CN109652419B - Sclerotinia sclerotiorum induced rape promoter pBnGH, identification method and application - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a sclerotinia sclerotiorum induced rape promoter pBnGH, an identification method and application. From Brassica napus (A)Brassica napus) Separating and cloning and identifying a sclerotinia sclerotiorum (a sclerotinia sclerotiorum)Sclerotinia sclerotiorum) Inducing the promoter. The nucleotide sequence of the promoter is the nucleotide sequence shown as SEQ ID No.1 in the sequence table. Experiments prove that the gene regulated and controlled by the promoter can generate specific response reaction to sclerotinia infection, but the expression level in leaves, stems, buds, horns and seeds of rape and arabidopsis thaliana is extremely low, and the gene only has certain expression in roots. In addition, the promoter is not induced by hormone treatment and most abiotic stresses, and only generates a certain response to high temperature. The expression of the resistance gene is regulated by utilizing a pBnGH promoter, and the expression of the resistance gene is only acutely induced when the sclerotinia is invaded, so that the resistance of the rape to the sclerotinia is enhanced.

Description

Sclerotinia sclerotiorum induced rape promoter pBnGH, identification method and application

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a sclerotinia sclerotiorum induced rape promoter pBnGH, an identification method and application. The promoter can be used for improving the sclerotinia sclerotiorum resistance of plants through plant genetic engineering technology.

Background

A promoter is a DNA sequence that RNA polymerase specifically recognizes, binds to, and initiates transcription of a gene, and serves as a "switch" for gene transcription, regulating the transcription time and the degree of expression of the gene. Promoters can be classified into constitutive promoters, tissue-specific promoters and inducible promoters according to their function and mode of action. Plant genetic engineering introduces exogenous genes into recipient plant cells and integrates the exogenous genes into a recipient chromosome, so that the recipient plant obtains a corresponding genetic phenotype. The traditional genetic engineering mostly uses constitutive expression promoters, such as (CaMV)35S, ubiquitin promoter and the like. However, the greatest disadvantages of this type of promoter are: it can make the exogenous gene continuously and strongly express in all tissues in the whole life cycle of the plant, and can result in excessive consumption of energy or toxic action on the plant. Therefore, the use of inducible promoters or tissue-specific promoters has been increasingly emphasized in plant genetic engineering. Especially, the stress-induced promoter is used for controlling the expression of the stress-resistant gene, the coding gene controlled by the promoter is expressed on the background or is not expressed under the condition of no stress, and once the stress occurs, the coding gene is rapidly expressed in a large quantity, so that the plant shows the stress-resistant phenotype and has important function in the stress-resistant breeding of crops. Several stress-inducible promoters have been identified and used, for example the RD29A promoter (Yamaguchi-Shinozaki and Shinozaki, propagation of the expression of a destination-responsive RD29gene of Arabidopsis and analysis of expression in transgenic plants, MGG,1993,236(2-3): 331-340; Kasuga et al, expression plant promoter 346, salt, and free expression gene transfer of a single expression-expression vector, Nature, 1999,17(3): 287; Kasuga et al, A binding of the expression EB1 expression vector and expression vector, 29 expression vector and expression vector, 36 expression vector, and expression vector, 35 (3): 350; 2004; Kasuga et al, A binding of the expression EB1 expression vector, expression-expression vector, expression, U.S. patent,2015, No.9,133,467), the Oshox24 promoter (Nakashima et al, Comparative functional analysis of six drug-responsive promoters in transgenic rice plant, 2014,239(1):47-60) and the ABRC1 promoter (Lee et al, Expression of Arabidopsis CBF1regulated by an ABA/stress induced promoter in transgenic rice plant, Cell Environ,2003,26(7): 1181-.

Sclerotinia sclerotiorum is a fungal disease caused by sclerotinia sclerotiorum, causes the yield loss of the rape by 10 to 20 percent every year, and is the most main disease restricting the rape production in China. The cultivation of disease-resistant rape is the most economic and effective way to prevent and cure sclerotinia rot of rape. However, the existing rape varieties and the related varieties lack effective resistance sources, so that the breeding progress of the rape sclerotinia rot resistance is slow. Therefore, after the disease-resistant related genes are identified, the disease-resistant genes are specifically and highly expressed under the regulation and control of the sclerotinia sclerotiorum inducible promoter in the crops by a plant genetic engineering technical means, so that the resistance of the crops to the sclerotinia sclerotiorum is improved, the yield loss is reduced, and the method is an important means for breeding the sclerotinia sclerotiorum resistant crops. However, the research on the sclerotinia sclerotiorum inducible promoter in the plant has not been reported. Therefore, the separation and identification of the sclerotinia sclerotiorum inducible promoter have very important significance for the breeding of the crop with the antibacterial sclerotinia sclerotiorum.

Disclosure of Invention

In order to overcome the defects, the invention discovers a gene BnGH (BnaC01g21880D) which is strongly induced and expressed by Sclerotinia sclerotiorum in Brassica napus through transcriptome analysis, and further discovers that the gene has extremely low expression level in each organ (rootage) of normally growing Brassica napus through quantitative PCR (qPCR) analysis, and the gene does not significantly induce and express under some abiotic stresses (such as cold, salt, PEG and the like) and hormone treatments such as methyl jasmonate, salicylic acid, ethephon and the like, and only significantly induces and expresses after high-temperature treatment. Therefore, the BnGH is considered as a sclerotinia sclerotiorum induced expression gene. The promoter of this gene was subsequently identified and cloned and was defined as the sclerotinia inducing promoter pBnGH. Furthermore, the promoter drives a GUS reporter gene (pBnGH:: GUS) to be transferred into Arabidopsis, and the GUS can be induced and expressed by sclerotinia sclerotiorum and methyl jasmonate, but has extremely low expression level in normal growth tissues of Arabidopsis such as leaves, stems, flowers, horny fruits and seeds, and has no obvious induced expression under the treatment of hormones such as salicylic acid, ethephon and the like.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a rape promoter pBnGH induced by sclerotinia sclerotiorum has a sequence shown in SEQ ID No. 1.

The invention also provides an expression vector containing the Sclerotinia sclerotiorum induced rape promoter pBnGH, which is named pBnGH 83.

The invention also provides a method for cloning the rape promoter pBnGH induced by the sclerotinia sclerotiorum. The promoter is cloned from a cabbage type rape antibacterial nuclear disease strain J964.

It is another object of the present invention to provide the use of the rape promoter pBnGH or the aforementioned expression vector for improving sclerotinia sclerotiorum resistance in plants. The specific method comprises the following steps: the target plant is transformed with an expression vector comprising the sclerotinia sclerotiorum-induced rape promoter pBnGH.

The method for identifying the rape promoter pBnGH induced by the sclerotinia sclerotiorum comprises the steps of analyzing the expression quantity of a rape promoter pBnGH gene in each organ of normally growing rape through quantitative PCR, and observing whether the expression is obviously induced under abiotic stress and hormone treatment;

cloning a rape promoter pBnGH gene, and transferring a GUS reporter gene driven by the promoter into arabidopsis thaliana; the expression quantity of the rape promoter pBnGH gene in each organ of normally growing arabidopsis thaliana is analyzed through quantitative PCR, and whether the expression is obviously induced or not is observed under abiotic stress and hormone treatment.

In conclusion, the applicant of the invention provides a plant promoter pBnGH for induction of sclerotinia sclerotiorum which is a sclerotinia sclerotiorum pathogen internationally for the first time. The promoter can be used for constructing a plant expression vector of the related gene of the sclerotinia sclerotiorum, and the sclerotinia sclerotiorum resistance of the plant is improved through genetic transformation.

Drawings

The sequence table SEQ ID NO.1 is the nucleotide sequence of the sclerotinia sclerotiorum induced promoter pBnGH separated and cloned from the cabbage type rape;

FIG. 1 is a graph showing the fold difference of expression levels of Brassica napus disease-resistant material (R-line) and susceptible material (S-line) at 24h, 48h and 96h after inoculation of Sclerotinia sclerotiorum relative to the non-inoculated control (Mock) BnGH gene; hpi (hour post encapsulation);

FIG. 2 shows the relative expression level of BnGH gene in various tissues of Brassica napus; the relative expression was calculated from transcriptome data, BnGH (FPKM)/BnUBQ10 (FPKM);

FIG. 3.qRT-PCR analysis of the expression changes of Brassica napus BnGH gene under abiotic stress and hormone treatment; fold differential expression is the relative expression of BnGH after treatment divided by its relative expression under the corresponding control conditions. Data are presented as mean ± standard deviation (3 biological replicates), internal reference gene BnUBQ10(BnaA10g 06670D);

FIG. 4 expression of pBnGH promoter and GUS fusion gene in different organs or tissues of Arabidopsis thaliana. FIGS. A-F: GUS staining results, and the scale represents 2 mm; and (G) in the figure: relative expression quantity detection of GUS gene, data are expressed as mean + -standard deviation (3 biological repetitions), and internal reference is AtEF-1 a;

FIG. 5 expression analysis of the pBnGH promoter fused with GUS gene in Arabidopsis after different hormone treatment (A, B) or sclerotinia sclerotiorum inoculation treatment (C, D); FIG. A: water (H)₂GUS staining results after 6h and 12h treatment of O), Ethylene (ET), Salicylic Acid (SA) and methyl jasmonate (MeJA); and (C) figure: GUS staining results after 12h and 24h of sclerotinia sclerotiorum (Ss) and control (sterile hypha block, Mock) treatment; scale represents 5 mm; FIGS. B and D: relative expression quantity detection of GUS gene, data are expressed as mean + -standard deviation (3 biological repetitions), and internal reference is AtEF-1 a;

FIG. 6 plasmid map of pBnGH83 vector.

Detailed Description

The present invention is further defined in the following examples, from which one skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. The invention adopts the prior art in the field except for special notes.

Example 1: expression change of BnGH gene in cabbage type rape disease-resistant material and susceptible material

The two materials used in the experiment are J964 (disease-resistant material, R-line) and J902 (susceptible material, S-line), which are pure materials of winter cabbage type rape and are obtained by screening sclerotinia sclerotiorum resistant materials in a laboratory for many years.

The pathogenic bacteria are from sclerotinia sclerotiorum SS-1 provided by the national institute of microbiology, Li national gentamist of Huazhong university of agriculture. The strain is stored in a refrigerator at 4 deg.C in dark, inoculated on potato culture medium (PDA, 25% potato extractive solution, 2.5% glucose, 1.5% agar, pH adjusted to 5.86) before use, activated twice in dark at 23 deg.C, and used for inoculation.

Inoculating the rape in the final flowering phase by adopting a living stem inoculation method in the adult stage, wherein the specific method comprises the following steps: and (4) punching new edge hyphae by using a 7mm puncher for inoculation, and fixing hypha blocks by using a preservative film. Each plant was inoculated separately in three different internodes from 30-60cm from the main stem, and inoculated with hypha-free PDA blocks in the same manner as a control. And (5) respectively taking stem tissues with the length, the width and the depth of the inoculation point of 10mm and 1mm by using a blade 24 hours, 48 hours and 96 hours after inoculation. Inoculation of R-and S-lines and controls at each sampling time point 5 individual pieces of stalk tissue (15 inoculation points in total) were mixed as one sample and 3 biological replicates were set. Immediately putting the sample into liquid nitrogen after sampling, and then storing the sample in a refrigerator at the temperature of minus 80 ℃.

The total RNA extraction kit (all-type gold, China) is adopted to extract RNA, and the experimental procedures refer to the instruction of the kit. RNase-free DNase I (Thermo Scientific, USA) was used to decontaminate genomic DNA. RNA was extracted from the control mixture of three time points in each biological replicate into one mixed control sample, so that each biological replicate for each material had 4 RNA samples (3 inoculated samples (24, 48, 96h) and 1 RNA sampleMix control). Mu.g of total RNA was taken from each sample, diluted to 50. mu.g, and used

TruSeq^TMThe RNA Sample Preparation Kit (illumina, USA) constructs the RNA-seq library for the 24 RNA samples, and the specific process is shown in the Kit instruction. All libraries were then double-ended sequenced at 2X 101bp using an Illumina HiSeq2000 sequencer (national emphasis laboratory for crop genetic improvement, university of agriculture in Huazhong).

Analysis of transcriptome data the reference inventors have published papers (Wu et al, Comparative transcription analysis systems for the complex genetic network to viral transcription in Brassica napus, Sci Rep,2016,6: 19007). Analyzing the expression quantity difference multiple of brassica napus disease-resistant material (R-line) and susceptible material (S-line) at 24h, 48h and 96h after the inoculation of sclerotinia sclerotiorum relative to the uninoculated control (Mock) BnGH gene according to transcriptome data.

Example 2: cloning of promoter pBnGH

A primer amplification promoter sequence is designed according to a reference genome sequence of the Brassica napus 'Darmor-bzh' to regulate about 2000bp upstream of an initiation codon of a BnGH gene (BnaC01g21880D), and the primer sequence is (BnGHpro-F: 5'-CCAAAGGAAGACAAGGAGCA-3' (SEQ ID No. 2); BnGHpro-R: 5'-AGCAAGAAGAAGATACAGTGGGA-3' (SEQ ID No. 3)).

Extracting the genome DNA of the cabbage type rape antibacterial nuclear disease strain J964 by using a CTAB method as a template, and carrying out PCR reaction, wherein the reaction program is as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 57 deg.C for 30s, extension at 72 deg.C for 2min, and reaction for 33 cycles and then extension at 72 deg.C for 7 min. After the end, DNA is purified and recovered by using a DNA recovery kit of Kangshi company, the purified DNA fragment is connected to a pEASY-Blunt simple vector (gold, China), the vector is incubated for 1 hour at 25 ℃, E.coli Trans T1 competent cells are transformed, positive clone plasmids are selected, sequencing is completed by the Procico company, and the preserved plasmid is named as BnGHpro-1. The sequencing result shows that a DNA fragment with the length of 1820bp, such as the nucleotide sequence shown in SEQ ID NO.1, is completely consistent with the reference genome sequence and is the expected promoter pBnGH sequence.

SEQ ID NO.1

Example 3: relative expression quantity of BnGH gene in various tissues of brassica napus

The test material J9712 was planted in the experimental field of Yangzhou university in Jiangsu. Obtaining cotyledon, seedling stage (1 month old) leaf, seedling stage (1 month old) root, stem leaf, apical meristem (SAM), flower bud, unpolished ovary, 14d seed, 14d horn peel, 24d seed, 24d horn peel, 34d seed, 34d horn peel, 50d seed and 50d horn peel respectively, obtaining and mixing all tissue samples from a plurality of plants to be used as a sample, setting three biological repetitions, immediately placing the sample into liquid nitrogen after sampling, and then storing the sample in a refrigerator at-80 ℃.

The total RNA extraction kit (all-type gold, China) is adopted to extract RNA, and the experimental procedures refer to the instruction of the kit. RNase-free DNase I (Thermo Scientific, USA) was used to decontaminate genomic DNA. The construction, sequencing and analysis of RNA-seq libraries were carried out in the same manner as in example 1, and the results are shown in FIG. 2 for relative expression. The BnGH gene has the highest expression level in rape seedling roots (0.1 of the expression level of the internal reference gene BnUBQ 10), and has weak expression level in the pericarp of the horn at different periods, and the expression level of the gene is very low in all the tissues of the rape.

Example 4 expression Change of Brassica napus BnGH Gene under abiotic stress and hormone treatment

Taking J9712 with a growth period of about 30d in a greenhouse as an experimental material to carry out abiotic stress and hormone treatment, wherein the hormone treatment comprises Salicylic Acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA) and Ethephon (ETH), and the abiotic stress treatment comprises PEG (polyethylene glycol), cold (4 ℃), heat (42 ℃) and hydrogen peroxide (H)₂O₂) Carrying out treatment; both abiotic stress and hormonal treatments were set up in parallel with control treatments.

The specific steps of hormone treatment are as follows: each hormone was prepared according to the principle of the present formulation, wherein the MeJA treatment concentration was 200. mu.M, the SA treatment concentration was 1mM, the ABA treatment concentration was 100. mu.M, and the ETH treatment concentration was 1 mM. Spraying living leaf surfaces, and carrying out the method that the leaves begin to drip water to a certain degree, covering the treated rape with a safety film for moisturizing, and sampling 3 h and 6h after treatment respectively. Samples sprayed with distilled water were also used as controls. One sample was taken of 5 leaves of consistent size and 3 biological replicates were set. The removed leaves were immediately put in liquid nitrogen and then stored in a refrigerator at-80 ℃.

The abiotic stress treatment comprises the following specific steps: performing simulated drought treatment by using 15% PEG, sampling 5d after treatment, and taking plants cultured in water for 5d as a control; carrying out salt stress treatment by using 200mM NaCl, sampling 6h after treatment, and simultaneously using a water-cultured plant as a control; carrying out cold stress treatment at 4 ℃, sampling 6h after treatment, and simultaneously using a plant which normally grows at 22 ℃ as a control; carrying out heat stress treatment at 42 ℃, sampling 1h after the treatment, and simultaneously taking a plant growing normally at 22 ℃ as a control; h₂O₂The treatment was performed in the same manner as the hormone treatment at a treatment concentration of 100. mu.M, and samples were taken 3 and 6 hours after the treatment. One sample was taken of 5 leaves of consistent size and 3 biological replicates were set. The removed leaves were immediately put in liquid nitrogen and then stored in a refrigerator at-80 ℃.

The total RNA extraction kit (all-type gold, China) is adopted to extract RNA, and the experimental procedures refer to the instruction of the kit. Reverse transcription of RNA was performed using a reverse transcription kit (Novovozan, China) for qRT-PCR template. The expression level of BnGH was detected in a quantitative manner, three technical replicates were set for each sample, and both SYBR GREEN MIX and consumables used for quantification were from ABI, USA. The primer sequence of the qnRT-PCR of BnGH is as follows: 5'-GAAACGCTGCGACTTTTGATAA-3' (SEQ ID No.4) BnGH-R5'-CATCGAACATGGCGAACAAATA-3' (SEQ ID No.5), the internal reference gene is BnUBC10, and the primer sequence is as follows: BnUBC10-F: 5'-TCCATCCGACAGCCCTTACTCT-3' (SEQ ID No.6) BnUBC10-R: 5'-ACACTTTGGTCCTAAAAGCCACC-3' (SEQ ID No. 7). The reaction system and the reaction procedure are shown in tables 1 and 2.

TABLE 1 qRT-PCR reaction System

TABLE 2 qRT-PCR reaction procedure

The relative expression of BnGH before and after treatment was calculated based on the reference gene, and the expression difference before and after treatment was finally expressed as the differential expression fold, which is the relative expression of BnGH after treatment divided by its relative expression under the corresponding control conditions, as shown in FIG. 3. Data are presented as mean ± standard deviation (3 biological replicates). The methods used are all prior art. The result shows that the BnGH gene can obviously induce expression under heat stress, but the expression change is much smaller compared with the expression change after the sclerotinia sclerotiorum is infected, and the BnGH gene can not be obviously differentially expressed under other abiotic stress and hormone treatment.

Example 5 construction of recombinant vector in which pBnGH promoter sequence was fused with GUS Gene and transformation of Arabidopsis thaliana (Columbia)

The plasmid BnGHpro-1DNA is used as a template, and BamH I recognition sites of restriction enzymes are added at both ends of a pBnGH promoter sequence through PCR. Detecting by using 1% agarose gel electrophoresis after the reaction is finished, recovering and purifying the target fragment, and carrying out enzyme digestion and connection on the purified target fragment and the plant expression vector pBI101 by using BamH I at the same time. The promoter segment obtained above is connected with the upstream of GUS gene to obtain recombinant vector named pBnGH:: GUS, and the recombinant vector is transformed into agrobacterium GV3101 by electric stimulation method.

Transforming Arabidopsis thaliana Columbia ecotype through agrobacterium mediation, spreading the received T0 generation seeds on a screening culture containing 50mg/L kanamycin after sterilization, transplanting the screened seedlings into soil for continuous culture, identifying the positivity through a PCR mode, and harvesting transgenic seeds. The transformation method adopts the prior art.

Example 6 pBnGH detection of GUS expression level in various tissues of GUS transgenic Arabidopsis

Samples of whole seedlings, true leaves (45d seedlings), roots (45d seedlings), stalks, flower buds and siliques grown in the greenhouse for 20d were taken and set for three biological replicates. One sample was used for GUS staining analysis and the other sample was used for quantitative analysis.

GUS staining experimental method: (1) GUS staining solution (1 XPBS, pH 7.4; 10mM EDTA; 0.5mM K3[ Fe (CN)6 ]; 0.5mM K4[ Fe (CN)6 ]; 0.1% TritonX-100 (v/v); 0.5mg/ml X-Gluc) was prepared in appropriate volume, and the sample to be stained was placed in the staining solution and treated under vacuum for 30 min. (2) Water bath at 37 ℃ for 6h to overnight. (3) And (3) decoloring the sample by using 70% ethanol, and replacing the decoloring solution once per hour until the color of the 70% ethanol is colorless and transparent. The GUS gene expression level was measured by qRT-PCR, and the RNA extraction, reverse transcription and qRT-PCR assay methods were the same as those in example 4. The primer sequence used by GUS gene qRT-PCR is as follows: GUS-F: 5'-AGTGAAGGGCGAACAGTTCCTGAT-3' (SEQ ID No. 8); GUS-R: 5'-TTCAGCGTAAGGGTAATGCGAGGT-3' (SEQ ID No. 9). The internal reference gene is AtEF-1a, (the primer sequence is AtEF-1a-F: 5'-TGAGCACGCTCTTCTTGCTTTCA-3' (SEQ ID No. 10); and AtEF-1a-R: 5'-GGTGGTGGCATCCATCTTGTTACA-3' (SEQ ID No. 11).

The GUS analysis results of the transgenic plants inoculated with the above different tissues and sclerotinia at different times are shown in FIG. 4. The results of GUS staining showed that GUS expression was relatively low in Arabidopsis thaliana at 20d seedling stage, 45d true leaves, stems and siliques, with relatively high expression at 45d roots (FIGS. 4A-F), which is roughly consistent with the results of BnGH expression in the various tissues of Brassica napus in example 3. The results of qRT-PCR were consistent with GUS staining (fig. 4G).

Example 7 pBnGH GUS transgenic Arabidopsis thaliana expression level detection after hormone treatment and Sclerotinia sclerotiorum inoculation

Taking 30d true leaves growing in a greenhouse, and carrying out hormone treatment on the transgenic plants in a living body spraying mode. Hormone treatments included MeJA (methyl jasmonate), SA (salicylic acid) and ETH (ethephon), where the MeJA treatment concentration was 100 μ M, the SA treatment concentration was 100 μ M, the ETH treatment concentration was 7mM, the sampling time points were 6 and 12h after treatment, and the treatment methods were the same as in example 4. Three biological replicates were set up. One sample was used for GUS staining analysis and the other sample was used for quantitative analysis.

Taking 30d true leaves growing in a greenhouse, and inoculating sclerotinia in vitro at a position deviated from the middle upper part of the main vein. Samples were taken 12 and 24h after inoculation while using hyphal-free PDA blocks as a control group and setting three biological replicates. One sample was used for GUS staining analysis and the other sample was used for quantitative analysis. The GUS staining test method, RNA extraction and qRT-PCR test method were the same as those in example 6.

The results of GUS analysis of transgenic plants treated with the above different hormones and inoculated with sclerotinia at different times are shown in FIG. 5. Both qRT-PCR and GUS staining results showed that GUS was not expressed after SA and ET treatment, and also not expressed at 6h but weakly expressed at 12h in MeJA treatment (fig. 5A, B). Both qRT-PCR and GUS staining results showed that GUS induced expression significantly after inoculation of sclerotinia sclerotiorum, and as the time of inoculation increased, it induced expression more strongly (fig. 5C, D).

Example 8 construction of the pBnGH83 vector

The Pme I and Spe I recognition sites of restriction enzymes are respectively added at both ends of a pBnGH promoter sequence by PCR by using a plasmid BnGHpro-1DNA as a template. On the basis of a plant expression vector pMDC83, the original 2 xCaMV 35S promoter is replaced by the pBnGH promoter of the invention in an enzyme digestion connection mode. The vector was constructed in the same manner as in example 5. The plasmid map of the pBnGH83 vector is shown in FIG. 6.

The experimental results show that the promoter pBnGH is induced and expressed by sclerotinia sclerotiorum and is a pathogen-related promoter, and the promoter cannot induce the expression of the sclerotinia sclerotiorum under normal growth conditions and after treatment of hormones and most abiotic stresses, so that a certain utilization resource is provided for improving the disease resistance of plants.

The invention separates, clones and identifies a sclerotinia sclerotiorum induction promoter pBnGH from the cabbage type rape. The nucleotide sequence of the promoter is the nucleotide sequence shown as SEQ ID No.1 in the sequence table. Experiments prove that the gene regulated and controlled by the promoter can generate specific response reaction to sclerotinia infection, but the expression level in leaves, stems, buds, horns and seeds of rape and arabidopsis thaliana is extremely low, and the gene only has certain expression in roots. In addition, the promoter is not induced by hormone treatment and most abiotic stresses, and only generates a certain response to high temperature. The invention also provides an expression vector containing pBnGH, which is named pBnGH 83. The expression of the resistance gene is regulated by utilizing a pBnGH promoter, and the expression of the resistance gene is only acutely induced when the sclerotinia is invaded, so that the resistance of the rape to the sclerotinia is enhanced.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

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<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agtgaagggc gaacagttcc tgat 24

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttcagcgtaa gggtaatgcg aggt 24

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgagcacgct cttcttgctt tca 23

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtggtggca tccatcttgt taca 24

Claims (4)

1. A Sclerotinia sclerotiorum induced rape promoter pBnGH is characterized in that the sequence of pBnGH is shown in SEQ ID No. 1; the sclerotinia sclerotiorum induced rape promoter pBnGH is used for improving the sclerotinia sclerotiorum resistance of plants.

2. An expression vector comprising the sclerotinia sclerotiorum-induced oilseed rape promoter pBnGH of claim 1.

3. The method for cloning the sclerotinia sclerotiorum induced rape promoter pBnGH of claim 1.

4. The use of the sclerotinia sclerotiorum-induced rapeseed promoter pBnGH according to claim 1 for increasing the resistance of a plant to sclerotinia sclerotiorum disease, characterized in that an expression vector comprising the sclerotinia sclerotiorum-induced rapeseed promoter pBnGH is transformed into a target plant.

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