CN114250233A

CN114250233A - Application of arabidopsis calcium ion channel gene AtCNGC3 in sclerotinia sclerotiorum prevention and control

Info

Publication number: CN114250233A
Application number: CN202111637084.6A
Authority: CN
Inventors: 蔡新忠; 刘梦娇; 易航
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2022-03-29
Anticipated expiration: 2041-12-29
Also published as: CN114250233B

Abstract

The invention provides application of an arabidopsis calcium ion channel gene AtCNGC3 in sclerotinia sclerotiorum prevention and control, which is application in obtaining arabidopsis and rape materials with changed disease resistance by creating transgenic arabidopsis and rape (Brassica napus). The invention clarifies the strong regulation and control function and mechanism of the gene on the resistance of the sclerotinia sclerotiorum for the first time. Discloses that the gene has positive regulation effect on sclerotinia sclerotiorum resistance and discloses that the gene regulates Ca²⁺The flow regulates the mechanism of action of disease resistance. Provides the situation that the AtCNGC3 gene respectively obtains the sclerotinia sclerotiorum resistance by creating over-expression Arabidopsis thaliana and rapeApplication of the materials of Arabidopsis thaliana and rape is disclosed. The AtCNGC3 gene provided by the invention is a new gene resource suitable for creating and breeding new materials and new varieties of sclerotinia sclerotiorum resistant rape, and has important significance for green prevention and control of crop sclerotinia sclerotiorum.

Description

Application of arabidopsis calcium ion channel gene AtCNGC3 in sclerotinia sclerotiorum prevention and control

Technical Field

The invention belongs to the technical field of biology, and relates to application of an arabidopsis calcium ion channel gene AtCNGC3 in sclerotinia sclerotiorum prevention and control, and application in sclerotinia sclerotiorum prevention and control through germplasm creation of an antibacterial sclerotinia sclerotiorum plant.

Background

1. Plant gene expression and 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 (photopype) or functional performance under normal expression. The supernormal expression of a gene includes two major classes of expression above normal levels and expression below normal levels. 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 determined by constructing gene over-expression 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 transduction and activation series defense reaction through the recognition of pathogen ligand by plant receptor. Genetically, genes involved in resistance regulation after receptor-ligand recognition include early disease resistance signaling genes and late defense-related genes. Generally, the effect of regulating the disease resistance of plants by using early disease resistance signal transduction genes is better. Calcium is a second messenger. The calcium signaling pathway is a recognized key early signaling pathway. The key Calcium signal driving plant disease resistance is from rapid and extensive Calcium influx from outside the cell membrane into the cytoplasm (Calcium influx). The mechanism for stimulating calcium influx is currently known to come from plasma membrane-localized calcium ion channels, in particular Cyclic nucleotide-gated ion channels (CNGC). CNGC is an early disease-resistant regulatory gene, and the advantage of good resistance regulatory effect is achieved by creating crop germplasm with enhanced disease resistance by using the CNGC gene.

3. Sclerotinia preventing and controlling technology

Plant sclerotinioses are caused by infection with Sclerotinia sclerotiorum (sclerotirotirus). Sclerotinia sclerotiorum is a necrotizing nutritional (necrotph) pathogenic fungus, has a wide host range, and is a main disease of oil crops, vegetable crops and the like. Causing huge economic losses every year. Chemical control remains an important tool due to the lack of high resistance varieties. 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

The plant breeding technology for disease resistance 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 application of an Arabidopsis thaliana (Arabidopsis thaliana) calcium ion channel gene AtCNGC3 in the prevention and control of sclerotinia sclerotiorum, wherein the nucleotide sequence of the gene AtCNGC3 is shown as SEQ ID: 1, and the coded protein sequences are respectively shown as SEQ ID: 2, respectively. The Arabidopsis thaliana (Arabidopsis thaliana) calcium channel gene AtCNGC3 has a regulation function on disease resistance, and can be applied to the creation of germplasm of antibacterial nuclear disease (pathogen: Sclerotinia sclerotiorum).

The application is the application of obtaining the Arabidopsis thaliana and rape materials with changed disease resistance by creating transgenic Arabidopsis thaliana (Arabidopsis thaliana) and rape (Brassica napus), obtaining the Arabidopsis thaliana materials with increased sclerotinia rot resistance by creating transgenic Arabidopsis thaliana with over-expression of AtCNGC3, or obtaining the rape materials with increased sclerotinia rot resistance by creating transgenic rape with over-expression of AtCNGC 3.

The invention uses arabidopsis Col-0cDNA as a template, and obtains an arabidopsis gene AtCNGC3 through PCR cloning, wherein the nucleotide sequence of the arabidopsis gene AtCNGC3 is shown in SEQ ID: 1, the Open Reading Frame (ORF) of the gene has the length of 2121bp, the coded protein consists of 706 amino acids, and the sequence of the coded protein is shown as SEQ ID: 2, respectively. The AtCNGC3 protein comprises a transmembrane domain, a cyclic nucleotide binding domain and a CaM binding domain, and is a calcium ion channel protein. The nucleotide sequence cloned in the invention is identical to the TAIR sequence AT2G 46430.

Prior to the present invention, the disease resistance function of the gene was not reported in any way. The invention firstly clarifies the regulation and control function and mechanism of the gene on the sclerotinia sclerotiorum resistance by constructing the overexpression of the gene, RNAi transgenic arabidopsis thaliana and the disease resistance analysis thereof. The results show that the mutant plants are more susceptible to sclerotinia (fig. 1) and the overexpressing transgenic plants are more resistant to sclerotinia (fig. 2, fig. 3) compared to the wild type arabidopsis plants, indicating that AtCNGC gc3 is regulating the resistance of arabidopsis to sclerotinia. Furthermore, Aequorin-based Ca²⁺Flow detection results show that three immune elicitors AtPep1, SsNLP1 and SsNLP2 express Ca generated by excitation of plants (Atcngc3-Aequorin) of the Aequorin gene on the background of Atcngc3 mutant²⁺The flow was significantly lower than that of the plant (Col-0-Aequorin) which simultaneously expressed the Aequorin gene against the background of the wild type (FIG. 4). Indicating that AtCNGC3 has Ca²⁺Channel activity and upregulation of immune elicitor-induced Ca²⁺And (4) streaming. Discloses that AtCNGC3 regulates Ca² ⁺Flow-regulated disease resistanceThe mechanism is used.

Based on the function and mechanism of the AtCNGC3 gene explained in the invention, the application of the invention aims to provide the application of the Arabidopsis AtCNGC3 gene in obtaining Arabidopsis thaliana with changed disease resistance and crop materials by creating transgenic Arabidopsis thaliana, including (1) the application in obtaining Arabidopsis thaliana materials with enhanced resistance to Sclerotinia sclerotiorum by creating transgenic Arabidopsis thaliana with over-expression of AtCNGC3 (see the description in example 1 specifically); and (2) application in obtaining rape material for enhancing sclerotinia disease resistance by creating transgenic rape over-expressing AtCNGC3 (see the description in example 2).

1. The application of the arabidopsis AtCNGC3 gene in obtaining an arabidopsis material for enhancing the resistance to sclerotinia sclerotiorum by creating transgenic arabidopsis with over-expression AtCNGC 3. The method is realized by the following steps:

(1) construction and acquisition of overexpression structure of AtCNGC3 gene

The Open Reading Frame (ORF) of the AtCNGC3 gene was cloned into a plant expression vector and driven to express by a strong promoter.

(2) Acquisition of Agrobacterium transformed with AtCNGC3 gene overexpression Structure

The constructed overexpression structure of the AtCNGC3 gene is transformed into an agrobacterium strain with strong infection capacity to arabidopsis thaliana by methods such as electric shock and the like.

(3) Transgenic arabidopsis thaliana for over-expressing AtCNGC3

The over-expression structure of the AtCNGC3 gene is introduced into Arabidopsis by an Agrobacterium dipping method, and the Arabidopsis with the over-expression structure of the AtCNGC3 gene is obtained.

(4) Acquisition of transgenic Arabidopsis homozygous line overexpressing AtCNGC3

Antibiotic resistance and AtCNGC3 gene expression are respectively used as detection indexes to detect the character separation condition of transgenic plant progeny, and a transgenic arabidopsis thaliana homozygous line of overexpression AtCNGC3, which has progeny characters which are not separated any more and can be stably inherited, is obtained.

(5) Screening, identifying and obtaining transgenic arabidopsis homozygous line for overexpression AtCNGC3 with enhanced sclerotinia sclerotiorum resistance

The transgenic arabidopsis thaliana homozygous system for over-expressing AtCNGC3 is taken as a material, the resistance to sclerotinia sclerotiorum is detected and analyzed, and the transgenic AtCNGC3 gene arabidopsis thaliana with enhanced disease resistance is obtained.

2. The application of the Arabidopsis thaliana AtCNGC3 gene in obtaining rape material with increased resistance to sclerotinia sclerotiorum disease by creating transgenic rape with over-expression AtCNGC 3. The method is realized by the following steps:

(1) construction and acquisition of overexpression structure of AtCNGC3 gene

(3) Transgenic rape for over-expressing AtCNGC3 creation and acquisition

The overexpression structure of the AtCNGC3 gene is introduced into the rape by an agrobacterium-mediated method, and the rape with the overexpression structure of the AtCNGC3 gene is obtained.

(4) Acquisition of transgenic rape homozygous line of over-expression AtCNGC3

And respectively using antibiotic resistance and AtCNGC3 gene expression as detection indexes to detect the character separation condition of transgenic plant progeny so as to obtain a transgenic rape homozygous line of overexpression AtCNGC3, the progeny character of which is not separated any more and can be stably inherited.

(5) Screening, identifying and obtaining transgenic rape homozygous lines of over-expression AtCNGC3 with enhanced sclerotinia sclerotiorum resistance

The transgenic rape homozygous system of over-expression AtCNGC3 is used as a material, the resistance to sclerotinia sclerotiorum is detected and analyzed, and the transgenic AtCNGC3 rape with enhanced sclerotinia sclerotiorum resistance is obtained.

The invention has the advantages that: (1) the AtCNGC3 gene provided by the invention is a high-quality antibacterial nuclear disease regulation gene resource, and a disease-resistant material obtained by using the gene has the advantages of strong disease resistance and the like. Resistance to Sclerotinia sclerotiorum caused by Sclerotinia sclerotiorum (sclerotirotinia sclerotiorum) is quantitative trait resistance, and is controlled by polygenes. In general, a single gene regulates this resistance to a lesser extent. Therefore, the arabidopsis sclerotinia sclerotiorum disease resistant materials are all deficient all over the world, and high-resistant materials are not available yet. The AtCNGC3 gene is a calcium ion channel gene and is an early disease-resistant regulatory gene, and the action of the AtCNGC3 gene is positioned at the upstream of signal conduction, so that the AtCNGC3 gene has good resistance regulatory effect and is a high-quality disease-resistant regulatory gene resource. The CNGC is used for creating disease-resistant varieties and germplasm, which is an economic, effective and safe way for green disease prevention and control. Therefore, the AtCNGC3 gene is a brand new gene resource suitable for creating and breeding new antibacterial nuclear disease crop materials and new varieties. (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 disease-resistant regulatory genes. 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 AtCNGC3 and adopts a genetic engineering method to create and culture materials of rape and the like with high resistance to sclerotinia rot, and has the characteristics of short period, rapid breeding and the like.

Drawings

Fig. 1 provides evidence of the regulatory function of atcnggc 3 gene for sclerotinia sclerotiorum resistance obtained based on gene mutant studies, and the results show that AtCNGC3 loss-of-function mutation weakens the resistance of arabidopsis thaliana to sclerotinia sclerotiorum, indicating that atcnggc 3 positively regulates the resistance of arabidopsis thaliana to sclerotinia sclerotiorum. The Arabidopsis Atcngc3 mutant ((SALK _066634C) was subjected to sclerotinia UF-1 inoculation analysis, the phenotype (A) 21h after inoculation, the statistical analysis result (B) of lesion area and the qRT-PCR detection result (C) of the relative content of sclerotinia biomass in diseased leaves are shown in the figure, the experiment is repeated three times, each time the experiment is repeated three times, the statistical analysis of Student's t-test is respectively carried out on the lesion area and the bacterial quantity data, and the data are expressed as the mean value +/-standard deviation, the difference is very significant (P)<0.005). The results show that the lesion area of the leaf blade of the Atcngc3 mutant plant is larger than that of the Col-0 wild type (A), and the lesion area of the mutant is 124.3mm²While the wild type is only 97.8mm²(B) In that respect And the bacterial quantity detection result shows that the sclerotinia sclerotiorum biomass in the mutant leaf is obviously more than that of the wild type and is 2.1 times (C) of that of the wild type. These results indicate that the Atcngc3 mutant plants are more susceptible and atcnggc 3 positively regulates the resistance of arabidopsis to sclerotinia sclerotiorum.

FIG. 2 provides evidence for obtaining transgenic Arabidopsis plants over-expressing AtCNGC3, and shows the results of detecting the expression of AtCNGC3 gene in transgenic Arabidopsis plants. Semi-quantitative PCR (A) and real-time fluorescent quantitative qRT-PCR (B) analysis are carried out on AtCNGC3 gene expression in AtCNGC3 gene overexpression (AtCNGC3-OE) plants, and AtACTIN8 is selected as an internal reference gene. The relative expression level of the wild type was set to 1. The experiment was repeated three times. Student's t-test statistical analysis was performed on the expression data, which are expressed as mean ± standard deviation. Indicates that the difference was extremely significant (P < 0.005). The results show that the expression level of AtCNGC3 in the two over-expression line plants is obviously higher than that of the wild type Col-0(A), and is 7.6 times and 6.1 times of that of the wild type (B) respectively. Indicating that these two strains are authentic AtCNGC3 overexpression strains.

FIG. 3 provides evidence of the regulation and control function of the resistance of AtCNGC3 gene to Sclerotinia sclerotiorum obtained based on the gene overexpression transgenic plant research obtained in the present patent, and the results show that the overexpression of AtCNGC3 gene enhances the resistance of Arabidopsis thaliana to Sclerotinia sclerotiorum, indicating that AtCNGC3 positively regulates the resistance of Arabidopsis thaliana to Sclerotinia sclerotiorum. The Arabidopsis thaliana AtCNGC3 gene overexpression (AtCNGC3-OE) plants are subjected to sclerotinia UF-1 inoculation analysis. The figure shows the phenotype (A) at 21h after inoculation, the statistical analysis of lesion area (B) and the qRT-PCR detection of the relative content of sclerotinia biomass in the diseased leaves (C). The experiment was repeated three times. Statistical analysis of Student's t-test was performed on the lesion area and the bacterial count data, respectively, and the data are expressed as mean ± standard deviation. The level of differential significance is indicated by different numbers (, P)<0.05；***，P<0.005). The results show that the leaf lesion area of the AtCNGC3-OE plant is smaller than that of the Col-0 wild type (A), and the leaf lesion area of the AtCNGC3-OE two strains is only 75.8mm respectively²And 78.9mm²While the wild type reaches 101.7mm²(B) In that respect And the results of the bacterial count detection show that the AtCNGC3-OE leavesThe sclerotinia biomass in the tablets was significantly less than the wild type, only about half of the wild type (C). These results indicate that the AtCNGC3-OE plants are more disease resistant, and AtCNGC3 positively regulates the resistance of Arabidopsis to Sclerotinia sclerotiorum.

FIG. 4 provides evidence of the mechanism of the antibacterial nuclear disease regulation of AtCNGC3 gene, showing that AtCNGC3 has Ca²⁺Channel activity, positive regulation of immune elicitor-induced Ca²⁺Flow, thereby modulating disease resistance. The invention obtains a plant (Atcngc3-Aequorin) which takes the Atcngc3 mutant as the background and expresses the Aequorin gene by hybridization with the Atcngc3 mutant as a male parent and the Col-0-Aequorin as a female parent. These plants were verified to be homozygous (A) using three-primer PCR amplification. Selecting Atcngc3-Aequorin homozygous plant, using wild type as background and plant (Col-0-Aequorin) expressing Aequorin gene as control, punching small round pieces with diameter of 3mm from the same position of the same leaf position, and detecting Ca excited by different immune elicitors²⁺And (4) streaming. Ca²⁺The final concentration of CTZ in the flow detection system was 10. mu.M, the final concentration of AtPep1 was 200nM, and the final concentration of SsNLP1/2 was 1. mu.M. The results show that three immune elicitors stimulate Ca production in Atcngc3-aequorin plants²⁺The flow is obviously lower than that of Col-0-aequorin plants. Indicating that AtCNGC3 has Ca²⁺Channel activity and upregulation of immune elicitor-induced Ca²⁺And (4) streaming.

Detailed Description

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

Example 1

The invention clones an arabidopsis gene AtCNGC3, clarifies the antibacterial nuclear disease regulation function of the gene by constructing overexpression transgenic arabidopsis for the first time, and reveals that the gene has an important positive regulation function on the resistance of sclerotinia sclerotiorum. Because the overexpression of the AtCNGC3 gene leads to the remarkable enhancement of the resistance of arabidopsis thaliana to sclerotinia sclerotiorum, a new arabidopsis thaliana material with enhanced resistance to sclerotinia sclerotiorum caused by sclerotinia sclerotiorum can be created by constructing the overexpression arabidopsis thaliana of the AtCNGC3 gene and used for gene function, action mechanism analysis and other purposes. The cloning, function and mechanism analysis of the AtCNGC3 gene, and the creation and acquisition of a novel Arabidopsis material with enhanced resistance to sclerotinia rot comprise the following main steps:

1) arabidopsis AtCNGC3 gene clone and construction and acquisition of overexpression structure thereof

The overexpression structure of the arabidopsis AtCNGC3 gene is constructed and obtained through the following steps. Primers CNGC3-1305-F (5'-ggacagcccagatcaactagtATGATGAATCCCCAAAGAAACAA-3') (sequence shown in SEQ ID: 3) and CNGC3-1305-R (5'-gcccttgctcaccatggatccGGTTTCATCCATAGGAAACTCAGG-3') (sequence shown in SEQ ID: 4) were first designed based on the sequence AtCNGC3 (AT2G46430) in the Arabidopsis genome database. Extracting total RNA of arabidopsis thaliana Col-0 leaves by using a TRIZOL reagent, amplifying by using an RT-PCR method to obtain AtCNGC3 cDNA, performing gel cutting and purification after electrophoresis of 1% agarose gel to recover a PCR product, inserting the PCR product into a pC1305 vector by using a one-step cloning method, wherein the vector drives the expression of a target gene by using a CaMV 35S promoter, and simultaneously carries a GFP label, thereby facilitating molecular identification and gene function research. And transforming the connecting product into escherichia coli DH5 alpha by heat shock, culturing overnight in an LB culture medium by shaking, extracting plasmids, checking whether the extracted plasmids contain AtCNGC3 genes by a PCR method, and finally sending the plasmids to a company for sequencing verification, thereby successfully cloning and obtaining the AtCNGC3 overexpression vector pC1305-AtCNGC 3. The nucleotide sequence of AtCNGC3 cloned by the invention is shown as SEQ ID: 1, the Open Reading Frame (ORF) of the gene has the length of 2121bp, the coded protein consists of 706 amino acids, and the sequence of the coded protein is shown as SEQ ID: 2, respectively. The gene coding product comprises a transmembrane domain, a cyclic nucleotide binding domain and a CaM binding domain, and is a calcium ion channel protein. The nucleotide sequence cloned in the invention is identical to the TAIR sequence AT2G 46430. Prior to the present invention, the disease resistance function of the gene was not reported in any way.

2) Acquisition of Agrobacterium transformed with AtCNGC3 gene overexpression Structure pC1305-AtCNGC3

The gene overexpression structure pC1305-AtCNGC3 of AtCNGC3 is transformed into an agrobacterium strain with strong infectivity to Arabidopsis thaliana, such as GV3101, by methods of electric shock and the like, transformants are screened on YEP culture medium containing kanamycin and rifampicin, and then the agrobacterium carrying the gene overexpression structure pC1305-AtCNGC3 of AtCNGC3 is obtained by PCR and sequencing identification. Used for the next step of genetic transformation of Arabidopsis thaliana.

3) Creation and acquisition of transgenic AtCNGC3 gene overexpression structure pC1305-AtCNGC3 Arabidopsis thaliana

The over-expression structure pC1305-AtCNGC3 of the AtCNGC3 gene is introduced into arabidopsis thaliana by an agrobacterium flower dipping method to obtain arabidopsis thaliana T transformed with pC1305-AtCNGC3₀And (4) generation. The specific operation steps are as follows:

(i) preparation of Arabidopsis thaliana

Cutting off flower buds at the top of the arabidopsis thaliana after one week of bolting, removing the top advantages to promote the lateral branch meristem, cutting off the pod and the blossom of the arabidopsis thaliana by the agrobacterium tumefaciens two days before the arabidopsis thaliana is transformed, only leaving the buds, watering the arabidopsis thaliana with enough water, and ensuring the good growth state of the arabidopsis thaliana.

(ii) Preparation of Agrobacterium infection solution

pC1305-AtCNGC3 Agrobacterium was streaked on YEB solid medium containing 50. mu.g/ml kanamycin and rifampicin antibiotic and incubated overnight in an incubator at 28 ℃. The single clone was picked up into liquid YEB medium containing the same concentration of antibiotic, shaking-cultured at 230rpm at 28 ℃ until turbid, and then transferred to 5ml YEB medium to continue shaking-culture. The Agrobacterium was shake-cultured to OD₆₀₀Transferring to 50ml centrifuge tube, centrifuging at room temperature 5000rpm for 8min, discarding supernatant, and adding sucrose-MgCl containing surfactant₂The buffer was resuspended and gently mixed.

(iii) Transformation of

The Arabidopsis buds are completely immersed in the Agrobacterium infection solution for about 1min, and the culture pot is horizontally placed in a plastic tray. And adding a small amount of water into the tray to play a moisturizing role, carrying the arabidopsis thaliana stems and the buds by using a wooden stick to prevent the arabidopsis thaliana stems and the buds from being wetted, covering all the agrobacterium-infected plants by using a black film, and carrying out photophobic co-culture for 24 hours to finish infection transformation.

(iv) Screening and identification

And normally culturing the plants subjected to the dipping transformation till seeds are harvested. The seeds were sown on 1/2MS solid medium containing 50. mu.g/ml hygromycin, and positive shoots were seen to root normally and begin to grow new leaves at about 10 days. Transferring the positive seedlings into a separate culture pot, placing the culture pot in a culture chamber for conventional management,finally harvesting to obtain T₀And (4) seeds.

4) Screening, identifying and obtaining of Arabidopsis homozygous line with overexpression structure pC1305-AtCNGC3 of AtCNGC3 gene

And (3) detecting the character separation condition of the transgenic plant progeny by respectively using hygromycin resistance and AtCNGC3 gene expression as detection indexes. Hygromycin resistance screening is carried out on hygromycin-containing plates aiming at the Arabidopsis seeds, and whether healthy seedlings can normally root and grow on the hygromycin-resistant plates is observed. The gene expression is detected by methods such as real-time fluorescence quantitative PCR and the like. An arabidopsis homozygous line which is transformed with the AtCNGC3 gene overexpression structure pC1305-AtCNGC3 and has the characteristics that the progeny traits are not separated any more and can be stably inherited is obtained.

The invention obtains two pC1305-AtCNGC3 arabidopsis pure synthetic lines which are named as CNGC3-OE-1 and CNGC3-OE-2, all the arabidopsis pure synthetic lines can normally grow into healthy seedlings on a hygromycin resistant plate, and the expression water average of AtCNGC3 gene is obviously higher than that of a transferred empty vector and is respectively 7.6 times and 6.1 times of that of the control (figure 2). The plants are shown to be true AtCNGC3 gene over-expression plants.

5) Disease resistance detection analysis of AtCNGC3 gene overexpression Arabidopsis homozygous line

The AtCNGC3 gene overexpression Arabidopsis thaliana homozygous system obtained in the step 4) is taken as a material, and the resistance of the AtCNGC3 gene overexpression Arabidopsis thaliana homozygous system to Sclerotinia sclerotiorum is detected and analyzed by inoculating Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), so that the regulation and control effect of the AtCNGC3 gene on the resistance of Sclerotinia sclerotiorum is determined, and a foundation is laid for establishing and obtaining the Sclerotinia sclerotiorum resistant crops by using the gene.

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 a sterile 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, using a puncher with the diameter of 4mm to punch mycelium blocks with the edges of bacterial colonies facing inwards by 3-5mm, inoculating the hypha-bearing surface facing downwards on a new PDA solid plate, and culturing at 23 ℃ in the dark place for about 36 hours to perform inoculation.

Inoculation of sclerotinia sclerotiorum: arabidopsis plants growing for 4-5 weeks with consistent growth were selected for inoculation. The leaves were sprayed with 0.1% Tween 20. And (3) punching a hypha block with the edge of a colony being inward 3-5mm by using a puncher with the diameter of 3mm, inoculating the hypha block to the middle position of a completely developed leaf with the hypha facing downwards, covering a film on the hypha block, preserving moisture, culturing the hypha block in a greenhouse at 23 ℃, photographing and recording after a proper time (about 24h), and analyzing the lesion area by using ImageJ software.

The inoculation experiment was repeated three times. Statistical analysis of the Student's t-test was performed on lesion areas. The results showed that the AtCNGC3 overexpressing (AtCNGC3-OE) plants were significantly more disease resistant than the non-transgenic plant controls (FIG. 3A). The result of quantitative analysis of the lesion area shows that the lesion area of the control plant reaches 101.7mm²The lesion areas of the two over-expression plants are respectively only 75.8mm²And 78.9mm²Significantly smaller than control plants (fig. 3B). Moreover, the bacteria amount detection result shows that the sclerotinia biomass in the AtCNGC3-OE leaves is obviously less than that of the wild type, and only about half of the wild type (C) exists.

These results indicate that the resistance of the AtCNGC3 overexpressing plants to sclerotinia is significantly higher than the non-transgenic plant controls. Overexpression of the AtCNGC3 gene results in significant enhancement of resistance of Arabidopsis to sclerotinia sclerotiorum, and therefore AtCNGC3 plays a positive role in regulating resistance of Arabidopsis to sclerotinia sclerotiorum. New crop materials and new varieties with high resistance to sclerotinia rot can be created by constructing an AtCNGC3 overexpression plant.

6) Ca of AtCNGC3²⁺Channel activity detection assay

In order to reveal the disease resistance regulation and control action mechanism of AtCNGC3, the invention constructs a fluorescence detection technology based on the Aequorin protein to detect whether AtCNGC3 has Ca or not²⁺Channel activity and whether there is a response to an immune exciton. Firstly, the invention adopts a hybridization method to construct a key material, namely a plant (Atcngc3-Aequorin) which takes an Atcngc3 mutant as a background and expresses an Aequorin gene at the same time. The method comprises the following specific steps:

(i) selecting a female parent: plants from which just a little white petals could be seen were selected as female parents.

(ii) Castration of female parent: the calyx (green part) of the female parent was carefully removed with forceps, the petals were spread open, and the stamen (yellowish) was removed. Firstly, carefully poking the buds for a few times along the direction of the buds by using the head of a pair of tweezers, poking the buds loose, and removing green calyxes; then, the white petals were spread with forceps, and the yellowish stamens were clipped off. Care was taken to disinfect the forceps and not touch the injured stigma.

(iii) Selecting a male parent: the plant of flower with completely spread flower, cross shape and yellow stamen is selected as male parent.

(iv) Artificial pollination: the handle of the stamen is clamped by a pair of tweezers to be taken down, the stamen is lightly wiped on the stigma of the female parent flower for a plurality of times, and the bag is sleeved and marked.

(v) Detection of Atcngc3-aequorin plants: the T-DNA insertion sequence of the Atcngc3 mutant was detected by three-primer PCR amplification to determine whether the plants were successfully crossed. The positive seedlings were subcultured until homozygous Atcngc3-aequorin plants were obtained.

The invention successfully obtains the Atcngc3-aequorin homozygous line through three-primer PCR amplification verification (figure 4A). Selecting Atcngc3-Aequorin homozygous plant, using wild type as background and plant (Col-0-Aequorin) expressing Aequorin gene as control, punching small round pieces with diameter of 3mm from the same position of the same leaf position, and detecting Ca excited by different immune elicitors²⁺And (4) streaming. Ca²⁺The final concentration of CTZ in the flow detection system was 10. mu.M, the final concentration of AtPep1 was 200nM, and the final concentration of SsNLP1/2 was 1. mu.M. The results show that three immune elicitors of AtPep1, SsNLP1 and SsNLP2 stimulate Ca produced by Atcngc3-aequorin plants²⁺The flow is obviously lower than that of Col-0-aequorin plants. Indicating that AtCNGC3 has Ca²⁺Channel activity and upregulation of immune elicitor-induced Ca²⁺And (4) streaming. Discloses that AtCNGC3 regulates Ca²⁺The flow regulates the mechanism of action of disease resistance.

Example 2

According to the positive regulation and control function of the arabidopsis gene AtCNGC3 on the resistance to sclerotinia rot, which is explained by the invention, a technical system for constructing the overexpression transgenic rape of the gene and creating and obtaining a new sclerotinia rot resistant rape material by adopting a genetic engineering technology is established. The method mainly comprises the following steps:

The method of 1) in example 1 is adopted to construct and obtain an arabidopsis AtCNGC3 gene overexpression structure pC1305-AtCNGC 3.

Agrobacterium transformed with the overexpression structure pC1305-AtCNGC3 of AtCNGC3 gene was obtained by the method of 2) in example 1. Used for the next step of rape genetic transformation.

3) Creation and acquisition of transgenic AtCNGC3 gene overexpression structure pC1305-AtCNGC3 rape

Introducing an AtCNGC3 gene overexpression structure pC1305-AtCNGC3 into rape by an agrobacterium-mediated method to obtain rape T transformed with pC1305-AtCNGC3₀And (4) generation. The specific operation steps are as follows:

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, 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;

pre-culturing: 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 2-3 d;

agrobacterium infection and co-cultivation: selecting Agrobacterium in infection solution to prepare OD₆₀₀The explants were inoculated in the agrobacterium suspension for 10min for infection, 0.2 agrobacterium resuspension. Inoculating the infected explants on sterile filter paper, airing, inoculating on a co-culture medium, and performing dark culture at 23 ℃ for 48-72 h;

bacteria removal (screening): inoculating the explants after co-culture on a degerming culture medium, and performing illumination culture at 23 ℃ for 6 d;

screening/differentiation: inoculating the explants subjected to the bacteria removal treatment on a screening/differentiation culture medium, wherein each dish contains 30 explants, and the explants are subjected to illumination culture at 23 ℃ and plate changing once after 15 days;

rooting culture: the differentiated bud seedlings are planted in a rooting culture medium and are subjected to illumination culture at the temperature of 23 ℃ until the bud seedlings root;

and (3) detection: extracting rape genome DNA by a CTAB method, and carrying out PCR detection on the antibiotic gene.

Transplanting and soil culture: the pot is filled with moderate humus soil. Removing the rape seedlings from the culture medium, washing the root culture medium with water, cleaning, transplanting into a pot, and culturing under 16h/8h photoperiod.

4) Screening, identifying and obtaining of rape homozygous line of transgenic AtCNGC3 gene overexpression structure pC1305-AtCNGC3

And (3) detecting the character separation condition of the transgenic plant progeny by respectively using hygromycin resistance and AtCNGC3 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 fluorescence quantitative PCR and the like. And acquiring the rape homozygous line of the transgenic AtCNGC3 gene overexpression structure pC1305-AtCNGC3, which has offspring traits not separated any more and can be stably inherited.

5) Disease resistance detection analysis of AtCNGC3 gene over-expression rape homozygous line

And (3) inoculating Sclerotinia sclerotiorum (sclerotiorum) to detect and analyze the resistance of the AtCNGC3 gene overexpression rape homozygosity system obtained in the step (4) to the Sclerotinia sclerotiorum to obtain the Sclerotinia sclerotiorum resistant rape.

Inoculation of sclerotinia sclerotiorum: arabidopsis plants growing for 4-5 weeks with consistent growth were selected for inoculation. The leaves were sprayed with 0.1% Tween 20. And (3) punching a hypha block with the edge of a colony being inward 3-5mm by using a puncher with the diameter of 4mm, inoculating the hypha block to the middle position of a completely developed leaf with the hypha facing downwards, covering a film on the hypha block, preserving moisture, culturing the hypha block in a greenhouse at 23 ℃, photographing and recording after a proper time (about 24 hours), and analyzing the lesion area by using ImageJ software.

Through the inoculation analysis, the resistance regulation and control effect of the AtCNGC3 on sclerotinia sclerotiorum is determined, and a novel rape material with high sclerotinia sclerotiorum resistance is obtained.

In summary, the present invention, in combination with the results of FIGS. 1 to 4, reveals for the first time the Arabidopsis thaliana calcium channel gene AtCNGC3Has positive regulation effect on sclerotinia sclerotiorum resistance and reveals that the gene regulates Ca²⁺The flow regulates the mechanism of action of disease resistance. More importantly, the invention provides an application approach and an application technology of AtCNGC3 in creating germplasm of a crop with sclerotinia sclerotiorum disease resistance by combining with an embodiment, and the application approach and the application technology have important significance for green prevention and control of sclerotinia sclerotiorum disease of the crop.

Sequence listing

<110> Zhejiang university

<120> application of Arabidopsis thaliana calcium ion channel gene AtCNGC3 in sclerotinia sclerotiorum prevention and control

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tttttctata tcccctatgt gaagcccgag cgattttgtc ttaatctaga caagaaactc 360

cagacaatcg cttgcgtttt ccgcactttc atcgacgctt tctatgtggt tcacatgctg 420

tttcagtttc ataccggttt catcactcct tcttctagtg gtttcggcag aggagagctt 480

aatgagaaac ataaagatat cgctttaaga tacctcggct catactttct aatcgatctt 540

ctttcgattc tccctattcc tcaggtagtt gttctagcta ttgttccaag gatgagacga 600

cccgcgtcgc tagtagcaaa agagctactg aaatgggtaa tcttttgcca atatgttccg 660

aggatagcga gaatctatcc gcttttcaag gaagtaacaa gaacttctgg tttggtaact 720

gaaactgctt gggctggtgc tgctttaaac ctattcctct acatgttagc tagccatgtt 780

tttgggtctt tttggtactt gatttcgata gaaaggaaag atagatgttg gcgtgaggcg 840

tgtgctaaga tacagaattg cactcatgca tacttatact gttcaccaac aggggaagac 900

aataggttat ttttaaacgg ttcttgtccg ttgatcgatc ccgaagaaat cacaaactcg 960

acggttttca actttggtat attcgctgat gcattgcagt ctggagttgt ggagtctaga 1020

gatttcccta agaagttttt ctactgtttc tggtggggtc tccgcaatct tagtgctttg 1080

ggccaaaact tgaagacgag tgcctttgaa ggagagatca tttttgcaat agtcatttgt 1140

atttctggac tagtcttgtt tgctctcctc attggaaata tgcagaaata tttgcaatca 1200

actactgtaa gagttgagga aatgagagtg aaaaggagag atgcagagca atggatgtct 1260

catcggatgt tgccagacga tctgagaaaa cgtatccgta aatacgaaca gtataaatgg 1320

caagaaacca aaggagttga ggaagaagct cttctctcta gtcttccaaa agatctcaga 1380

aaggacatta aacgccatct ttgtctcaag ttgctcaaaa aggtgccttg gttccaagct 1440

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aatttaatca gcaccactac ttatggtggc agaactggtt tcttcaattc tgttgactta 1620

gttgctggtg atttctgtgg agatcttctc acttgggcat tagatcctct cagttcccaa 1680

ttccctatct ctagtagaac cgttcaagct ttgacagaag tcgaaggctt tcttctctca 1740

gctgatgatc tcaagtttgt cgctactcag tatcgtcgcc tccatagcaa gcaactccga 1800

cacatgttca ggttttattc agtgcaatgg cagacatggg cagcatgttt tatacaagca 1860

gcatggaaga gacattgtag aaggaagctc tcaaaggctt tacgtgaaga agaaggcaaa 1920

ctacataaca ctcttcagaa cgatgattca ggaggcaata aacttaactt aggtgctgcg 1980

atttatgcgt cgaggtttgc ttctcatgct ctgaggaatc taagagcaaa tgcagcagcc 2040

cgaaattcta gattcccgca catgttaact ttgttgcctc agaaaccagc cgatcctgag 2100

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Phe Lys Arg Pro Leu Ser Val His Ser Asn Lys Asn Lys Glu Asn Asn

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Lys Lys Lys Lys Ile Leu Arg Val Met Asn Pro Asn Asp Ser Tyr Leu

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Gln Ser Trp Asn Lys Ile Phe Leu Leu Leu Ser Val Val Ala Leu Ala

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Phe Asp Pro Leu Phe Phe Tyr Ile Pro Tyr Val Lys Pro Glu Arg Phe

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Cys Leu Asn Leu Asp Lys Lys Leu Gln Thr Ile Ala Cys Val Phe Arg

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Thr Phe Ile Asp Ala Phe Tyr Val Val His Met Leu Phe Gln Phe His

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Thr Gly Phe Ile Thr Pro Ser Ser Ser Gly Phe Gly Arg Gly Glu Leu

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Asn Glu Lys His Lys Asp Ile Ala Leu Arg Tyr Leu Gly Ser Tyr Phe

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Leu Ile Asp Leu Leu Ser Ile Leu Pro Ile Pro Gln Val Val Val Leu

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Ala Ile Val Pro Arg Met Arg Arg Pro Ala Ser Leu Val Ala Lys Glu

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Leu Leu Lys Trp Val Ile Phe Cys Gln Tyr Val Pro Arg Ile Ala Arg

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Ile Tyr Pro Leu Phe Lys Glu Val Thr Arg Thr Ser Gly Leu Val Thr

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Glu Thr Ala Trp Ala Gly Ala Ala Leu Asn Leu Phe Leu Tyr Met Leu

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Ala Ser His Val Phe Gly Ser Phe Trp Tyr Leu Ile Ser Ile Glu Arg

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Lys Asp Arg Cys Trp Arg Glu Ala Cys Ala Lys Ile Gln Asn Cys Thr

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His Ala Tyr Leu Tyr Cys Ser Pro Thr Gly Glu Asp Asn Arg Leu Phe

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

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Thr Val Phe Asn Phe Gly Ile Phe Ala Asp Ala Leu Gln Ser Gly Val

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Val Glu Ser Arg Asp Phe Pro Lys Lys Phe Phe Tyr Cys Phe Trp Trp

340 345 350

Gly Leu Arg Asn Leu Ser Ala Leu Gly Gln Asn Leu Lys Thr Ser Ala

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Phe Glu Gly Glu Ile Ile Phe Ala Ile Val Ile Cys Ile Ser Gly Leu

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Thr Thr Val Arg Val Glu Glu Met Arg Val Lys Arg Arg Asp Ala Glu

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Gln Trp Met Ser His Arg Met Leu Pro Asp Asp Leu Arg Lys Arg Ile

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Arg Lys Tyr Glu Gln Tyr Lys Trp Gln Glu Thr Lys Gly Val Glu Glu

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Glu Ala Leu Leu Ser Ser Leu Pro Lys Asp Leu Arg Lys Asp Ile Lys

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Arg His Leu Cys Leu Lys Leu Leu Lys Lys Val Pro Trp Phe Gln Ala

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Leu Tyr Thr Glu Lys Ser Tyr Ile Val Arg Glu Gly Glu Pro Val Glu

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Asp Met Leu Phe Ile Met Arg Gly Asn Leu Ile Ser Thr Thr Thr Tyr

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<212> DNA

<213> Artificial sequence (Unknow)

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Claims

1. The application of an arabidopsis thaliana calcium ion channel gene AtCNGC3 in sclerotinia sclerotiorum prevention and control is characterized in that the nucleotide sequence of the gene AtCNGC3 is shown as SEQ ID: 1, and the coded protein sequences are respectively shown as SEQ ID: 2, respectively.

2. Use according to claim 1, for obtaining Arabidopsis and oilseed rape material with altered disease resistance by creating transgenic Arabidopsis thaliana (Arabidopsis thaliana) and oilseed rape (Brassica napus).

3. Use according to claim 2, characterized in that in arabidopsis material for increasing the resistance to sclerotinia sclerotiorum is obtained by creating transgenic arabidopsis thaliana overexpressing AtCNGC 3.

4. Use according to claim 2, characterized in that it is used for obtaining rape material with increased resistance to sclerotinia sclerotiorum by creating transgenic rape overexpressing AtCNGC gc 3.

5. Use according to claim 3, characterized in that the use in obtaining an Arabidopsis thaliana material with increased resistance to sclerotinia sclerotiorum disease is achieved by:

(1) construction and acquisition of overexpression structure of AtCNGC3 gene

Cloning an Open Reading Frame (ORF) of the AtCNGC3 gene into a plant expression vector, and enabling the plant expression vector to be driven by a strong promoter to express;

Transforming the constructed overexpression structure of the AtCNGC3 gene into an agrobacterium strain with strong infection capacity to arabidopsis thaliana by methods such as electric shock and the like;

(3) transgenic arabidopsis thaliana for over-expressing AtCNGC3

Introducing the overexpression structure of the AtCNGC3 gene into arabidopsis thaliana by an agrobacterium flower dipping method to obtain the arabidopsis thaliana with the overexpression structure of the AtCNGC3 gene;

Respectively taking antibiotic resistance and AtCNGC3 gene expression as detection indexes, detecting the character separation condition of transgenic plant progeny, and obtaining a transgenic arabidopsis thaliana homozygous line of overexpression AtCNGC3, wherein the progeny characters are not separated any more and can be stably inherited;

6. Use according to claim 4, characterized in that the use in obtaining rape material with increased resistance to sclerotinia sclerotiorum is achieved by:

(1) construction and acquisition of overexpression structure of AtCNGC3 gene

(3) transgenic rape for over-expressing AtCNGC3 creation and acquisition

Introducing the overexpression structure of the AtCNGC3 gene into the rape by an agrobacterium-mediated method to obtain the rape with the overexpression structure of the AtCNGC3 gene;

(4) acquisition of transgenic rape homozygous line of over-expression AtCNGC3

Respectively taking antibiotic resistance and AtCNGC3 gene expression as detection indexes, detecting the character separation condition of transgenic plant progeny, and obtaining a transgenic rape homozygous line of overexpression AtCNGC3, the progeny character of which is not separated any more and can be stably inherited;