CN114292317B - Disease-resistant regulation function application of rape gene BnCAMTA3 - Google Patents

Abstract

The invention provides an application of a disease resistance regulation function of a rape gene BnCAMTA3, in particular to an application of the rape gene BnCAMTA3 in the prevention and control of sclerotinia sclerotiorum, which is an application in obtaining a rape material with changed disease resistance by creating transgenic rape (Brassica napus). The invention constructs the overexpression and RNAi transgenic rape of BnCAMTA3, discloses the positive regulation and control function of the gene to the resistance of sclerotinia sclerotiorum for the first time, and provides the application of obtaining the rape material with reduced resistance to sclerotinia sclerotiorum by creating BnCAMTA3-RNAi rape and the application of obtaining the anti-sclerotinia sclerotiorum rape material by creating BnCAMTA3 overexpression rape. The BnCAMTA3 gene provided by the invention is a new gene resource suitable for creating and breeding new materials and new varieties of sclerotinia sclerotiorum resistant rapes, and has important significance for green prevention and control of sclerotinia sclerotiorum.

Description

Disease-resistant regulation function application of rape gene BnCAMTA3

Technical Field

The invention belongs to the field of plant disease resistance biotechnology, relates to the application of a disease resistance regulation function of a rape gene BnCAMTA3, and particularly relates to the application of the rape gene BnCAMTA3 in the prevention and control of sclerotinia rot.

Background

1. Plant gene function analysis technology

Plant gene function is performed by gene expression and protein accumulation. Therefore, gene function is usually judged and clarified by comparing the abnormal expression of the gene with the Phenotype (Phenotype) or functional condition under the normal expression condition. Overexpression of a gene includes both higher than normal expression and lower than normal expression. Expression above normal levels is mainly Over-expression/overexpression (Over-expression), which is achieved mainly by linking a strong promoter to drive expression of the gene of interest. Expression at levels lower than normal is achieved mainly by means of RNA interference (RNAi), knock-out (Knock-out), and the like. RNAi is achieved by constructing and transforming a hairpin structure containing a fragment of the same sequence inserted in the opposite direction into an intron or other unexpressed sequence, with the result that the expression of the gene of interest is reduced. Knock-out is achieved by inserting a long non-plant sequence into the target gene in the plant genome or by excising the target gene, resulting in complete or near complete suppression of the expression of the target gene. The regulation function of the target gene to the character can be defined by constructing gene overexpression plants and/or RNAi plants and gene knockout mutants and comparing the phenotype and the character of the plants with the difference of wild type/normal plants.

2. Plant disease-resistant genetics regulation and control technology

The plant disease resistance is the result of the disease resistance signal transmission activated by the recognition of pathogenic ligand by plant receptor and the activation of serial defense reactions. Genetically, plant disease resistance is controlled by genes. The identification and utilization of the disease-resistant regulatory gene have important significance for the creation and breeding of disease-resistant crop germplasm and variety, thereby green prevention and control of plant diseases. The calcium signal channel is widely involved in the regulation and control of various plant disease resistance. CAMTA3 (Calmodulin-binding transcription activator 3) is Ca ²⁺ The important transcription factor of the signal path is an important regulation node of plant disease resistance, so that the transcription factor is a high-quality disease-resistant regulation gene resource.

3. Sclerotinia preventing and controlling technology

Plant sclerotinioses are caused by infection with Sclerotinia sclerotiorum (sclerotirotirus). Sclerotinia sclerotiorum is a necrotrophic (necrotrop) pathogenic fungus, has a very wide host range, and is a main disease of oil crops, vegetable crops and the like. Causing huge economic losses every year. Because the research on the disease resistance mechanism is not deep enough and high-resistance varieties are lacked, chemical prevention and treatment are still important means. As some pesticides have the problems of ecological pollution, human and animal toxicity, easy pathogenic substances to generate drug resistance and the like, the identification of important sclerotinia sclerotiorum regulation genes and the creation and utilization of disease-resistant varieties are of great importance for green prevention and control of sclerotinia sclerotiorum.

4. Plant disease-resistant breeding technology

Mainly comprises traditional breeding for disease resistance, breeding for disease resistance through genetic engineering, and the like. The traditional disease-resistant breeding has natural genetic isolation phenomenon, so that the available range of disease-resistant resources is obviously limited, only disease-resistant resources with relatively close genetic relationship can be applied, and multiple times of hybridization, backcross and the like are needed, so that the breeding period is long, and a large amount of manpower and material resources are needed. The genetic engineering breeding method is to introduce exogenous disease resistance regulating gene into plant via agrobacterium mediating process and other technological process to obtain disease resistance. Therefore, the genetic engineering breeding method breaks through the limit of natural genetic isolation phenomenon, widens the available range of disease-resistant resources, and has the characteristics of relatively simple and convenient operation, short breeding period and no need of a large amount of manpower and material resources. In addition, a variety with broad-spectrum disease resistance can be created by introducing a broad-spectrum disease resistance regulation gene or a plurality of genes with different disease resistance spectrums. Therefore, the method has the advantages of being especially suitable for culturing broad-spectrum and durable disease-resistant varieties and the like.

Disclosure of Invention

The invention aims to provide the application of a disease-resistant regulation function of a rape gene BnCAMTA3, in particular to the application of the rape gene BnCAMTA3 in the prevention and control of Sclerotinia sclerotiorum, wherein the function is the regulation function of a rape (Brassica napus) calcium signal pathway gene BnCAMTA3 on disease resistance, and the application can be used for creating germplasm of Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) with resistance to Sclerotinia sclerotiorum.

The application is the application of obtaining the rape material with changed disease resistance by creating transgenic rape. The method is an application in obtaining rape material for increasing the resistance of the sclerotinia sclerotiorum by creating transgenic rape of the BnCAMTA3 over-expression, or an application in obtaining rape material for reducing the resistance of the sclerotinia sclerotiorum by creating BnCAMTA3-RNAi transgenic rape.

The invention uses double 11 varieties cDNA in rape as a template, obtains rape gene BnCAMTA3 by PCR cloning, and the nucleotide sequence is shown as SEQ ID:1, the Open Reading Frame (ORF) of the gene has the length of 3096bp, the coded protein consists of 1031 amino acids, and the sequence is shown as SEQ ID:2, respectively. The BnCAMTA3 protein comprises a nuclear localization signal and DNA binding domain CG-1, an ANK (ankyrin) repetitive domain involved in protein interaction, and an IQ/CaMBD domain binding to CaM. The nucleotide sequence of the cloned BnCAMTA3 is similar to the nucleotide sequences of BnaC04g34700D of a rape variety Darmor-bzh gene and LOC106375930 at a ZS11 site of the rape variety, and the nucleotide sequences are sequentially and respectively 107 bases and 111 bases, so that 23 amino acids are changed.

Prior to the present invention, the function of the gene was not reported in any public. The invention firstly clarifies the regulation and control function and mechanism of the gene on the sclerotinia rot resistance of rape by constructing the overexpression of the gene, RNAi transgenic rape and the disease resistance analysis thereof. The results show that the overexpressing transgenic canola plants are more resistant to sclerotinia than the parental ZS11 canola plants used for the transgenesis, whereas the RNAi transgenic plants are more susceptible to sclerotinia, suggesting that BnCAMTA3 is regulating the resistance of canola to sclerotinia.

Based on the function of the BnCAMTA3 gene elucidated in the invention, the application of the invention aims to provide the application of the BnCAMTA3 gene in obtaining rape materials with changed disease resistance by creating transgenic rape, including (1) the application in obtaining rape materials with increased resistance to sclerotinia sclerotiorum by creating transgenic rape over-expressing the BnCAMTA3 (see the description in example 1 specifically); and (2) application of the transgenic rape with BnCAMTA3-RNAi creation to obtain rape material with reduced resistance to sclerotinia sclerotiorum (see the description in example 2).

1. Application of rape BnCAMTA3 gene in preparing transgenic rape with over-expressed BnCAMTA3 to obtain rape material with increased resistance to sclerotinia rot. The method is realized by the following steps:

(1) Construction and acquisition of BnCAMTA3 gene overexpression structure

Cloning BnCAMTA3 gene Open Reading Frame (ORF) into a plant expression vector, and allowing the expression to be driven by a strong promoter;

(2) Acquisition of Agrobacterium transformed with BnCAMTA3 gene overexpression Structure

Transforming the constructed BnCAMTA3 gene overexpression structure into an agrobacterium strain with strong infection capacity to rape by methods such as electric shock and the like;

(3) Creation and acquisition of transgenic rape over-expressing BnCAMTA3

Introducing the BnCAMTA3 gene overexpression structure into the rape by an agrobacterium-mediated method to obtain the rape with the BnCAMTA3 gene overexpression structure;

(4) Acquisition of transgenic rape homozygous line of overexpression BnCAMTA3

Respectively taking antibiotic resistance and BnCAMTA3 gene expression as detection indexes, detecting the character segregation condition of transgenic plant progeny, and obtaining a transgenic rape homozygous line of overexpression BnCAMTA3, wherein the progeny character of the transgenic plant progeny is not separated any more and can be stably inherited;

(5) Screening, identifying and obtaining transgenic rape homozygous line of overexpression BnCAMTA3 with increased sclerotinia sclerotiorum resistance

The transgenic rape homozygous line of overexpression BnCAMTA3 is taken as a material, the resistance to sclerotinia sclerotiorum is detected and analyzed, and the transgenic rape with enhanced disease resistance, namely BnCAMTA3, is obtained.

2. Application of rape BnCAMTA3 gene in preparing BnCAMTA3-RNAi transgenic rape to obtain rape material with reduced resistance to sclerotinia sclerotiorum. The method is realized by the following steps:

(1) Construction and acquisition of BnCAMTA3 gene RNAi structure: cloning a specific sequence fragment of the BnCAMTA3 gene into an intermediate expression vector pKANNIBAL in the positive and negative directions respectively, and then inserting the recombinant structure into an RNAi vector (pART 27) to obtain an RNAi structure pART27-BnCAMTA3;

(2) Obtaining agrobacterium for transforming BnCAMTA3 gene RNAi structure: the RNAi structure of the BnCAMTA3 gene (pART 27-BnCAMTA 3) is transformed into an agrobacterium strain with strong infection capacity to rape by methods such as electric shock and the like;

(3) Creation and acquisition of BnCAMTA3 gene transfer RNAi structure rape: introducing the RNAi structure of the BnCAMTA3 gene into the rape by an agrobacterium-mediated method to obtain the rape with the RNAi structure of the BnCAMTA3 gene;

(4) Obtaining a rape homozygous line with a BnCAMTA3 gene RNAi structure: respectively taking antibiotic resistance and BnCAMTA3 gene expression as detection indexes, detecting the character segregation condition of transgenic plant progeny, and obtaining a rape homozygous line of a BnCAMTA3 gene RNAi structure, wherein the progeny character of the rape homozygous line is not separated any more and can be stably inherited;

(5) Screening, identifying and obtaining the BnCAMTA3 gene-transferred RNAi structure rape homozygous lines for the sclerotinia rot resistance: the BnCAMTA3 gene RNAi structure (pART 27-BnCAMTA 3) transferred rape homozygospore system is used as a material to detect and analyze resistance to sclerotinia sclerotiorum and obtain the BnCAMTA3 gene RNAi rape for resisting the sclerotinia sclerotiorum.

The invention has the advantages that: (1) The BnCAMTA3 gene provided by the invention is a high-quality sclerotinia sclerotiorum resistance regulation gene resource, and a disease-resistant material obtained by utilizing the gene has the advantages of strong disease resistance and the like. Rape sclerotiniose resistance is quantitative trait resistance, controlled by multiple genes. In general, a single gene regulates this resistance to a lesser extent. Therefore, the sclerotinia sclerotiorum resistant materials are all deficient and high resistant materials are not available all over the world. The BnCAMTA3 gene is a key transcription factor gene of a calcium signal channel, is a central node for controlling plant disease resistance, and is a key disease resistance control gene resource. The method for creating disease-resistant varieties and germplasm by using BnCAMTA3 is an economic, effective and safe way for green disease prevention and control. Therefore, the BnCAMTA3 gene is a brand new gene resource suitable for creating and breeding new materials and new varieties of the sclerotinia rot resistant rape. And (2) the period for obtaining the disease-resistant material is short. The method for obtaining disease-resistant plant materials and varieties mainly comprises a conventional traditional breeding method and a genetic engineering breeding method utilizing a disease-resistant regulatory gene. The traditional breeding method has the defects that the range of available disease-resistant resources is limited by natural genetic isolation, the breeding period is long, a large amount of manpower and material resources are needed, and the like. The genetic engineering breeding method has the advantages of wide range of available disease-resistant resources, relatively simple and convenient operation, short breeding period, no need of a large amount of manpower and material resources, and is particularly suitable for breeding broad-spectrum, durable and high disease-resistant varieties. The invention utilizes the disease-resistant regulatory gene BnCAMTA3, adopts a genetic engineering method, creates and cultures the sclerotinia rot resistant rape material, and has the characteristics of short period, rapid breeding and the like.

Drawings

FIG. 1 provides evidence of various transgenic rape plants obtained in the present patent, showing the results of the expression detection of BnCAMTA3 gene in transgenic rape plants. The RNAi plant (A) for silencing the BnCAMTA3 gene and the OE plant (B) for over-expressing the BnCAMTA3 gene are detected by using real-time fluorescent quantitative PCR (polymerase chain reaction). Rape BnACTN 7 is selected as an internal reference gene, and the relative expression quantity of the non-transgenic parent ZS11 is set to be 1. The experiment was repeated three times, and Student's t-test statistical analysis was performed on the expression result data, which are expressed as mean ± standard deviation. Significance of differences is indicated by different numbers (, P <0.01;, P < 0.001). The result shows that the expression level of BnCAMTA3 in RNAi plants is obviously reduced and is only 25 percent (A) of that of control plants, while the expression level of BnCAMTA3 in over-expression strains is obviously improved and is 4.9 times (B) of that of the control plants. The plants are shown to be real BnCAMTA3 gene RNAi plants and over-expression plants.

FIG. 2 provides evidence of the anti-sclerotic disease regulatory function of the BnCAMTA3 gene, showing that BnCAMTA3 positively regulates the resistance of oilseed rape to sclerotinia sclerotiorum. The inoculation analysis of Sclerotinia sclerotiorum UF-1 is carried out on rape BnCAMTA3 transgenic plants. The graph shows the results of quantitative statistical analysis of phenotype (A) and lesion area 24h after inoculation (B). The experiment was repeated three times. Statistical analysis of Student's t-test was performed on lesion area data, which are expressed as mean ± standard deviation. Significance of differences is indicated by different numbers (, P)<0.01；***，P<0.005). The results show that RNAi plants show a more disease-sensitive phenotype than the non-transgenic (ZS 11) control, whereas over-expressed plants are significantly more disease-resistant than the lighting (A). The result of quantitative analysis of the lesion area shows that the lesion area of the ZS11 control plant is 115.6mm ² The lesion area of RNAi plants averagely reaches 151.1mm ² Significantly greater than control plants; the lesion area of the over-expression OE plant is only 95.9mm ² Significantly less than control (B). These results indicate that BnCAMTA3 positively regulates resistance of oilseed rape to sclerotinia sclerotiorum.

Detailed Description

The invention is further explained by the accompanying drawings and examples.

Example 1

The invention clones a rape gene BnCAMTA3, clarifies the sclerotinia rot resistance regulation function of the gene by constructing the overexpression transgenic rape for the first time, and reveals that the gene has an important positive regulation function on the sclerotinia rot resistance of the rape. Because the overexpression of the BnCAMTA3 gene leads the remarkable enhancement of sclerotinia sclerotiorum resistance of rape, a new sclerotinia sclerotiorum resistance rape material can be created and obtained by constructing the overexpression transgenic rape of the gene and adopting a gene engineering technology. The method mainly comprises the following steps:

1) Cloning and preservation of rape BnCAMTA3 gene

The rape BnCAMTA3 gene provided by the invention is obtained by cloning through the following steps. Firstly, primers BnCAMTA3-F (5'-atg gcg gaa gca aga cga ttt-3') (the sequence is shown as SEQ ID: 3) and BnCAMTA3-R (5'-agc gtt cca caa agt tga gga-3') (the sequence is shown as SEQ ID: 4) are designed according to the sequence of BnCAMTA3 in the rape genome database. Extracting total double-11-leaf RNA in rape varieties by using TRIZOL reagent, amplifying by using an RT-PCR method to obtain BnCAMTA3 cDNA, performing gel cutting and purification after electrophoresis of 1% agarose gel to recover PCR products, directly connecting target fragments to pGWB5 overexpression vectors by using Golden gate endonuclease, thermally shocking and transforming Escherichia coli DH5 alpha, recovering bacteria in LB culture medium by shaking for 1h, absorbing bacterial liquid to coat on a kanamycin plate for overnight culture, selecting single clone, extracting plasmid, performing PCR amplification by using BnCAMTA3-F/BnCAMTA3-R as primers, checking whether the extracted plasmid contains BnCAMTA3 gene, and finally sending to a company for sequencing verification, thereby successfully cloning and obtaining the BnCAMTA3 gene cDNA full-length sequence. The nucleotide sequence of BnCAMTA3 is shown as SEQ ID:1, the Open Reading Frame (ORF) of the gene has the length of 3096bp, the coded protein consists of 1031 amino acids, and the sequence is shown as SEQ ID:2, respectively. The gene coding product comprises a nuclear localization signal and DNA binding structural domain CG-1, an ANK (ankyrin) repetitive structural domain involved in protein interaction, and an IQ/CaMBD structural domain combined with CaM. BLAST analysis shows that the nucleotide sequence cloned by the invention has 107 base differences with the BnaC04g34700D nucleotide sequence of the rape variety Darmor-bzh, which results in 23 amino acid changes; the nucleotide sequence of the mutant has 111 base differences with LOC106375930 nucleotide sequence of rape variety ZS11, and 23 amino acid changes are also caused. Prior to the present invention, there was no public report of the function of this gene

Coli transformed with a vector carrying the BnCAMTA3 gene sequence was stored in a freezer at-80 ℃. The BnCAMTA3 gene can be subcloned into a target vector by activating a strain at any time, extracting plasmids and amplifying by PCR (polymerase chain reaction) and is used for researches such as transgenosis and the like. Meanwhile, the pGWB5 overexpression vector drives the expression of a target gene by a CaMV 35S strong promoter and carries a GFP label, thereby facilitating molecular identification and gene function research.

2) Acquisition of Agrobacterium transformed with BnCAMTA3 gene overexpression Structure pGWB5-BnCAMTA3

The BnCAMTA3 gene overexpression structure pGWB5-BnCAMTA3 is transformed into agrobacterium strain with strong infectivity to rape, such as EHAl05, transformants are screened on YEP culture medium containing kanamycin and rifampicin antibiotics, and then positive strains are identified by PCR to obtain agrobacterium carrying the BnCAMTA3 gene overexpression structure pGWB5-BnCAMTA 3. Used for the next step of rape genetic transformation.

3) Creation and acquisition of BnCAMTA3 gene-transferred overexpression structure pGWB5-BnCAMTA3 rape

Introducing BnCAMTA3 gene overexpression structure pGWB5-BnCAMTA3 into rape by an agrobacterium-mediated method, and finally obtaining regenerated rape T transferred with pGWB5-BnCAMTA3 ₀ And (4) generation. The specific operation steps are as follows:

(i) Seed cleaning and germination

75% ethanol for 30-60s, sterile water for 1 time, 1 min/time; 0.15% mercuric chloride for 10min, and cleaning with sterile water for 2 times and 1 min/time; cleaning with sterile water for 30min, inoculating to sterile filter paper, and air drying; inoculating the seeds into a culture bottle, and performing dark culture at 23 ℃ for 5-6 days;

(ii) Preculture

Cutting the hypocotyl of germinated rape seedling into 0.4-0.6cm segments, inoculating to a pre-culture medium, and culturing at 23 deg.C under illumination for 2-3d;

(iii) Agrobacterium infection and co-culture

Selecting Agrobacterium in infection solution to prepare OD ₆₀₀ =0.2 Agrobacterium resuspension, explants were inoculated into Agrobacterium suspension for 10min of infection. Inoculating the infected explants on sterile filter paper, airing, inoculating on a co-culture medium, and performing dark culture at 23 ℃ for 48-72h;

(iv) Bacteria-free (delay screen)

Inoculating the explants after co-culture on a degerming culture medium, and performing illumination culture at 23 ℃ for 6d;

(v) Screening/differentiation

Inoculating the explants subjected to the bacteria-removing treatment on a screening/differentiation culture medium, inoculating 30 explants on each dish, performing illumination culture at 23 ℃, and changing the culture medium once for 15 days;

(vi) Rooting culture

And (3) inoculating the differentiated bud into a rooting culture medium, and culturing at 23 ℃ under illumination until the bud grows roots.

4) Screening, identifying and obtaining rape homozygous lines of BnCAMTA3 gene overexpression structure pGWB5-BnCAMTA3

And (3) detecting the character segregation condition of the transgenic plant progeny by respectively using hygromycin resistance and BnCAMTA3 gene expression as detection indexes. Hygromycin resistance screening is carried out on a hygromycin-containing plate aiming at rape seeds, and whether healthy seedlings can normally grow on the hygromycin-resistant plate or not is observed. The gene expression is detected by methods such as real-time fluorescent quantitative PCR and the like. And acquiring the rape homozygous line of the transgenic BnCAMTA3 gene overexpression structure pGWB5-BnCAMTA3 which has the progeny character which is not separated any more and can be stably inherited.

The BnCAMTA3 overexpression transgenic rape homozygous line obtained by the invention can normally grow into healthy seedlings on a hygromycin resistant plate, and the BnCAMTA3 gene expression level is obviously higher than that of a control and is 4.9 times that of the control (figure 1B). The plants are proved to be true BnCAMTA3 gene over-expression plants.

5) Disease resistance detection analysis of BnCAMTA3 gene overexpression rape homozygous line

The BnCAMTA3 gene overexpression rape homozygosis system obtained in the step 4) is taken as a material, and the resistance of the BnCAMTA3 gene overexpression rape homozygosis system to rape Sclerotinia sclerotiorum is detected and analyzed by inoculating Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), so that the regulation and control effect of the BnCAMTA3 gene on the rape Sclerotinia sclerotiorum resistance is determined, and whether a new Sclerotinia sclerotiorum resistance rape material can be created and obtained by constructing overexpression transgenic rape of the gene and adopting a genetic engineering technology.

Activation culture of sclerotinia sclerotiorum: the method comprises the steps of selecting plump and pollution-free sclerotinia sclerotiorum sclerotia, cutting the sclerotinia sclerotiorum into two halves by using an aseptic blade burned by an alcohol lamp, placing the sclerotinia sclerotiorum with a section facing downwards on a PDA solid plate, culturing at 23 ℃ in a dark place for 3 days, punching a hypha block with the diameter of 4mm from the edge of a bacterial colony to the inner side by using a puncher, inoculating the hypha block to a new PDA solid plate with the side facing downwards with the hypha, and culturing at 23 ℃ in the dark place for about 36 hours to perform inoculation.

Inoculation of sclerotinia sclerotiorum: and (4) selecting the rape plants with consistent growth vigor for inoculation. Using a puncher with the diameter of 4mm to punch hypha blocks with the edges of colonies inward by 3-5mm, inoculating the hypha blocks to fully developed leaves with the hypha surfaces facing downwards, respectively inoculating one hypha block to the left half and the right half She Duichen of each leaf, covering films to preserve moisture, placing the hypha blocks in a greenhouse for culture at 23 ℃, taking pictures after a proper time (about 24 hours) and analyzing the lesion area by ImageJ software.

The inoculation experiment was repeated three times. Student's t-test statistical analysis was performed on lesion area and data are presented as mean ± standard deviation. Significance of differences was indicated by different number (P < 0.005). The results show that BnCAMTA3 overexpression plants show a remarkably more disease-resistant phenotype than non-transgenic parent plants, and the lesion area is remarkably reduced compared with a control. The over-expression of BnCAMTA3 gene is shown to remarkably enhance the resistance of rape to sclerotinia sclerotiorum (figure 2).

These results indicate that BnCAMTA3 overexpressing plants are significantly more resistant to sclerotinia than wild type plant controls. Overexpression of the BnCAMTA3 gene leads to remarkable improvement of sclerotinia sclerotiorum resistance of rape, so that the BnCAMTA3 plays a positive control role in sclerotinia sclerotiorum resistance of rape. The result also proves that the new antibacterial sclerotinia rot rape material can be created and obtained by constructing the overexpression transgenic rape of the BnCAMTA3 and adopting the gene engineering technology.

Example 2

The embodiment 1 of the invention clarifies the positive regulation function of the rape gene BnCAMTA3 on the resistance of sclerotinia sclerotiorum by constructing an over-expression plant of the BnCAMTA3 gene. This example illustrates the RNAi rapeseed construction technology of BnCAMTA3 gene, and further proves the positive regulation function of BnCAMTA3 to sclerotinia sclerotiorum resistance by the material. This example provides a technique for creating a new material for obtaining oilseed rape with reduced resistance to sclerotinia sclerotiorum by constructing RNAi oilseed rape of BnCAMTA3 gene, and the material can be used for gene function and mechanism of action analysis. The method mainly comprises the following steps:

(1) Construction and acquisition of BnCAMTA3 gene RNAi structure

Designing a primer BnCAMTA3-F3 (5'-ggagaggacacgctcgag atg gcg gaa gca aga cgt-3', the italic part is a sequence which comprises an Xho I enzyme cutting site and is consistent with a linearized vector pKANNABAL) (the sequence is shown as SEQ ID: 5) and BnCAMTA3-R3 (5'-tttccttaccaattggggtacc atg tag aac atc aac gct tcc ag-3', the italic part is a sequence which comprises a Kpn I enzyme cutting site and is consistent with a linearized vector pKANNABAL) (the sequence is shown as SEQ ID: 6) according to the sequence of the cloned BnCAMTA3; bnCAMTA3-F4 (5'-ttcgaaatcgataagctt atg tag aac atc aac gct tcc ag-3', the italic part is the sequence containing the Hind III cleavage site, identical to the linearized vector pKANNIBAL) (sequence shown in SEQ ID: 7), and BnCAMTA3-R4 (5'-ttaaagcaggactctaga atg gcg gaa gca aga cgt-3', the italic part is the sequence containing the Xba I cleavage site, identical to the linearized vector pKANNIBAL) (sequence shown in SEQ ID: 8). Taking pGWB5-BnCAMTA3 plasmid obtained in 1) of example 1 as a template, and BnCAMTA3-F3 and BnCAMTA3-R3 as primer pairs, obtaining a 300bp BnCAMTA3 forward fragment (fragment 1) through PCR amplification, then obtaining an intermediate expression vector pKANNIBAL-BnCAMTA3-1 with a 300bp BnCAMTA3 forward fragment subcloned through steps of Xho I/Kpn I double digestion pKANNIBAL vector (fragment 2), recombinant ligation of the fragments 1 and 2 by recombinant ligase, transformation, kanamycin culture medium plate screening, digestion, PCR identification, sequencing identification and the like. The correctly sequenced plasmid was double digested with Xba I and Hind III and the large fragment (fragment 3) was recovered. Meanwhile, pGWB5-BnCAMTA3 plasmid is used as a template, bnCAMTA3-F4 and BnCAMTA3-R4 are used as primer pairs, a BnCAMTA3 reverse fragment (fragment 4) with the length of 300bp is obtained through PCR amplification and is recovered, and the fragments 3 and 4 are subjected to recombination and connection by using recombination ligase, transformation, kanamycin culture medium plate screening, enzyme digestion, PCR, sequencing identification and other steps to obtain the pKANNABAL-BnCAMTA 3-RNAi vector containing the positive and reverse BnCAMTA3 fragments. pKANNIBAL-BnCAMTA3-RNAi vector and pART27 were digested with Not I and ligated with T4. Then Xba I and Xho I double enzyme digestion, PCR and sequencing are used for identifying whether a 300bp BnCAMTA3 fragment is inserted into the two ends of the PDK intron of the recombinant vector in a positive and negative two-way manner, so that an RNAi structure pART27-BnCAMTA3 is obtained.

(2) Acquisition of Agrobacterium transformed with BnCAMTA3 Gene RNAi Structure

The RNAi structure of the BnCAMTA3 gene (pART 27-BnCAMTA 3) is transformed into an agrobacterium strain with strong infection capacity to rape, such as EHA105, transformants are screened on a YEP culture medium containing spectinomycin, and the agrobacterium carrying the RNAi structure of the BnCAMTA3 gene pART27-BnCAMTA3 is obtained through Xba I and Xho I double digestion and PCR identification. And (4) carrying out the next step of genetic transformation of rape.

(3) Creation and acquisition of BnCAMTA3 gene-transferred RNAi structure rape

Introducing BnCAMTA3 gene RNAi structure pART27-BnCAMTA3 into rape by agrobacterium-mediated method, and finally obtaining the rape T transferred with pART27-BnCAMTA3 ₀ And (4) generation. The specific procedures were as described in 3) of example 1.

(4) Acquisition of BnCAMTA3 gene-transferred RNAi structure rape homozygous line

And respectively detecting the character separation condition of the transgenic plant progeny by taking the antibiotic resistance and the BnCAMTA3 gene expression as detection indexes. Kanamycin resistance screening is carried out on a kanamycin-containing plate aiming at rape seeds, and whether healthy seedlings can normally grow on the kanamycin-resistant plate or not is observed. The gene expression is detected by methods such as real-time fluorescence quantitative PCR and the like. And obtaining the rape homozygous line of the transgenic BnCAMTA3 gene RNAi structure pART27-BnCAMTA3 which is not separated from the progeny character and can be stably inherited. These rape homozygous lines all grew normally into healthy plantlets on kanamycin resistant plates, and the gene expression level of BnCAMTA3 was significantly lower than that of the empty vector control.

The invention obtains BnCAMTA3-RNAi rape homozygous lines, healthy seedlings can normally grow on a kanamycin-resistant plate, the expression level of the BnCAMTA3 gene is obviously lower than that of a wild type control, and only 25 percent of the control plant (figure 1A) is obtained. The plants are shown to be real BnCAMTA3 gene RNAi plants.

(5) Screening, identifying and obtaining BnCAMTA3 gene-transferred RNAi structure rape homozygous lines for resisting sclerotinia rot

The BnCAMTA3 gene RNAi structure (pART 27-BnCAMTA 3) transferred rape homozygosis system is used as a material to detect and analyze the resistance to sclerotinia sclerotiorum. The inoculation method and evaluation of disease resistance were as described in 5) of example 1).

The result of sclerotinia inoculation analysis of the BnCAMTA3-RNAi plant constructed by the invention shows that the BnCAMTA3-RNAi plant is obviously more susceptible than a non-transgenic plant control (figure 2). The lesion area of the BnCAMTA3-RNAi plant was significantly larger than the control (figure 2). The BnCAMTA3-RNAi plant is shown to have significantly lower resistance to sclerotinia sclerotiorum than the control. Inhibition of the expression of the BnCAMTA3 gene results in a significant reduction in resistance of oilseed rape to sclerotinia sclerotiorum. The invention successfully creates and obtains a new rape material with obviously reduced sclerotinia sclerotiorum resistance by constructing BnCAMTA3-RNAi rape.

In summary, the results of the invention combined with the results of fig. 1-2 reveal that the rape calcium signal pathway key gene BnCAMTA3 plays a positive control role in rape sclerotinia rot resistance for the first time. The invention provides an application approach and an application technology of BnCAMTA3 in creating germplasm of an antibacterium nucleopathy crop, provides an embodiment and successfully obtains the antibacterium nucleopathy rape.

Sequence listing

<110> Zhejiang university

<120> application of disease resistance regulation function of rape gene BnCAMTA3

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atggcggaag caagacgttt tggccttaat aacgaactag atgttggcca aatactttca 60

gaagcacgga atcgatggct tcgtcctcct gaaatctgcg aaatcttaca gaattaccaa 120

aagtttcaaa tttctaccga gccacctacc acaccggcca gtggatctgt ttttctattt 180

gatcgaaagg tgctcagata cttcaggaaa gacggtcaca actggaggaa gaaaagagat 240

ggaaagacag ttaaagaagc tcatgagagg ttgaaggctg gaagcgttga tgttctacat 300

tgttactatg cgcatggaca agacaatgaa aattttcaaa gacgcagtta ttggatgctt 360

caagaagagc tttcccacat tgtttttgtc cattatctcg aagttaaggg tagtagagtt 420

tctacttctt ataatcggat gcaaaggact gaagattcta ctcgatcttc tcaagaaact 480

ggggaagtct acaccagtga acgtaatggt tatgcttctg gcagcattaa ccaatatgat 540

cacagcaaca atcagtcaca agctactgac tcagcaagtg tcaatggtgt tcacacccca 600

gaacttgaag atgcacaatc agcatacaat cagcagggga gccccatact ttactcacat 660

caagcacttc agcagcctcc agctactagt tttgatcctt actatcagat gtctttgacg 720

ccgagggata gctatcagaa agagattcac acaatctcct cgtccactat ggtagaaaaa 780

ggcagaacta tcaacggtcc tgttgtaaca aatagcataa aaaacaaaaa atccattgat 840

tcccaaacct gggaagagat actgggaaac tgtggttctg gaggtgaagg tttacctatg 900

cagcctcaca gtgagcatga agggcttgat caaatgctcc aaagctactc ttttactatg 960

caagattttg ccagtctaca ggagtccatt gtcaaaagcc agaatcagga gttaaattca 1020

gggcttacat ctgatcgttc actgtggtta caaggacaag ctgtagatat agagccaaat 1080

gcgctaagca atttagcttc aagtgaaaaa gctccatatc tatctacgat gaaacagcat 1140

ctgttagacg gtgcattagg tgaagaaggc ttgaaaaaaa tggacagttt caaccgctgg 1200

atgagcaaag agctcggaga actcggagat gttggtgtta ctgccgatgc aaacgagtct 1260

tttactcatt cgagttccac agcctactgg gaagaagttg agagtgaaga tgtgtctaat 1320

ggtggatatg ttatgagtcc ttccttatca aaggaacagc tctttagcat cattgacttt 1380

gctccgaact ggacttatgt gggctgtgaa gtgaaggttc ttgttagtgg aaagttctta 1440

aagatggctg agagtggaga gtggtgttgc atgtttgggc aaacagaagt tccagcggat 1500

attatagcta atggtatact cgagtgcgtt gcccctatgc atgaggctgg aagagttccc 1560

ttttatgtaa catgttccaa caggttagca tgcagcgaag tgcgtgagtt cgagtacaag 1620

gttttggagt ctcaaggctt tgatagagaa acatatgatt cttccaccgg ttgcaactct 1680

attgagagtc tggaggcaag atttgttaaa ctgctgtgct cgaaatctga ttgcacgaac 1740

tcttctcttc ccggggggaa cgacagtgat ttgtcccaag tgagcgagaa gattagctta 1800

ctgcttttcg agaacgatga ccagctggat cagatgctga tgaatgaaat ctctcaagag 1860

aatatgaaga acaacctctt gcaggaagct ctgaaggaaa gcttacactc atggcttctg 1920

caaaagatag cagaaggtgg gaaaggtccg aacgtgttgg acgaaggtgg acaaggtgta 1980

ctacactttg ctgctgctct tggctacaac tgggcgttag aaccgacgat agtcgctggt 2040

gtaagcgttg attttcgcga cgtgaatggc tggactgcac ttcactgggc agctttcttt 2100

ggcagggagc tgataatagg ttctctcata gctctcggtg catctcccgg aactttgact 2160

gatccgaatc cggacttccc atcaggaagc acaccttctg atctagccta cgctaatggt 2220

tacaaaggaa tcgctggtta tctctcggaa tacgccttga gaacacatgt ttctttgctc 2280

agtctgaatg agaaaaacgc ggaaacatct ctaggaggag cggttgaggc agctcctagc 2340

ccgtccagct cggcgttaac agactctctc acagctgtgc gcaacgctag ccaagcggcg 2400

gctcggattc atcaggtttt cagggctcag tctttccaga agaagcagat gaaagagttt 2460

ggtgatagga agctggggat gtcggaagag cgtgctcttt caatgcttgc tccaaaaaca 2520

cacaaacaag gacgagggca tagtgatgat tccgtgcaag ctgctgcgat caggattcag 2580

aacaagttcc gtggttacaa gggaaggaaa gattatctga ttactcgtca aagaatcatc 2640

aagatacagg ctcatgtaag aggttatcag gttaggaaaa actacaggaa gataatttgg 2700

tctgttggga tactagagaa ggtgatactg cgttggagaa ggaaaggagc tggtttgcgt 2760

gggtttaagt cagacgcact tgttaccaaa atgcaagatg gaacagagaa agaagaagat 2820

gatgatttct tcaagcaagg tagaaagcaa acagaggaaa ggcttgaaaa ggctcttgca 2880

agagttaagt caatggttca gtatcctgaa gctagagatc aataccgcag attactaaat 2940

gtggtcaacg acatccaaga aagcaaggtt gaaaaggctc ttgcaaattc agaagaagca 3000

acttgttttg atgatgatct gatagatatt gaggcattgt tgggagatga tgacactttg 3060

atgatgccta tgtcctcaac tttgtggaac gcttga 3096

<210> 2

<211> 1031

<212> PRT

<213> rape (Brassica napus)

<400> 2

Met Ala Glu Ala Arg Arg Phe Gly Leu Asn Asn Glu Leu Asp Val Gly

1 5 10 15

Gln Ile Leu Ser Glu Ala Arg Asn Arg Trp Leu Arg Pro Pro Glu Ile

20 25 30

Cys Glu Ile Leu Gln Asn Tyr Gln Lys Phe Gln Ile Ser Thr Glu Pro

35 40 45

Pro Thr Thr Pro Ala Ser Gly Ser Val Phe Leu Phe Asp Arg Lys Val

50 55 60

Leu Arg Tyr Phe Arg Lys Asp Gly His Asn Trp Arg Lys Lys Arg Asp

65 70 75 80

Gly Lys Thr Val Lys Glu Ala His Glu Arg Leu Lys Ala Gly Ser Val

85 90 95

Asp Val Leu His Cys Tyr Tyr Ala His Gly Gln Asp Asn Glu Asn Phe

100 105 110

Gln Arg Arg Ser Tyr Trp Met Leu Gln Glu Glu Leu Ser His Ile Val

115 120 125

Phe Val His Tyr Leu Glu Val Lys Gly Ser Arg Val Ser Thr Ser Tyr

130 135 140

Asn Arg Met Gln Arg Thr Glu Asp Ser Thr Arg Ser Ser Gln Glu Thr

145 150 155 160

Gly Glu Val Tyr Thr Ser Glu Arg Asn Gly Tyr Ala Ser Gly Ser Ile

165 170 175

Asn Gln Tyr Asp His Ser Asn Asn Gln Ser Gln Ala Thr Asp Ser Ala

180 185 190

Ser Val Asn Gly Val His Thr Pro Glu Leu Glu Asp Ala Gln Ser Ala

195 200 205

Tyr Asn Gln Gln Gly Ser Pro Ile Leu Tyr Ser His Gln Ala Leu Gln

210 215 220

Gln Pro Pro Ala Thr Ser Phe Asp Pro Tyr Tyr Gln Met Ser Leu Thr

225 230 235 240

Pro Arg Asp Ser Tyr Gln Lys Glu Ile His Thr Ile Ser Ser Ser Thr

245 250 255

Met Val Glu Lys Gly Arg Thr Ile Asn Gly Pro Val Val Thr Asn Ser

260 265 270

Ile Lys Asn Lys Lys Ser Ile Asp Ser Gln Thr Trp Glu Glu Ile Leu

275 280 285

Gly Asn Cys Gly Ser Gly Gly Glu Gly Leu Pro Met Gln Pro His Ser

290 295 300

Glu His Glu Gly Leu Asp Gln Met Leu Gln Ser Tyr Ser Phe Thr Met

305 310 315 320

Gln Asp Phe Ala Ser Leu Gln Glu Ser Ile Val Lys Ser Gln Asn Gln

325 330 335

Glu Leu Asn Ser Gly Leu Thr Ser Asp Arg Ser Leu Trp Leu Gln Gly

340 345 350

Gln Ala Val Asp Ile Glu Pro Asn Ala Leu Ser Asn Leu Ala Ser Ser

355 360 365

Glu Lys Ala Pro Tyr Leu Ser Thr Met Lys Gln His Leu Leu Asp Gly

370 375 380

Ala Leu Gly Glu Glu Gly Leu Lys Lys Met Asp Ser Phe Asn Arg Trp

385 390 395 400

Met Ser Lys Glu Leu Gly Glu Leu Gly Asp Val Gly Val Thr Ala Asp

405 410 415

Ala Asn Glu Ser Phe Thr His Ser Ser Ser Thr Ala Tyr Trp Glu Glu

420 425 430

Val Glu Ser Glu Asp Val Ser Asn Gly Gly Tyr Val Met Ser Pro Ser

435 440 445

Leu Ser Lys Glu Gln Leu Phe Ser Ile Ile Asp Phe Ala Pro Asn Trp

450 455 460

Thr Tyr Val Gly Cys Glu Val Lys Val Leu Val Ser Gly Lys Phe Leu

465 470 475 480

Lys Met Ala Glu Ser Gly Glu Trp Cys Cys Met Phe Gly Gln Thr Glu

485 490 495

Val Pro Ala Asp Ile Ile Ala Asn Gly Ile Leu Glu Cys Val Ala Pro

500 505 510

Met His Glu Ala Gly Arg Val Pro Phe Tyr Val Thr Cys Ser Asn Arg

515 520 525

Leu Ala Cys Ser Glu Val Arg Glu Phe Glu Tyr Lys Val Leu Glu Ser

530 535 540

Gln Gly Phe Asp Arg Glu Thr Tyr Asp Ser Ser Thr Gly Cys Asn Ser

545 550 555 560

Ile Glu Ser Leu Glu Ala Arg Phe Val Lys Leu Leu Cys Ser Lys Ser

565 570 575

Asp Cys Thr Asn Ser Ser Leu Pro Gly Gly Asn Asp Ser Asp Leu Ser

580 585 590

Gln Val Ser Glu Lys Ile Ser Leu Leu Leu Phe Glu Asn Asp Asp Gln

595 600 605

Leu Asp Gln Met Leu Met Asn Glu Ile Ser Gln Glu Asn Met Lys Asn

610 615 620

Asn Leu Leu Gln Glu Ala Leu Lys Glu Ser Leu His Ser Trp Leu Leu

625 630 635 640

Gln Lys Ile Ala Glu Gly Gly Lys Gly Pro Asn Val Leu Asp Glu Gly

645 650 655

Gly Gln Gly Val Leu His Phe Ala Ala Ala Leu Gly Tyr Asn Trp Ala

660 665 670

Leu Glu Pro Thr Ile Val Ala Gly Val Ser Val Asp Phe Arg Asp Val

675 680 685

Asn Gly Trp Thr Ala Leu His Trp Ala Ala Phe Phe Gly Arg Glu Leu

690 695 700

Ile Ile Gly Ser Leu Ile Ala Leu Gly Ala Ser Pro Gly Thr Leu Thr

705 710 715 720

Asp Pro Asn Pro Asp Phe Pro Ser Gly Ser Thr Pro Ser Asp Leu Ala

725 730 735

Tyr Ala Asn Gly Tyr Lys Gly Ile Ala Gly Tyr Leu Ser Glu Tyr Ala

740 745 750

Leu Arg Thr His Val Ser Leu Leu Ser Leu Asn Glu Lys Asn Ala Glu

755 760 765

Thr Ser Leu Gly Gly Ala Val Glu Ala Ala Pro Ser Pro Ser Ser Ser

770 775 780

Ala Leu Thr Asp Ser Leu Thr Ala Val Arg Asn Ala Ser Gln Ala Ala

785 790 795 800

Ala Arg Ile His Gln Val Phe Arg Ala Gln Ser Phe Gln Lys Lys Gln

805 810 815

Met Lys Glu Phe Gly Asp Arg Lys Leu Gly Met Ser Glu Glu Arg Ala

820 825 830

Leu Ser Met Leu Ala Pro Lys Thr His Lys Gln Gly Arg Gly His Ser

835 840 845

Asp Asp Ser Val Gln Ala Ala Ala Ile Arg Ile Gln Asn Lys Phe Arg

850 855 860

Gly Tyr Lys Gly Arg Lys Asp Tyr Leu Ile Thr Arg Gln Arg Ile Ile

865 870 875 880

Lys Ile Gln Ala His Val Arg Gly Tyr Gln Val Arg Lys Asn Tyr Arg

885 890 895

Lys Ile Ile Trp Ser Val Gly Ile Leu Glu Lys Val Ile Leu Arg Trp

900 905 910

Arg Arg Lys Gly Ala Gly Leu Arg Gly Phe Lys Ser Asp Ala Leu Val

915 920 925

Thr Lys Met Gln Asp Gly Thr Glu Lys Glu Glu Asp Asp Asp Phe Phe

930 935 940

Lys Gln Gly Arg Lys Gln Thr Glu Glu Arg Leu Glu Lys Ala Leu Ala

945 950 955 960

Arg Val Lys Ser Met Val Gln Tyr Pro Glu Ala Arg Asp Gln Tyr Arg

965 970 975

Arg Leu Leu Asn Val Val Asn Asp Ile Gln Glu Ser Lys Val Glu Lys

980 985 990

Ala Leu Ala Asn Ser Glu Glu Ala Thr Cys Phe Asp Asp Asp Leu Ile

995 1000 1005

Asp Ile Glu Ala Leu Leu Gly Asp Asp Asp Thr Leu Met Met Pro Met

1010 1015 1020

Ser Ser Thr Leu Trp Asn Ala

1025 1030

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence (Unknow)

<400> 3

atggcggaag caagacgatt t 21

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence (Unknow)

<400> 4

agcgttccac aaagttgagg a 21

<210> 5

<211> 36

<212> DNA

<213> Artificial sequence (Unknow)

<400> 5

ggagaggaca cgctcgagat ggcggaagca agacgt 36

<210> 6

<211> 45

<212> DNA

<213> Artificial sequence (Unknow)

<400> 6

tttccttacc aattggggta ccatgtagaa catcaacgct tccag 45

<210> 7

<211> 41

<212> DNA

<213> Artificial sequence (Unknow)

<400> 7

ttcgaaatcg ataagcttat gtagaacatc aacgcttcca g 41

<210> 8

<211> 36

<212> DNA

<213> Artificial sequence (Unknow)

<400> 8

ttaaagcagg actctagaat ggcggaagca agacgt 36

Claims (3)

1. Rape geneBnCAMTA3Characterized in that the function is rape (rape)Brassica napus) Calcium signaling pathway genesBnCAMTA3Regulatory function against disease resistance, the geneBnCAMTA3The nucleotide sequence of (a) is shown in SEQ ID:1, and the sequence of the encoded protein is shown as(ii) SEQ ID:2, the application refers to the overexpression by creatingBnCAMTA3To obtain rape material with increased resistance to sclerotinia or by creatingBnCAMTA3RNAi transgenic oilseed rape to obtain oilseed rape material with reduced resistance to sclerotinia rot.

2. Use according to claim 1, characterized in that the use in obtaining rape material with increased resistance to sclerotinia sclerotiorum is achieved by:

（1）BnCAMTA3construction and acquisition of Gene overexpression constructs

Will be provided withBnCAMTA3Cloning the gene open reading frame into a plant expression vector, so that the gene open reading frame is driven by a strong promoter to express;

(2) Transformation ofBnCAMTA3Acquisition of Agrobacterium with Gene overexpression Structure

Will be constructedBnCAMTA3The gene overexpression structure is transformed into an agrobacterium strain with strong infection capacity to rape by methods such as electric shock and the like;

(3) OverexpressionBnCAMTA3Creation and acquisition of transgenic oilseed rape

By Agrobacterium mediated methodBnCAMTA3Introducing the gene over-expression structure into rape to obtain transgenic rapeBnCAMTA3Rape with gene over-expression structure;

(4) OverexpressionBnCAMTA3Obtaining of transgenic rape homozygous lines

Respectively with antibiotic resistance andBnCAMTA3the gene expression is used as a detection index to detect the character separation condition of the transgenic plant progeny, and obtain the overexpression which can stably inherit and has the progeny character which is not separated any moreBnCAMTA3The transgenic rape homozygous line of (1);

(5) Overexpression with increased resistance to sclerotinia sclerotiorumBnCAMTA3Screening, identifying and obtaining of transgenic rape homozygous line

To overexpressBnCAMTA3The transgenic rape pure system is used as material for detecting and analyzing the resistance to sclerotinia sclerotiorum to obtain transgenic rape with high disease resistanceBnCAMTA3A genetically modified rape.

3. Use according to claim 1, characterized in that the use in obtaining reduced sclerotinia sclerotiorum resistant rape material is achieved by the following steps:

（1）BnCAMTA3construction and acquisition of Gene RNAi constructs

Will be provided withBnCAMTA3A specific sequence fragment of the gene is respectively cloned into the intermediate expression vector pKANNIBAL in the positive and negative directions, and then the recombination structure is inserted into the RNAi vector pART27 to obtain the RNAi structure pART27-BnCAMTA3；

(2) Transformation ofBnCAMTA3Acquisition of Agrobacterium of Gene RNAi Structure

Will be provided withBnCAMTA3Gene RNAi Structure pART27-BnCAMTA3Transforming agrobacterium strains with strong infection ability to rape by methods such as electric shock and the like;

(3) Rotating deviceBnCAMTA3Creation and acquisition of Gene RNAi Structure rape

Through Agrobacterium mediated transformationBnCAMTA3Introduction of Gene RNAi Structure into oilseed rape to obtain transformantsBnCAMTA3Rape with a gene RNAi structure;

(4) Rotating shaftBnCAMTA3Obtaining of Gene RNAi Structure rape homozygous line

Respectively with antibiotic resistance andBnCAMTA3the gene expression is used as detection index to detect the character separation condition of the transgenic plant progeny and obtain the transgenic plant progeny which can not separate the progeny character and can be stably inheritedBnCAMTA3Rape homozygous lines of gene RNAi structure;

(5) Transformation of antibacterial nuclear diseaseBnCAMTA3Screening, identifying and obtaining of gene RNAi structure rape homozygous line

To rotateBnCAMTA3Gene RNAi Structure pART27-BnCAMTA3The rape pure clone is used as material, the resistance to sclerotinia sclerotiorum is detected and analyzed, and the sclerotinia sclerotiorum resisting strain is obtainedBnCAMTA3Gene RNAi rape.

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