CN113337519B - Application of Different Copies of BrMYC2/3/4 Gene in Plant Growth - Google Patents

Abstract

The invention discloses the application of different copies of BrMYC2/3/4 genes in plant growth, and the application of BrMYC2 and BrMYC4-1 in promoting early flowering of plants, increasing plant seed yield, and improving resistance to fungal pathogens; BrMYC3-1 in promoting The application of BrMYC3-2 in promoting plants to delay flowering, increasing plant seed yield and improving resistance to fungal pathogens; BrMYC4-2 in promoting delayed flowering and improving resistance to fungal pathogens Applications in pathogen resistance. The invention provides new genetic resources for improved breeding of cabbage, thereby promoting the breeding process of cabbage.

Description

Application of Different Copies of BrMYC2/3/4 Gene in Plant Growth

technical field

The invention relates to the technical field of plant genetic engineering, in particular to the application of different copies of BrMYC2/3/4 genes in plant growth.

Background technique

Chinese cabbage (Brassica rapa ssp.pekinensis) belongs to Cruciferae and Brassica leaf vegetables, also known as 'heading cabbage' and 'yellow sprout'. It is native to North China and has a long history of cultivation. Now widely cultivated in various places, it is the leaf vegetable with the largest cultivation area of Brassica. Cabbage not only has a wide variety, but also has the characteristics of low temperature resistance, crisp taste, etc. It is also rich in minerals, carotenoids and glucosinolates. Chinese cabbage is rich in glucosinolates, which are caused by its unique flavor and important secondary metabolites glucosinolates and their hydrolyzed products' anti-disease and insect resistance, anti-cancer, anti-cancer, and anti-oxidation effects in botany, medicine, biology, food attention of researchers in many disciplines.

As an important member of the bHLH IIIe family, MYC2/3/4 transcription factors play an important role in the regulation of biological metabolism mediated by jasmonic acid. At present, most researches on the function of MYC2/3/4 transcription factors are based on the model plant Arabidopsis thaliana. host. Although Chinese cabbage shares a common ancestor with Arabidopsis, due to the complexity of its allotriploid genome, the application of glucosinolates defense function in Chinese cabbage is affected to a certain extent. It is necessary to deeply understand the role of BrMYC2/3/4 in plants Functional differentiation in growth and development and metabolism of indole glucosinolates.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of different copies of BrMYC2/3/4 gene in plant growth, so as to solve the deficiencies of the prior art.

The present invention adopts following technical scheme:

Application of different copies of BrMYC2/3/4 genes in plant growth: BrMYC2 and BrMYC4-1 are used in promoting early flowering, increasing plant seed yield, and improving resistance to fungal pathogens; BrMYC3-1 in promoting early flowering, Application of BrMYC3-2 in improving resistance to fungal pathogens; application of BrMYC3-2 in promoting plant delayed flowering, increasing plant seed yield, and improving resistance to fungal pathogens; BrMYC4-2 in promoting delayed flowering and improving resistance to fungal pathogens Applications.

Further, the plant is Arabidopsis.

Further, the fungal pathogen is Sclerotinia sclerotiorum.

Beneficial effects of the present invention:

The invention discovers different applications of different copies of BrMYC2/3/4 genes in plant growth. BrMYC2 and BrMYC4-1 can be applied to promote plants to bloom in advance, increase plant seed yield, and improve resistance to fungal pathogens; BrMYC3-1 can be applied To promote early flowering of plants and improve resistance to fungal pathogens; BrMYC3-2 can be used to promote delayed flowering of plants, increase plant seed yield, and improve resistance to fungal pathogens; BrMYC4-2 can be used to promote delayed flowering of plants and improve resistance to fungi Pathogen resistance.

The invention provides new genetic resources for improved breeding of cabbage, thereby promoting the breeding process of cabbage.

Description of drawings

Figure 1 shows the construction process of the plant expression vector pCAMBIA2302 vector.

Figure 2 shows the comparison results of the amplification and sequencing of the plant expression vector pCAMBIA2301 vector nptII; (A) the comparison results of DNA amplification and sequencing of the pCAMBIA2301 vector nptII; (B) the amino acid comparison results of the amplification and sequencing of the pCAMBIA2301 vector nptII.

Fig. 3 is the enzyme digestion detection of the plant expression vector pCAMBIA2302; Abbreviations: Lane M, DNA marker; Lane H, ddH ₂ O.

Figure 4 shows the results of the sequencing and alignment of the plant expression vector pCAMBIA2302 vector plasmids.

Figure 5 shows the electrophoresis bands of total RNA in Chinese cabbage.

Figure 6 is the analysis of the results of PCR electrophoresis of overexpression vectors; (A) p2302MYC4-1 overexpression vector; (B) overexpression vectors from left to right are: p2302MYC3-2, p2302MYC4-2, p2302MYC3-1, p2302MYC2; abbreviations : Lane M, _DNAmarker ; Lane H, ddH2O.

Figure 7 is the sequencing comparison result of the PCR products of the p2302MYC2/3/4 overexpression vector transfected into E. coli bacteria liquid.

Figure 8 is the seed growth index of T ₃ generation BrMYC2/3/4 transgenic Arabidopsis lines; (A) phenotype of mature seeds; (B) phenotype of dissected mature siliques; (C) comparison of seed lengths ; (D) comparison of seed width; (E) comparison of thousand-grain weight; (F) comparison of the number of seeds per silique; tested transgenic lines are Ctrl, BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE ; seeds and siliques with healthy and consistent growth status of each genotype were randomly selected and photographed under the same conditions; data are shown as the mean ± from three independent biological replicates SD; Statistical analysis was performed using ANOVA followed by Tukey's multiple comparison test (p<0.05); scale bar = 500 μm.

Figure 9 is the total seed weight per plant and thousand kernel weight of T ₃ generation BrMYC2/3/4 transgenic Arabidopsis lines.

Figure 10. Root and hypocotyl lengths of T ₃ generation BrMYC2/3/4 transgenic Arabidopsis lines; (A) phenotype of 6-day-old seedlings; (B) comparison of root lengths; (C) hypocotyls Comparison of axis lengths; transgenic lines are Ctrl, BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE ; data are shown as mean ± SD from three independent biological replicates ; Statistical analysis was performed using ANOVA followed by Tukey's multiple comparison test (p<0.05); scale bar = 1 cm.

Figure 11 is the difference in the vegetative stage transition time of T ₃ generation BrMYC2/3/4 transgenic Arabidopsis lines; (A) 28-day-old transgenic Arabidopsis phenotype; (B) leaf phenotype, the numbers show the appearance The leaf position of the first rosette leaf of fur; (C) Leaf growth rate of transgenic Arabidopsis in a short time; the number of leaves was counted on the 12th, 16th, 20th, 24th and 28th days after planting; transgenic lines were Ctrl , BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE ; data shown as mean ± SD from three independent biological replicates; statistical analysis was performed using ANOVA followed by Tukey Multiple comparison test of (p<0.05); scale bar = 1 cm.

Figure 12 shows plant height and flowering time of T ₃ generation BrMYC2/3/4 transgenic Arabidopsis lines; (A) phenotype of 3-week-old Arabidopsis; (B) bolting time; (C) rosette at bolting Number of leaves; (D) phenotype of 7-week-old Arabidopsis; (E) plant height; (F) number of branches; transgenic lines are Ctrl, BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE ; data are shown as mean ± SD from three independent biological replicates; statistical analysis was performed using ANOVA followed by Tukey's multiple comparison test (p<0.05); scale bar = 1 cm .

Figure 13 is the inhibition of Sclerotinia sclerotiorum growth by lyophilized powder of rosette leaves of T ₃ generation BrMYC2/3/4 transgenic Arabidopsis strain on PDA; from left to right: ddH ₂ O, Ctrl, BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE ; a sclerotinia sclerotiorum was placed in the center of each petri dish; sclerotinia sclerotiorum mycelia were white cottony mycelial plaques; scale bar = 1 cm.

Detailed ways

The present invention will be further explained below with reference to the embodiments and the accompanying drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of implementation of the present invention.

1 Construction of BrMYC2/3/4 overexpression vector

1.1 Test material

1.1.1 Plant material

The cabbage used in this study was 'Chiifu' (the strain of Zhejiang Provincial Key Laboratory of Agricultural Product Quality Improvement Technology Research, that is, the strain of this laboratory). Plants were planted in the light culture room of our laboratory under the conditions of light intensity of 600 μmol·m ^-2 ·s ^-1 , photoperiod of 16h light/8h darkness, 65% humidity, and 16h 28°C/8h 20°C.

1.1.2 Carriers and strains

The plasmid vectors used in this study are pCAMBIA2301, pCAMBIA1302, and pCAMBIA2302. Among them, plasmids pCAMBIA2301 and pCAMBIA1302 are the vectors for constructing pCAMBIA2302, which are preserved by our laboratory.

The strain of Sclerotinia Sclerotiorum was gifted by Mr. Shi Haojie, School of Agriculture and Food Science, Zhejiang Agriculture and Forestry University.

1.1.3 Preparation of solution and medium

CTAB extract: Weigh CTAB 20g (2%W/V), PVP40 20g (2%W/V), NaCl 55.84g (58.44g·mol ^-1 ), 1M Tris-HCl 100mL (pH 8.0), 0.5M EDTA 40mL (pH 8.0), distilled water to 1L. When using, add 1 μL of β-mercaptoethanol to every 1 mL of extract.

1L 1M Tris-HCl (pH 8.0): Weigh 121.1g Tris into a 1L beaker, add about 800mL deionized water, and stir well. After dissolving, add an appropriate amount of HCL to the desired pH value and make up to 1L.

100mL of 0.5M EDTA (pH=8.0): Weigh 18.612g of EDTA, dissolve it in deionized water, add NaOH particles (about 2.0g) while dissolving, and adjust the volume to 100mL after complete dissolution, and adjust the pH to 8.0.

100 mL of 50 mg·mL ^-1 kanamycin (Kanamycin, kana): Weigh 5 g of Kana and dissolve it in 100 mL of ddH ₂ O, completely dissolve it, filter sterilize it, and store it in aliquots at -20°C.

20 mL of 50 mg·mL ^-1 Gentamicin (Gentamicin, Gen): Weigh 1 g of Gen and dissolve it in 20 mL of ddH ₂ O, completely dissolve it, filter sterilize it, and store it in aliquots at -20°C.

20 mL of 50 mg·mL ^-1 Rifampicin (Rifampicin, Rif): Weigh 1 g of Rif and dissolve it in 20 mL of DMSO, completely dissolve it, filter sterilize it, and store it in aliquots at -20°C.

LB solid medium: Tryptone 10g·L ^-1 , NaCl 10g·L ^-1 , Yeastextract 5g·L ^-1 , Agar 8g·L ^-1 , pH 7.0, autoclaved at 121°C 20min.

LB liquid medium: Tryptone 10g·L ^-1 , NaCl 10g·L ^-1 , Yeast extract 5g·L ^-1 , pH 7.0, autoclaved at 121°C for 20min.

1L 1×TAE electrophoresis solution: Weigh 242g Tris Base, 372g EDTA, 57.1mL glacial acetic acid, make up to 1L to make 50×TAE electrophoresis solution, and dilute 50 times when using.

20mL agarose electrophoresis gel: Weigh 0.24g of Agarose G-50 and add 20mL of 1×TAE, boil at high temperature in microwave oven, and add 1μl of Gelstain dye when it cools down to not hot on the back of the hand.

1.1.4 Primer synthesis and sequencing analysis

Primer synthesis and sequencing analysis were performed by Zhejiang Youkang Biotechnology Co., Ltd.

1.1.5 Software and Data Analysis

Primer design software is Primer Primer 5.0; sequence analysis software is DNAMAN 6.0 and SnapGenev5.0.5; image processing software is Photoshop CS6 and Power Point 2019.

1.2 Test method

1.2.1 Acquisition of BrMYC2/3/4 gene sequence and its coding sequence

BrMYC3-1 and BrMYC3-2, BrMYC4-1 and BrMYC4-2 are BrMYC2 paralogous genes. Gene numbers of BrMYC2/3/4 BraA05g023030.3C(BrMYC2), BraA09g022310.3C(BrMYC3-1), BraA06g041690.3C(BrMYC3-2), BraA01g009460.3C(BrMYC4-1), BraA01g009470.3C(BrMYC4) and BraA08g000150.3C (BrMYC4-3), obtained from Brassica database annotations (http://brassicadb.cn/#/Annotations/). Its coding sequence was obtained from the Brassica database gene sequence (http://brassicadb.cn/#/GeneSequence/).

1.2.2 Reconstituted pCAMBIA2302 vector

HS DNA Polymerasev201扩增pCAMBIA2301质粒npt II基因；npt II基因的扩增引物为p2301 npt II RC-f：TACAAATCTATCTCTCTCGAGATGGGGATTGAACAAGATG；p2301 npt II RC-r：ATTATTATGGAGAAACTCGAGCTTGTCGATCGACTCTAGC(Tm＝47℃)。上述PCR反应程序为：98℃10s，47℃5s，72℃1min，共30cycles。用XhoI单酶切pCAMBIA1302质粒得到线性化克隆载体。插入片段扩增产物和线性化克隆载体各取2μL进行琼脂糖凝胶电泳以检验扩增产量和特异性，若PCR产物电泳条带单一且大小正确，按照TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0提供的方法进行胶回收：紫外灯下切取含有插入片段扩增产物和线性化克隆载体的琼脂糖凝胶于1.5mL离心管中，加入4个凝胶体积量的Buffer GM，均匀混合后将离心管置于37℃金属浴令胶块充分溶解。将溶液转移至Spin Column，12,000pm离心1min，弃滤液；重复此步骤2次以提高DNA回收率。加入700μL DNA Buffer WB，室温12,000pm离心30s，弃滤液，并重复此步骤一次。空离2min，将Spin Column转移到新的1.5mL离心管中，加入30μL ddH₂0，室温静置2min后12,000pm离心1min，得到纯化产物(可保存于-20℃)。The plasmids of pCAMBIA2301 and PCAMBIA1302 were extracted, respectively. use

HS DNA Polymerasev201 amplifies the npt II gene of the pCAMBIA2301 plasmid; the amplification primers of the npt II gene are p2301 npt II RC-f: TACAAATCTATCTCTCTCGAGATGGGGATTGAACAAGATG; p2301 npt II RC-r: ATTATTATGGAGAAACTCGAGCTTGTCGATCGACTCTAGC (Tm=47°C). The above PCR reaction program was: 98°C for 10s, 47°C for 5s, 72°C for 1 min, a total of 30 cycles. The pCAMBIA1302 plasmid was digested with XhoI to obtain a linearized cloning vector. Take 2 μL each of the amplified product of the insert and the linearized cloning vector for agarose gel electrophoresis to check the amplification yield and specificity. The provided method is used for gel recovery: cut the agarose gel containing the amplified product of the insert fragment and the linearized cloning vector into a 1.5 mL centrifuge tube under UV light, add 4 gel volumes of Buffer GM, mix evenly, and then centrifuge. Place the tube in a 37°C metal bath to fully dissolve the glue block. The solution was transferred to Spin Column, centrifuged at 12,000pm for 1 min, and the filtrate was discarded; this step was repeated twice to improve DNA recovery. Add 700μL DNA Buffer WB, centrifuge at 12,000pm for 30s at room temperature, discard the filtrate, and repeat this step once. Evacuate for 2 min, transfer the Spin Column to a new 1.5 mL centrifuge tube, add 30 μL of ddH ₂ 0, stand at room temperature for 2 min, and then centrifuge at 12,000 pm for 1 min to obtain the purified product (can be stored at -20°C).

II One Step Cloning Kit试剂盒进行同源重组，构建植物表达载体pCAMBIA2302(图1)。重组产物转化DH5α，转化方法如下：20μL冷却反应液加入到50μL冰上融化的感受态细胞中，轻弹管壁数下混匀，冰上静置30min。42℃金属浴热激45s，迅速转移至冰水浴孵育2min。向离心管中加入1mL不含抗生素的液体LB培养基，37℃200rpm复苏60min。取100μL复苏液均匀涂布于含有50mg·L^-1kana的LB固体培养基上，37℃倒置培养12h。挑取重组产物转化平板上若干个单菌落于含有相同浓度kana的LB液体培养基中过夜培养，用于菌落PCR鉴定。pCAMBIA2302的鉴定通过扩增的npt II基因进行，测序及菌落PCR引物为p2302 npt II-f：CTTCGCAAGACCTTCCTCTA；p2302 npt II-r；CTGGGAACTACTCACACATT(Tm＝50℃)。上述PCR反应程序为：94℃3min；94℃3min，50℃30s，72℃1min，共35cycles；72℃10min。同时设空白对照(未加模板DNA)。选择菌落PCR鉴定为阳性的菌落，将菌液进行一代测序。测序结果无误后，采用冻融法将质粒pCAMBIA2302导入GV3101感受态细胞中，具体转化步骤如下：then use

The II One Step Cloning Kit was used for homologous recombination to construct the plant expression vector pCAMBIA2302 (Figure 1). The recombinant product was transformed into DH5α, and the transformation method was as follows: 20 μL of the cooling reaction solution was added to 50 μL of competent cells thawed on ice, and the tube wall was flicked for several times to mix, and let stand on ice for 30 min. Heat shock in a metal bath at 42°C for 45s, then quickly transfer to an ice-water bath and incubate for 2min. Add 1 mL of antibiotic-free liquid LB medium to the centrifuge tube, and recover at 37°C at 200 rpm for 60 min. Take 100 μL of the recovery solution and spread it evenly on LB solid medium containing 50 mg·L ^-1 kana, and invert at 37°C for 12 h. Pick several single colonies on the transformation plate of the recombinant product and cultivate overnight in LB liquid medium containing the same concentration of kana for colony PCR identification. The identification of pCAMBIA2302 was carried out by the amplified npt II gene, sequencing and colony PCR primers were p2302 npt II-f: CTTCGCAAGACCTTCCTCTA; p2302 npt II-r; CTGGGAACTACTCACACATT (Tm=50°C). The above PCR reaction program was: 94°C for 3 min; 94°C for 3 min, 50°C for 30 s, 72°C for 1 min, a total of 35 cycles; 72°C for 10 min. At the same time, a blank control (no template DNA was added) was set. The colonies identified as positive by colony PCR were selected, and the bacterial liquid was subjected to next-generation sequencing. After the sequencing results were correct, the plasmid pCAMBIA2302 was introduced into GV3101 competent cells by freeze-thaw method. The specific transformation steps were as follows:

(1) Take the competent Agrobacterium GV3101 stored at -80°C and thaw naturally on ice.

(2) Add 1 μg of recombinant plasmid DNA per 100 μL of competent cells (preferably the volume should not exceed 10 μL), flick the tube wall, and let stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes.

(4) Add 1 mL of antibiotic-free liquid LB medium, and shake and culture on a shaker at 28°C and 200 rpm for 3 hours.

(5) Centrifuge at 6000 rpm for 1 min to collect bacterial cells, take 100 μL of supernatant, and gently pipette with a pipette to resuspend the bacterial mass. It was spread on the LB plate containing kana and placed upside down in a 28°C incubator for 48h.

(6) Pick a single colony and inoculate it in liquid LB medium (containing 50mg·L ^-1 Gen, 50mg·L ^-1 Rif and 50mg·L ^-1 Kana), 28 ℃, 200rpm shaking culture for 48h, the bacterial liquid with The strains with positive results were identified by PCR of bacterial liquid and stored at -20°C. One transformed single colony was randomly selected for plasmid sequencing analysis.

1.2.3 BrMYC2/3/4 transcription factor coding region cloning

1.2.3.1 Total RNA extraction and cDNA synthesis of cabbage

The 'Chiifu' wild-type cabbage seedlings in good growth condition were selected, and the fifth functional leaf (fifth from the top) of each plant was selected as the RNA extraction material when the seedlings grew to the five-leaf and one-heart stage. The plant genomic RNA was extracted by the Trizol method, and the specific method was as follows:

(1) Prepare test materials. The mortar, grinding rod, medicine spoon, etc. were wrapped in tin foil for high temperature sterilization in advance, and dried at 180°C for 12 hours; the large, medium and small pipette tips and 2mL centrifuge tubes were treated with DEPC water and dried at 72°C overnight; 75% ethanol was dried with DEPC Prepared in deionized water, pre-chilled with chloroform, isopropanol, and Total RNA Extractor.

(2) Take 100 mg of fresh samples and grind them in liquid nitrogen at least three times to ensure that the leaf cells are broken. After adding an appropriate amount of Total RNA Extractor, the homogenate was placed in a 2mL centrifuge tube, and the homogenate was allowed to stand at room temperature for 5 minutes, and then centrifuged at 12,000 rpm for 5 minutes at 4°C to completely separate nucleoproteins and nucleic acids.

(3) Transfer the supernatant to a new 2mL centrifuge tube. Add 0.2 mL of chloroform, vortex for 15 s, and leave at room temperature for 3 min. Centrifuge at 12,000rpm and 4°C for 10min.

(4) Transfer the upper aqueous phase to a clean centrifuge tube, add an equal volume of isopropanol, mix well, and place at room temperature for 20 minutes.

(5) Centrifuge at 12,000rpm at 4°C for 10min, and discard the supernatant.

(6) Add 1 mL of 75% ethanol prepared with DEPC-treated water to wash the precipitate. Centrifuge at 12,000 rpm at 4°C for 3 min, and discard the supernatant. Dry at room temperature for 5 min.

(7) Add 30 μL of RNase-free ddH ₂ O to dissolve the RNA fully. To detect the concentration and purity of RNA, the ratio of RNA purity A260/A280 should be between 1.8 and 2.1. Then take 2 μL for agarose gel electrophoresis detection. After the detection is correct, the obtained RNA solution is stored at -80°C or used for subsequent experiments. PrimeScript ^TM II 1st Strand cDNA Synthesis Kit was used for DNA digestion and cDNA synthesis. The reverse transcription reaction system is shown in Table 1:

Table 1 Reverse transcription reaction system

After mixing with a pipette, incubate at 65°C for 5 min, and cool on ice for 2 min, then use the above reaction solution to prepare the following mixture (Table 2).

Table 2

Mix the above samples slowly with a pipette and perform reverse transcription by PCR.

The above PCR reaction program was: 45°C for 60 min and 95°C for 5 min. After the reaction, it was immediately placed on ice to cool, and the product obtained by RT-PCR amplification was stored at -20°C or used for subsequent experiments.

1.2.3.2 DNA extraction from cabbage

Plant genomic DNA was extracted by CTAB method, and the selection of DNA extraction materials was the same as that in 1.2.3.1. The specific method is as follows:

(1) After washing and drying the leaves, take 0.2 g of the leaf and put it into a 1.5 mL centrifuge tube. After grinding with liquid nitrogen, add 700 μL of 2% CTAB preheated at 65 °C to mix well, and then take a water bath at 65 °C for 1 hour. Shake gently every 10 minutes during this period. once.

(2) Take out the above centrifuge tube and cool to room temperature, add 650 μL of 24:1 chloroform isoamyl alcohol solution, invert and mix, and let stand for 10 min.

(3) Centrifuge at 13000 rpm for 10 min, take the supernatant and continue to add 550 μL of 24:1 chloroform isoamyl alcohol solution, invert and mix, and let stand for 10 min.

(4) Centrifuge at 13,000 rpm for 10 min, take the supernatant, add 400 μL of isopropanol in an ice bath, and store at -20°C for 20 min.

(5) Centrifuge at 13,000 rpm for 10 min, discard the supernatant, add 500 μL of 80% ethanol to the remaining precipitate to wash twice (mix by suction and beating), and blow dry on an ultra-clean bench.

(6) Before use, add 30 μL of ddH ₂ O to the dried precipitate to dissolve it, let it stand at room temperature for 5 minutes, mix it with a pipette, and let it stand for another 5 minutes.

(7) Take 1.5 μL for DNA concentration and purity detection, and then take 2 μL for agarose gel electrophoresis detection. After the detection was correct, the obtained DNA solution was stored at -20°C for subsequent experiments.

1.2.3.3 Primer design and synthesis

Primers were designed using Primer Primer 5.0. Finally, primers were synthesized by Zhejiang Youkang Biotechnology Co., Ltd. Primer designs are shown in Table 3.

Table 3 Gene cloning primer design

1.2.3.4 PCR amplification of inserts and acquisition of linearized vectors

Using plant genomic DNA or cDNA obtained by reverse transcription as the amplification template, the insert fragment was amplified with high-fidelity KOD enzyme, and the PCR amplification reaction system was shown in Table 4:

Table 4 PCR amplification reaction system

The PCR program was set as follows: 94°C for 3 min; 98°C for 10 s, 52°C for 30 s, 68°C for 2 min, a total of 40 cycles; 68°C for 10 min. The PCAMBIA2302 plasmid was digested with NcoI to obtain a linearized cloning vector. Perform agarose gel electrophoresis on the amplified product of the insert and the linearized cloning vector to check the amplification yield and specificity. method for glue recovery.

1.2.4 Construction of p2302MYC overexpression vector

II One Step Cloning Kit同源重组方法，反应体系如表5所示：Overexpression vector using

II One Step Cloning Kit homologous recombination method, the reaction system is shown in Table 5:

Table 5 Homologous recombination system

After the system is prepared, mix the components by suction. After reacting at 37°C for 30 min, the reaction tube was immediately placed in an ice bath to cool for 5 min. The heat shock method was used to transform 20 μL of each gene cooling reaction solution into an appropriate amount of DH5α. The cells were cultured at 37°C overnight, and positive clones were picked for colony PCR verification and agarose gel electrophoresis detection.

The verification primers are p2302MYC-f: TCCCACTATCCTTCGCAAGA; p2302MYC-r: GAATTGGGACAACTCCAGTG (Tm=52°C). PCR products were subjected to next-generation sequencing. If the sequencing results were correct, the overexpression vector plasmids of each gene were extracted and transformed into GV3101 competent cells by freeze-thaw method.

1.3 Results and Analysis

1.3.1 Construction and identification of pCAMBIA2302 plant expression vector

The sequence analysis results of the amplified pCAMBIA2301 npt II gene are shown in FIG. 2 . After homologous recombination, the product of E. coli transformed with plasmid pCAMBIA2302 was detected by enzyme digestion. Bands 1-13 were 13 single colonies picked randomly, and a 982bp fragment was cut during cutting, and ddH ₂ O was added to the control (lane H). . The gel electrophoresis results were as expected (Figure 3). The pCMABIA2302 plasmid was transformed into Agrobacterium, and single clones were picked for activation. The results of plasmid sequencing and comparison are shown in Figure 4. This indicates that the pCAMBIA2302 plant expression vector was successfully constructed, the GUS gene of the vector was successfully replaced by the GDP gene, and the T-DNA segment only contained the npt II gene.

1.3.2 Acquisition of the target gene in cabbage and amplification of the target gene

The cabbage RNA extracted by the Trizol method was immediately detected by agarose gel electrophoresis. The detection result showed three bands, 5s, 18s and 28s bands from bottom to top, and the brightness of the 18s band was about the brightness of the 28s band. 1/2 times of , the RNA bands are bright and clear. Nucleic acid molecules contain bases that make the nucleic acid have maximum absorption at 260nm. The average concentration of RNA extracted this time measured by NanoDrop2000 nucleic acid analyzer is 2367.1ng·μL ^-1 , the A260/A280 value is 1.98, and the A260/A230 value is 2.21 , indicating that the sample is of good purity (Figure 5). The cDNA was reverse transcribed according to the measured RNA concentration, and the cabbage genomic DNA and cDNA extracted by CTAB method were detected by agarose gel electrophoresis. Linear vector recombination.

1.3.3 Construction and identification of p2302MYC overexpression vector

The amplified product was constructed into the pCAMBIA2302 vector and then transferred into E. coli and single clones were picked for colony PCR identification. The electrophoresis band was consistent with the target gene band and had higher brightness, which could be preliminarily determined as positive colonies. The sizes of the target bands amplified by MYC2, MYC3-1, MYC3-2, MYC4-1 and MYC4-2 were 1860 bp, 1734 bp, 1785 bp, 1317 bp and 936 bp, respectively (Fig. 6). However, BrMYC4-3 has only 163 bp, so it was not amplified. Then send the PCR product of the target band to the company for sequence alignment. Colony PCR and sequencing results were in line with expectations (Figure 7), and the plasmid was extracted and transformed into Agrobacterium.

2 Acquisition and functional verification of BrMYC2/3/4 overexpressing transgenic Arabidopsis

2.1 Test material

2.1.1 Plant material

In this study, the genetic transformation of the heterologous expression vector was the Columbia ecotype Arabidopsis thaliana. Plant planting conditions are the same as 1.1.1.

2.1.2 Test strains

Agrobacterium transformed with the p2302MYC2/3/4 overexpression vector constructed in 1.2.4.

2.1.3 Preparation of solution and medium

MS suspension: 1/4 MS + 6% sucrose + ddH ₂ O + 0.02% silwet + L-77

100 mL of 1M mannitol: 18.217 g of mannitol was dissolved in 100 mL of ddH ₂ O, and heated at 25°C to dissolve.

20mL of 5mM Sinigrin: Dissolve 41.548mg in 20mL of sonicated water, and store in aliquots at -20°C.

100mL DEAE Sephadex A25: 2.5g DEAE Sephadex A25 + 200mL distilled water, boil for 2h, and store in aliquots at 4°C.

25 mL SiO ₂ : 1 g SiO ₂ + 25 mL distilled water.

20 mL of 0.1% sulfatase: 0.02 g of sulfatase + 20 mL of distilled water, and stored in aliquots at -20°C.

The rest of the solution and medium are prepared with the same method as 1.1.3.

2.2 Test method

2.2.1 Transformation and screening of Arabidopsis thaliana

A 5-w-old single plant of Arabidopsis thaliana with robust growth and multiple inflorescences was selected for inflorescence infection. The overexpression vector Agrobacterium needs to be activated in advance, centrifuge the liquid LB with the target strain to obtain the bacterial block, and resuspend the solution with MS suspension to make the solution OD ₆₀₀ ≈ 0.8, and pour the suspension into a suitable graduated cylinder to prepare Arabidopsis thaliana infection. dye. Proceed as follows:

(1) Remove Arabidopsis seed pods and fully opened flower buds.

(2) Wrap the Arabidopsis cave pot with plastic wrap to prevent the matrix from leaking out during the upside down infection.

(3) Insert the budding Arabidopsis inflorescence into the bacterial suspension, infect for 60s, and all inflorescences must be immersed in the suspension.

(4) After the infection is completed, absorb the excess bacterial liquid on the sprigs of Arabidopsis thaliana, put the Arabidopsis thaliana into a clean plug covered with newspaper and moisten in advance, cover the transparent tray cover and dry newspaper, and place it in the dark Infection in a cool place for 24h.

(5) Remove the wrapped plastic wrap the next day, straighten the infected Arabidopsis thaliana, and place it in an artificial climate box or an artificial climate chamber (do not place the plants in high temperature or strong light to avoid burning them).

(6) If Arabidopsis grows well, infect the second time after one week (repeat the above steps).

(7) Keep the soil moist before the fruit clips mature and fall, and keep the watering to see the principle of dryness and wetness.

(8) Harvest seeds per plant and mark them well.

2.2.2 Molecular detection and screening of transgenic Arabidopsis plants

(1) PCR detection of transgenic Arabidopsis. The rosette leaves of the positive plants p2302MYC2/3/4 and pCAMBIA2302 were used as materials for DNA extraction, using the detection primers p2302 npt II-f and p2302 npt II-r (same as 1.2.2) of the marker gene npt II in the pCAMBIA2302 vector. Positive PCR test.

(2) In the presence of Kana, homozygous plants with a single copy of the transgenic insert were selected in the T ₃ generation based on a 3:1 segregation ratio of the T ₂ line. Leaf samples from T3 transgenic plants _were harvested, frozen in liquid nitrogen, and stored at -80°C until later use. Three independent biological replicates were used for each experiment.

2.2.3 Yield evaluation of overexpressed transgenic Arabidopsis seeds

To determine seed yield, thirty plants _were extracted from the T3 line of each genotype. After the siliques were ripe, the first five incompletely developed siliques were removed from the main inflorescence rachis. The number of seeds per silique was then assessed using the first whole silique. Ripe siliques were spread on A4 paper and the carpel walls were removed with a dissecting needle, and then photographed with a Leica stereo microscope (MZ16FA, Leica, Germany) to count the number of seeds per silique. The remaining siliques were allowed to ripen and the seeds were selected for observation from the siliques located at the base of the main inflorescence. Approximately 2,000 mature seeds were observed and photographed, randomly selected from p2302MYC2/3/4 and pCAMBIA2302 transgenic lines using a Leica stereo microscope. Seed length and width were estimated using ImageJ software (ImageJ, 1.47v, NIH, Bethesda, USA). Use an electronic scale to measure the seed weight. Data represent the mean of three biological replicates.

2.2.4 Evaluation of growth stages of overexpressed transgenic Arabidopsis plants

To assess the difference in growth stage transition between different genotypes of overexpressing Arabidopsis, approximately ₃₀ seedlings were randomly selected from each T3 line and transplanted to sterilized peat and vermiculite (1:1) substrates in the greenhouse for about 2-3 weeks. To assess whether the seedlings had reached maturity, leaf abaxial epidermal hairs were observed using a Leica stereomicroscope and the appearance of epidermal hairs was scored as a marker for the time to transition to the vegetative growth stage. For leaf shape analysis, fully expanded leaves were removed, taped to cardboard with double-sided tape, flattened with scotch tape, and scanned using an Epson V700 Professional scanner (Epson, Suwa, Japan). The bolting time and the number of rosette leaves at the time of bolting were recorded to determine the flowering time of Arabidopsis. Plant height and number of branches were determined after flowering. Photographs were taken from plants near a ruler used to calibrate ImageJ software to accurately measure distances. Maximum height was estimated from the outlines of Arabidopsis thaliana of different sizes (from the growth point to the highest leaf tip, as indicated by the vertical line). All measurements were performed in three independent biological replicates.

2.2.5 Determination of glucosinolate content in overexpressed transgenic Arabidopsis

Homozygous T ₃ generation seedling rosette leaves of transgenic Arabidopsis lines were used to determine glucosinolate content. HPLC analysis was performed using an Agilent 1200 system (Agilent Technologies, Inc., Santa Clara, USA) and a C18 reversed-phase column (250×4 mm, 5 μm, Bischoff, Germany). Chromatography was performed in the following sequence at a flow rate of 1 mL min ^-1 over 60 min: 100% H2O ( ₂ min), a linear gradient of 0%-20% ACN (32 min), 20% ACN (6 min), then an injection of 20 %-100% ACN (5 min) and 0% ACN before injecting the next sample. The eluate was monitored with a UV detector at 229 nm. The measured glucosinolate components were identified according to the peak time of the liquid chromatogram, and the content was calculated according to the amount of internal standard sinigrin ^and the corresponding response factor. ¹ DW) as a unit. The experiment was repeated three times biologically and three times technically, and the data were analyzed by SPSS software.

2.2.6 Bioassay for antifungal activity of overexpressed transgenic Arabidopsis

Freeze-dried leaf powder from each transgenic Arabidopsis line was used to study the effect of different glucosinolate compositions and contents on the visible growth of Sclerotinia sclerotiorum. Sclerotinia sclerotiorum was stored at 4°C and then reactivated in petri dishes containing potato dextrose agar (PDA) medium (Becton Dickinson, Columbia, MD). The mycelium was inoculated to the center with a 5mm hole punch. The dishes were then incubated at 22°C for 72h to provide actively growing mycelium for subsequent experiments. The edge of the mycelium in the PDA medium cultivated for 72h was cut with a hole punch to obtain an agar screen with a diameter of 5mm with a uniform growth of Sclerotinia sclerotiorum. The agar screen was placed in the center of the surface of the new PDA medium, and then 3 filter paper discs with a diameter of 10 mm were placed evenly on the edge of the PDA medium. Each filter disc received only 25 mg of lyophilized powder and 100 μL of ddH ₂ O, with 100 μL of ddH ₂ O as a control. After incubating the dishes containing Sclerotinia sclerotiorum and lyophilized powder for 72 h at 22°C, the growth of Sclerotinia sclerotiorum was assessed by observing visible mycelial growth and the number of sclerotia per dish. The experiment was performed with three biological replicates and three technical replicates.

2.3 Results and Analysis

2.3.1 The effect of overexpression of BrMYC2/3/4 on Arabidopsis seed yield

BrMYC2/3/4-overexpressing transgenic Arabidopsis (named BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE and BrMYC4-2 ^OE , respectively) and the empty vector pCAMBIA2302 as control were analyzed. Seed yield of mustard (named Ctrl) plants, including seed size (length and width), thousand-kernel weight, number of seeds per silique and total seed weight per plant. To minimize the impact of environmental factors on seed development, keep all plants under the same growing conditions, including temperature, light, moisture and nutrients. As shown in Figures 8 and 9, there were significant differences in the seed yield of the different transgenic plants. BrMYC2 ^OE lines had the smallest seed lengths; BrMYC2 ^OE , between BrMYC3-2 ^OE and BrMYC4-1 ^OE , between BrMYC3-2 ^OE , BrMYC4-1 ^OE and BrMYC4-2 ^OE , and between BrMYC4-1 ^OE , BrMYC4-2 There were no significant differences in seed length between ^OE and BrMYC3-1 ^OE , all of which were significantly smaller than controls (Fig. 8C). BrMYC3-2 ^OE and BrMYC4-1 ^OE lines had the smallest seed width, followed by BrMYC4-2 ^OE , BrMYC2 ^OE , BrMYC3-1 ^OE , Ctrl, BrMYC3-2 ^OE , BrMYC4-1 ^OE , and BrMYC4-2 ^OE between And there were no significant differences between BrMYC4-2 ^OE , BrMYC2 ^OE and BrMYC3-1 ^OE and between BrMYC3-1 ^OE and control ( FIG. 8D ). Thousand kernel weights were significantly reduced in all transgenic lines compared to controls (Figure 8E). BrMYC2 ^OE line had the smallest seed weight, followed by BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE and BrMYC3-1 ^OE . The 1000-kernel weight of BrMYC3-1 ^OE was slightly higher than that of BrMYC3-2 ^OE and BrMYC4-1 ^OE , while the 1000-kernel weight of BrMYC3-2 ^OE , BrMYC4-1 ^OE and BrMYC4-2 ^OE and between BrMYC4-2 ^OE and BrMYC3-1 ^OE There was no significant difference in the 1000-kernel weight of the seeds. The BrMYC2 ^OE line had the least number of seeds per silique, while all other transgenic lines had significantly higher number of seeds per silique than the control (Figures 8B and 8F). The transgenic BrMYC4-1 ^OE line had the highest number of seeds per silique, followed by BrMYC3-2 ^OE , BrMYC3-1 ^OE , and BrMYC4-2 ^OE . As shown in Figure 9, the BrMYC2 ^OE line had the highest total seed weight per plant, followed by BrMYC3-2 ^OE and BrMYC4-1 ^OE . BrMYC2 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE were all higher than the control. There was no significant difference between BrMYC4-2 ^OE and control, and BrMYC3-1 ^OE was significantly lower than control.

2.3.2 Effects of overexpression of BrMYC2/3/4 on Arabidopsis plant development

To determine whether BrMYC2/3/4 expression affects vegetative growth and reproductive development, the phenotype of BrMYC2/3/4-overexpressing transgenic Arabidopsis plants was observed and analyzed at the Arabidopsis seedling and bolting stages (Figures 10-12).

During juvenile vegetative growth, we investigated the root and hypocotyl length of seedlings. Root and hypocotyl lengths were significantly different between control and transgenic plants (Figure 10). The BrMYC2 ^OE line exhibited the shortest root length, followed by BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE and BrMYC4-2 ^OE (Fig. 10B). Root lengths of BrMYC2 ^OE and BrMYC3-1 ^OE lines were shorter than controls. Although there was no significant difference between the BrMYC3-2 ^OE , BrMYC4-1 ^OE and BrMYC4-2 ^OE lines, their root lengths were significantly increased compared to controls. The transgenic BrMYC2 ^OE also had the shortest hypocotyl length, followed by BrMYC3-1 ^OE , BrMYC4-2 ^OE , BrMYC4-1 ^OE and BrMYC3-2 ^OE (Fig. 10C). The hypocotyl length of BrMYC2 ^OE was shorter than that of control, whereas the hypocotyl length of BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE and BrMYC4-2 ^OE was longer than that of control. Thus, the expression of BrMYC2 ^OE and BrMYC3-1 ^OE inhibited root elongation in Arabidopsis, and the expression of BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE promoted root elongation in Arabidopsis. The expression of BrMYC2 ^OE inhibited the hypocotyl elongation of Arabidopsis thaliana, and the expression of BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE and BrMYC4-2 ^OE promoted the hypocotyl elongation of Arabidopsis thaliana.

About 3-4 weeks after seed sowing, Arabidopsis seedlings transition from juvenile to adult vegetative growth (Figure 11). The leaves of the transgenic plants were normal in shape and were indistinguishable from the control leaves. BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE overexpressing transgenic plants produced the first leaf with epidermal hairs with lower leaf position compared to the control, while BrMYC4-2 ^OE was not significantly different from the control (FIG. 11B). For leaf growth rates, BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE were significantly faster than the control, but BrMYC3-2 ^OE was similar to the control ( FIG. 11C ).

As for the reproductive growth period, we studied the bolting time, the number of rosette leaves, the plant height and the number of tillers at the bolting stage (Fig. 12). The bolting time of BrMYC2 ^OE was significantly faster, while the bolting time of BrMYC3-2 ^OE and BrMYC4-2 ^OE was slower than that of the control. Bolting times for both BrMYC3-1 ^OE and BrMYC4-1 ^OE were similar to controls (Figures 12A and 12B). Compared with the control, the number of rosette leaves was significantly decreased in BrMYC2 ^OE , BrMYC3-1 ^OE , and BrMYC4-1 ^OE overexpressing transgenic plants when bolted, while BrMYC3-2 ^OE , BrMYC4-2 ^OE increased (Figures 12A and 12C). The number of rosette leaves during bolting can be used as a basis for judging flowering time, and the rosette leaves of late-flowering plants are more. Thus, in Arabidopsis, heterologous overexpression of BrMYC2 ^OE , BrMYC3-1 ^OE and BrMYC4-1 ^OE promoted flowering time, whereas heterologous overexpression of BrMYC3-2 ^OE and BrMYC4-2 ^OE promoted flowering in Arabidopsis time delay. In terms of plant height, compared with the control, the plant height of BrMYC2 ^OE was significantly increased, while that of BrMYC3-2 ^OE , BrMYC4-1 ^OE and BrMYC4-2 ^OE was significantly decreased. Furthermore, there were no significant differences between BrMYC3-2 ^OE and BrMYC4-2 ^OE and between BrMYC3-1 ^OE and control (Figures 12A and 12E). The number of branches of BrMYC2 ^OE and BrMYC3-1 ^OE was significantly higher than that of control, while there was no significant difference between BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE and control ( FIGS. 12A and 12F ).

2.3.3 Analysis of glucosinolate content in overexpressed transgenic Arabidopsis

In order to study the effect of heterologous overexpression of BrMYC2/ ₃ /4 on glucosinolate metabolism, the types and contents of glucosinolates in leaves of T3 line were determined by HPLC. The levels of most short and long chain aliphatic glucosinolates and indole glucosinolates were significantly increased in the transgenic lines compared to controls (Table 6). Homomethionine-derived glucoiberin (GBR) was increased 2.5-fold in the BrMYC2 ^OE line. The aliphatic glucosinolates glucoerucin (GEC) and glucoraphanin (GRN) derived from dihomomethionine were increased by 3.0-fold and 2.1-fold, respectively, in the BrMYC2 ^OE strain. Glucoalyssin (GAS) from trihomomethionine was increased 2.2-fold in the BrMYC2 ^OE line. Pentamethylene-derived glucohirsutin (GHT) was increased 3.9-fold in BrMYC2 ^OE , 1.9-fold in BrMYC3-1 ^OE , and 1.8-fold in BrMYC4-1 ^OE . BrMYC2 ^OE indole glucosinolates dominated, such as GBC, 4MeGBC and NeoGBC, which were increased by 1.5-fold, 8.7-fold and 3-fold, respectively. The 4MeGBC of BrMYC3-1 ^OE was increased by a factor of 4.6. In BrMYC3-1 ^OE , BrMYC3-2 ^OE , BrMYC4-1 ^OE , BrMYC4-2 ^OE overexpressing transgenic Arabidopsis thaliana, NeoGBC increased 2.8-fold, 1.8-fold, 2.6-fold and 2.0-fold, respectively. As shown in Table 7, aliphatic glucosinolates were increased by 2.9, 1.7 and 1.6-fold in BrMYC2 ^OE , BrMYC3-1 ^OE , and BrMYC4-1 ^OE overexpressing transgenic Arabidopsis, respectively. In Arabidopsis thaliana overexpressing BrMYC2 ^OE and BrMYC3-1 ^OE , the indole glucosinolates were increased by 4.6-fold and 2.9-fold, respectively. In BrMYC2 ^OE , BrMYC3-1 ^OE , BrMYC4-1 ^OE overexpressing transgenic Arabidopsis, the total GS was increased by 3.0-fold, 1.7-fold and 1.6-fold, respectively. Therefore, the levels of most aliphatic glucosinolates and indole glucosinolates were significantly increased in all strains except BrMYC4-2 ^OE .

Table 6 GS content of single glucosinolate (μmol·g ^-1 DW)

Note: Data represent mean ± standard deviation of triplicate samples. Statistical analysis was performed using ANOVA followed by Tukey's multiple comparison test (p<0.05). Abbreviations: 4MeGBC, 4-methoxyglucobrassicin; 4-OHGBC, 4-hydroxyglucobrassicin; GAS, glucoalyssin; GBC, glucobrassicin; GB, gluconapin; GRN, glucoraphanin; NeoGBC, neoglucobrassicin; GBR, glucoiberin; GBV, glucoiberverin; GEC, glucoerucin; GHT, glucohirsutin; GNA, gluconapin; GRN, glucoraphanin; NeoGBC, neoglucobrassicin.

Table 7 Total glucosinolate GS content (μmol·g ^-1 DW)

Note: Data represent mean ± standard deviation of triplicate samples. Statistical analysis was performed using ANOVA followed by Tukey's multiple comparison test (p<0.05). Abbreviations: AGS, aliphatic glucosinolate; IGS, indoleglucosinolate.

2.3.4 In vitro antifungal activity analysis of overexpressed transgenic Arabidopsis

In order to study the resistance of transgenic Arabidopsis to Sclerotinia sclerotiorum, 25 mg×3 lyophilized powder of rosette leaves of each transgenic plant was taken and evenly distributed around the mycelium. After 72 hours of culture, patchy cotton mycelium grew with visible changes (Figure 13). The thinnest Sclerotinia plaques were found in BrMYC2 ^OE , followed by BrMYC3-1 ^OE , BrMYC4-1 ^OE , BrMYC3-2 ^OE and BrMYC4-2 ^OE . The control line expression vector control showed much thinner S. sclerotiorum plaques than the ddH ₂ O control, indicating that the endogenous basal glucosinolate level in the presence of Ctrl inhibited the growth of S. sclerotiorum to some extent. Notably, the control showed thicker Sclerotinia plaques than the BrMYC2/3/4 overexpressing transgenic lines. This may be due to the accumulation of higher levels of glucosinolates in BrMYC2/3/4 overexpressing transgenic lines, which in turn inhibited the growth of Sclerotinia sclerotiorum hyphae.

Claims (1)

1. Application of BrMYC2 gene overexpression in improving plant resistance to fungal pathogens, BrMYC2 gene number is BraA05g023030.3C, the plant is Arabidopsis thaliana, and the fungal pathogen is Sclerotinia sclerotiorum.

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