA inhibitor (20U/μl) 1μl, 10mM dNTP 2μl, RevertAid ^TM M-MuLV reverse transcriptase (200U/μl) 1μl, mix gently, and centrifuge . Incubate at 42°C for 60min. Finally, the reaction was terminated by treating at 70°C for 5 min.

The PCR product of the previous step was used as a template, primers OXDCFP (5'GGTACCTGATGCATTCCAAAACTTTCC3') and primer OXDCRP (5'GAGCTCTCCCACAACTCTACTCATAAGCAC 3') were used as primers, and the coding region of the gene SsOXDC2 was cloned. The PCR system is as follows: a total reaction volume of 50 μl, 2 μl of the reaction product from the previous step, 5 μl of 10×PCR buffer, 1 μl of 10 mM dNTP, 1 μl of primer OXDCFP, 1 μl of primer OXDCRP, 39.5 μl of ddH ₂ O, and 0.5 μl of 5U/μl Taq. PCR program: 95°C for 3min, 95°C for 30s, 56°C for 30s, 72°C for 2min, 30cycles, 72°C for 7min.

The above PCR products were recovered with the AxyPrep DNA Gel Recovery Kit (purchased from Axygen), and connected to the pMD18-T vector (purchased from Bao Bio-Engineering Dalian Co., Ltd.). The system was: 2 μl of PCR recovered products, 1 μl of pMD18-T, ₂ O2 μl, Solution I 5 μl. 16°C water bath overnight. Add to 50μl competent cells, mix gently and place in ice bath for 20-30min. After heating in a water bath at 42°C for 45 seconds, immediately put it back on the ice, and then place it in the ice for 2 minutes. Add 890 μl of SOC medium, shake and culture at 37°C for 60min. The carrier T-OXDC was obtained by screening on LB-agar plates containing ampicillin (Amp, 50 μg/ml).

Among them: LB medium: peptone 10g, yeast powder 5g, NaCl 10g, adjust the pH to 7.0 with 5M NaOH, add distilled water to 1000ml, add 2% agar to the solid medium;

SOB medium: tryptone 20g, yeast powder 5g, NaCl 0.5g, add 10ml 250mM KCl after complete dissolution, add 5M NaOH 0.2ml to adjust to pH 7.0, add water to 1000ml, add 5ml 2M MgCl ₂ before use; 1L SOB add 20ml 1M Glucose is the SOC medium.

Example 3: Functional verification of the oxalate decarboxylase gene SsOXDC2 of Sclerotinia sclerotiorum

pGR106 is an infection vector transformed from a plant single-stranded RNA virus-Potato Virus X (PVX) (see Figure 3B). Proteins are expressed as fusion proteins. After pGR106 is integrated into the plant genome through Agrobacterium-mediated transformation, it will express the coding potato X virus, and the foreign gene will be expressed at the same time. The exogenous gene can be packaged together with the viral genome, and expressed in other plant tissues along with the replication and transmission of the virus. The specific steps are as follows:

1. Transformation vector construction

The virus-mediated expression vector of S. sclerotiorum oxalate decarboxylase gene SsOXDC2 was constructed on the basis of plasmid pGR106 (see FIG. 3B ). Firstly, T-OXDC was used as a template and OXDC-AscI (5'A GGCGCGCC ATGCATTCCAAAACTTTC CTG 3') and OXDC-NotI (5'A GCGGCCGC TACTCATAAGCACCACTTCCCTTTCCA 3') were used as primers to clone the coding region of the oxalate decarboxylase gene. The amplification system was as follows: T-OXDC 0.5 μl, 10×PCR buffer 5 μl, 10 mM dNTP 1 μl, OXDC-AscI 1 μl, OXDC-NotI 1 μl, ddH ₂ O 41 μl, 5U/μl Taq 0.5 μl. The PCR program is as follows: 95°C for 3min, 95°C for 30s, 55°C for 30s, 72°C for 2min, 30 cycles, 72°C for 7min. The PCR product was ligated to pMD18-T (see FIG. 3A ), and the obtained vector was named T-AON (see FIG. 3A ).

The vectors pGR106 (see FIG. 3B ) and T-AON (see FIG. 3A ) were digested with enzyme Not I at 37°C for 3 h, purified and recovered, and then digested with Asc I (New England Biolabs) at 37°C for 3 h. Purification and recovery, T ₄ -DNA ligase ligation, the ligation system is as follows: 10×T ₄ -DNA ligase buffer 1 μl, pGR106 recovered fragment 1 μl, T-AON recovered fragment 3 μl, ddH ₂ O 4.5 μl, T ₄ -DNA ligase 0.5 μl. After overnight at 16°C, transform Escherichia coli DH5α, and PCR verified that positive clones were picked to extract the plasmid, which was named pGR106O+. The plasmids pGR106 and pGR106O+ (see Figure 3C) were transformed into Agrobacterium EHA105 by electric shock, the specific method is as follows:

Transfer 2 μl of TE-dissolved plasmid samples (pGR106 and pGR106O+) to 1.5ml sterile EP tubes, respectively. Place on ice. Prepare 1ml LB medium for each sample and store at room temperature. The 0.1cm shock cup was pre-cooled on ice. Dissolve Agrobacterium EHA105 competent cells on ice, add 20 μl competent cells to each EP tube containing samples, and mix gently. Turn the BioRadmicropulser (Bio-Rad) to "Agr". The adjustment parameters are voltage 2.5kv, capacitance 25mF and resistance 200 ohms. Transfer the mixture of plasmid and competent cells to the bottom of the electric shock cup, and place the electric shock cup on the chute of the electric shock chamber, push the chute into the electric shock chamber, so that the electric shock cup is in good contact with the electrodes. Press the pulse key. Take out the electric shock cup and quickly add the prepared LB medium. After mixing, transfer to EP tube again. Incubate at 28°C with shaking at 250r/min for 3h. Take 200 μl of the culture solution and spread it on the LB plate containing corresponding antibiotics (kanamycin 50 μg/ml, rifampin 50 μg/ml, streptomycin 50 μg/ml). After culturing at 28°C for 2 days, a single colony was picked for PCR verification. Finally, two corresponding strains of Agrobacterium Agr (containing pGR106 plasmid) and Agr (containing pGR106O+ plasmid) were obtained. The two Agrobacterium strains were stored in 15% glycerol at -70°C for future use. the

2. Transformation of soybean

2.1 Preparation of soybean transformation medium

According to the components of various culture media shown in Table 3, take an appropriate amount of mother liquor and dilute it to prepare. Add an appropriate amount of agar to the solid medium according to the hardness of the medium to dissolve and mix evenly, adjust the pH value and distribute it into 500ml Erlenmeyer flasks, and sterilize by high pressure steam (121°C, 30min). Organic components were added prior to pouring the media plates. the

Table 3 The composition of various media

Among them, the components of MS macroelement mother liquor, B5 macroelement mother liquor, MS trace element mother liquor, B5 trace element mother liquor, and MS iron salt mother liquor are shown in Table 4-Table 8 respectively, each component is dissolved in distilled water respectively, and mixed step by step in order, 4 ℃ refrigerator. the

Table 4 Composition of MS macroelement mother liquor

After the reagents are complete, add distilled water to 1L. the

Table 5B5 The composition of the macroelement mother solution of the medium

After the reagents are complete, add distilled water to 1L. the

Table 6 Composition of MS trace element mother liquor

After the reagents are complete, add distilled water to 1L. the

Table 7B5 Composition of trace element mother liquor

After the reagents are complete, add distilled water to 1L. the

The preparation of table 8MS iron salt mother liquor

After the reagents are complete, add distilled water to 1L. the

In order to avoid incomplete chelation of FeSO ₄ and disodium ethylenediaminetetraacetic acid (Na ₂ -EDTA), FeSO ₄ crystallizes when refrigerated, FeSO ₄ and Na ₂ -EDTA should be heated and dissolved separately and then mixed to adjust the pH value to 5.5. Let cool at room temperature, then refrigerate.

Table 9 Vitamin Mother Liquid Components

Table 10 other ingredients

Put 200 soybean "Zhonghuang No. 18" (soybean variety approved by the Institute of Crop Science, Chinese Academy of Agricultural Sciences) seeds into a sealed container, and use appropriate amount of sodium hypochlorite solution (about 10ml) and concentrated hydrochloric acid (about 3.5ml) according to the size of the container Prepare chlorine gas, introduce chlorine gas into a container containing seeds, and treat for 5 hours; transfer to GM medium plate, 6-8 seeds per Petri dish (90mm). Cultivate at 25°C for 14 days until the first leaf emerges from the cotyledons and turns green, as shown in Figure 6. the

2.2 Agrobacterium-mediated soybean transformation

Agrobacteria carrying plasmids pGR106 and pGR106+ were added to 100ml LB liquid medium (containing Kanamycin (Kan) 50 μg/ml, Rifampicin (Rif) 50 μg/ml, Streptomycin (Str) 50 μg/ml, respectively. ml) in a 250ml Erlenmeyer flask, 28°C, shake culture at 250r/min until _OD650 =0.8-1.0, centrifuge 50ml of medium at 7000r/min for 10min, suspend the precipitate with 25ml of liquid CCM, and incubate at room temperature for 30min.

Cut off the hypocotyl and radicle at about 2-5mm below the cotyledon node of the germinated seeds, split the two pieces at the cotyledon node, use a blade to draw about 5-10 scars on the vertical longitudinal axis of the cotyledon node, and remove the epigerm axis. About 50 treated explants were placed in 25ml of Agrobacterium, and incubated at 25°C for 30min. Move the explants to the co-culture plate, with the cotyledon section down, and culture in the dark at 25°C for 5 days; wash the Agrobacterium liquid on the surface of the explants with liquid SIM (without adding hygromycin), and transfer to SIM (Amp 50ug /ml, cephalosporin 500ug/ml) plate, in order to ensure the normal growth of the tissue, the cotyledon hypocotyls should be inserted into the culture medium to induce germination. Cultured at 25°C, 90-150μmol photons m ^-2 s ^-1 , 18h (light)/6h (dark) for 14 days, removed the hypocotyls, and transferred the explants (callus or buds) to the added The solid SIM (containing hygromycin 5ug/ml, Amp 50ug/ml, cephalosporin 500ug/ml) plate was used for screening, and after 14 days of culture, the dosage of hygromycin was further increased to 10ug/ml, and cultured for 14 days. After induction culture 32d, remove cotyledon, the new shoot that obtains and callus are transferred on the SEM (containing cephalosporin 500ug/ml) that contains 10ug/ml hygromycin, further screening transformant, because plant is to hygromycin The tolerance is weak, and the dosage of hygromycin should be reduced (10ug/ml, 5ug/ml, 0ug/ml) when the culture medium is changed every 14 days. When the length of the shoots reached 4 cm, the explants were transferred to RM medium (the formulation was the same as GM medium, but the content of NH ₄ NO ₃ was 4950 mg/L, and KH ₂ PO ₄ was 510 mg/L) to induce rooting. The new plants after rooting are transferred to the soil for cultivation.

2.3. Detection of gene ScOXDC2 expression in soybean transformed plants

The extraction and reverse transcription of total RNA from transformed plants refer to the method described in Example 1. Using the reverse transcription product as a template, using the specific primer SF (5'CTGGATGAGGGCTGTGAGTT 3') and primer SR (5'AATAACATCATCCGGCAAGC 3') designed according to the ScOXDC2 mRNA as a template, the expression of the gene SsOXDC2 in the transformed plants was detected by PCR . The PCR system is as follows: a total reaction volume of 50 μl, 2 μl of the reaction product from the previous step, 5 μl of 10×PCR buffer, 1 μl of 10 mM dNTP, 1 μl of primer SF, 1 μl of primer SR, 39.5 μl of ddH ₂ O, and 0.5 μl of 5U/μl Taq. PCR program: 95°C for 3min, 95°C for 30s, 56°C for 30s, 72°C for 2min, 30cycles, 72°C for 7min. Electrophoresis detection found that the gene ScOXDC2 in the four transformed plants could detect the accumulation of mRNA, the results are shown in Figure 4.

2.4. Evaluation of the resistance of soybean transformed plants to Sclerotinia sclerotiorum

Activate the Sclerotinia strain Ep-1PNAa367 on the PDA plate, transfer the activated Sclerotinia to a 1/2 PDA plate (half of the normal PDA concentration) and cultivate it on the plate for 2 days, use a 5mm hole punch to punch out the edge of the colony and grow vigorously mycelium blocks. Inoculate mycelium clumps onto living leaves. Gently spray a layer of water on the surface of the leaves with a watering can, moisturize and cultivate, and record the area of the lesion after 48 hours. It can be seen that compared with the plant sex of the blank vector pGR106, the plants of the S. sclerotinia oxalate decarboxylase gene SsOXDC2 can significantly inhibit soybean leaf bacteria The expansion of sclerotinia lesions reduces the area of sclerotinia by 12 times, and the sclerotinia inoculated on the petioles of living plants cannot expand to adjacent petioles, preventing the disease from spreading further. The results are shown in Figure 6 and accompanying drawing 7. the

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Claims (3)

1. A separation sclerotium disease is produced the sclerotinite oxalate decarboxylase gene SsOXDC2 of resistance, its nucleotide sequence is shown in sequence table SEQ ID NO:1.
2. A separation sclerotium disease is produced the sclerotinite oxalate decarboxylase gene SsOXDC2 of resistance, the sequence of the protein of its coding is shown in sequence table SEQ IDNO:3.
3. 3. claim 1 or 2 described genes are increasing soybean to the application in the resistance to sclerotinia sclerotiorum.

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