CN114250233B

CN114250233B - Application of arabidopsis calcium ion channel gene AtCNGC3 in sclerotinia sclerotiorum prevention and control

Info

Publication number: CN114250233B
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: 2023-02-10
Anticipated expiration: 2041-12-29
Also published as: CN114250233A

Abstract

The invention provides an application of an arabidopsis calcium ion channel gene AtCNGC3 in sclerotinia sclerotiorum prevention and control, which is an application in obtaining arabidopsis and rape materials with changed disease resistance by creating transgenic arabidopsis and rape (Brassica napus). The invention firstly clarifies the strong regulation and control function and mechanism of the gene on the sclerotinia sclerotiorum resistance. Discloses that the gene has positive regulation function on sclerotinia sclerotiorum resistance and discloses that the gene regulates Ca ²⁺ The flow regulates the mechanism of action of disease resistance. Provides the application of the AtCNGC3 gene in respectively obtaining the arabidopsis thaliana and rape materials with reduced sclerotinia sclerotiorum resistance by creating over-expression arabidopsis thaliana and rape. 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 rapes, 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. The expression higher than the normal level is mainly Over-expression/Over-expression (Over-expression), and is mainly achieved by connecting a strong promoter to drive the expression of a target gene. 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 critical early signaling pathway. The key Calcium signal driving plant disease resistance arises 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 sclerotiorum prevention and control technology

Plant Sclerotinia sclerotiorum is caused by infection with Sclerotinia sclerotiorum (sclerotiotinia sclerotiorum). 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. 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 disease-resistant breeding technology comprises traditional disease-resistant breeding, disease-resistant breeding 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 encoded 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 nucleopathy (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 disease resistance by creating transgenic Arabidopsis thaliana with over-expression of AtCNGC3, or obtaining the rape materials with increased sclerotinia disease resistance by creating transgenic rape with over-expression of AtCNGC3.

The invention takes 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 resistance of sclerotinia sclerotiorum 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 c3 positively regulates the resistance of arabidopsis to sclerotinia. Furthermore, aequorin-based Ca ²⁺ The flow detection result shows that the three immune stimulators AtPep1, ssNLP1 and SsNLP2 are Ca generated by the excitation of a plant (Atcngc 3-Aequorin) which takes the Atcngc3 mutant as the background and expresses the Aequorin gene at the same time ²⁺ 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 Ca ²⁺ Channel activity and upregulation of immune elicitor-induced Ca ²⁺ And (4) streaming. Revealing that AtCNGC3 regulates Ca ² ⁺ The flow regulates the mechanism of action of disease resistance.

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 sclerotiorum resistance by creating transgenic rape over-expressing AtCNGC3 (see the description in example 2 specifically).

1. The application of the arabidopsis AtCNGC3 gene in preparing an arabidopsis material for enhancing the resistance to sclerotinia sclerotiorum by creating transgenic arabidopsis for over-expressing AtCNGC3. 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 is cloned into a plant expression vector and driven to express by a strong promoter.

(2) Acquisition of agrobacterium for transforming overexpression structure of AtCNGC3 gene

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 thaliana by an agrobacterium flower dipping method, and the Arabidopsis thaliana with the over-expression structure of the AtCNGC3 gene is obtained.

(4) Acquisition of transgenic arabidopsis homozygous line overexpressing AtCNGC3

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

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

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

2. The application of the arabidopsis AtCNGC3 gene in obtaining rape materials with increased resistance to sclerotinia rot by creating transgenic rape over-expressing AtCNGC3. 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.

(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

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

(5) Screening, identifying and obtaining transgenic rape homozygous lines of overexpression AtCNGC3 with enhanced resistance to sclerotinia sclerotiorum

The transgenic rape homozygous system for over-expressing AtCNGC3 is taken as a material, the resistance to sclerotinia sclerotiorum is detected and analyzed, and the AtCNGC3 transgenic 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 thaliana sclerotinia sclerotiorum resistant materials are scarce all over the world, and high-resistant materials are not available. The AtCNGC3 gene is a calcium ion channel gene, is an early disease-resistant regulatory gene, and has the function of being positioned at the upstream of signal conduction, so that the AtCNGC3 gene has a good resistance regulatory effect and is a high-quality disease-resistant regulatory gene resource. The development of disease-resistant varieties and germplasm by using the CNGC is an economic, effective and safe way for green prevention and control of diseases. Therefore, the AtCNGC3 gene is a brand new gene resource suitable for creating and breeding new materials and new varieties of anti-sclerotic disease crops. 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 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 the like, 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 AtCNGC3 gene on sclerotinia sclerotiorum resistance obtained based on gene mutant research, and the results show that Atcngc3 loss-of-function mutation weakens the resistance of Arabidopsis thaliana to sclerotinia sclerotiorum, indicating that AtCNGC3 positively regulates the resistance of Arabidopsis thaliana to sclerotinia sclerotiorum. The Arabidopsis Atcngc3 mutant ((SALK _ 066634C) is subjected to sclerotinia UF-1 inoculation analysis, the phenotype (A) of 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, student's t-test statistical analysis is respectively carried out on the lesion area and the bacterial quantity data, and the data are expressed as the average value +/-standard deviation, the difference is extremely obvious (P)<0.005). The results show that the leaf lesion area of the Atcngc3 mutant plant is larger than that of the Col-0 wild type (A), and the lesion area of the mutant is 124.3mm ² While the wild type is only 97.8mm ² (B) .1. The 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 overexpressing AtCNGC3, and shows the results of detecting the expression of AtCNGC3 gene in transgenic Arabidopsis plants. And (3) carrying out semi-quantitative PCR (A) and real-time fluorescent quantitative qRT-PCR (B) analysis on the AtCNGC3 gene expression in the AtCNGC3 gene overexpression (AtCNGC 3-OE) plant, and selecting AtACTIN8 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 respectively 7.6 times and 6.1 times of that of the wild type (B). Indicating that the two lines are true AtCNGC3 overexpression lines.

FIG. 3 provides evidence of regulation and control functions of AtCNGC3 gene on sclerotinia sclerotiorum resistance obtained based on research on gene overexpression transgenic plants obtained in the patent, and results show that AtCNGC3 gene overexpression enhances the resistance of Arabidopsis thaliana on sclerotinia sclerotiorum, and that AtCNGC3 positively regulates and controls the resistance of Arabidopsis thaliana on sclerotinia sclerotiorum. The Arabidopsis thaliana AtCNGC3 gene overexpression (AtCNGC 3-OE) plants are subjected to sclerotinia UF-1 inoculation analysis. The figure shows the phenotype 21h after inoculation (A), the statistical analysis result of lesion area (B) and the qRT-PCR detection result of the relative content of the sclerotinia sclerotiorum biomass in the diseased leaf (C). The experiment was repeated three times. Student's t-test statistical analysis is carried out on the lesion area and the bacterial quantity data respectively, and the data are expressed as the mean value +/-standard deviation. The level of differential significance is indicated by different numbers (, P)<0.05；***，P<0.005). The results show that the leaf spot area of the AtCNGC3-OE plant is smaller than that of the Col-0 wild type (A), and the leaf spot areas of the AtCNGC3-OE plants are only 75.8mm respectively ² And 78.9mm ² While the wild type reaches 101.7mm ² (B) In that respect Moreover, the bacteria quantity 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 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 regulation of sclerotiniose resistance of AtCNGC3 gene, showing that AtCNGC3 has Ca ²⁺ Channel activity, positive regulation of immune elicitor-induced Ca ²⁺ Flow, thereby modulating disease resistance. The invention obtains a plant (Atcngc 3-Aequorin) which expresses the Aequorin gene by taking the Atcngc3 mutant as a background through hybridization by taking the Atcngc3 mutant as a male parent and taking Col-0-Aequorin as a female parent. The plants are verified to be homozygote by adopting three-primer PCR amplification (A) In that respect Selecting Atcngc3-Aequorin homozygous plant, using wild type as background and plant (Col-0-Aequorin) expressing Aequorin gene as contrast, punching small round pieces with diameter of 3mm from the same position of the same leaf position, and detecting Ca excited by different immune elicitors ²⁺ And (4) streaming. Ca ²⁺ The final concentration of CTZ in the flow detection system is 10 muM, the final concentration of AtPep1 is 200nM, and the final concentration of SsNLP1/2 is 1 muM. The results show that three immune elicitors stimulate Ca production in Atcngc3-aequorin plants ²⁺ The flow is obviously lower than that of Col-0-aequorin plants. Indicating that AtCNGC3 has Ca ²⁺ Channel activity and upregulation of immune elicitor-induced Ca ²⁺ 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 and obtained by constructing the overexpression arabidopsis thaliana of the AtCNGC3 gene, and the novel arabidopsis thaliana material is used for analyzing gene functions, action mechanisms and the like. 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 sclerotiorum comprise the following main steps:

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

The arabidopsis AtCNGC3 gene overexpression structure provided by the invention is constructed and obtained through the following steps. Firstly, primers CNGC3-1305-F (5) -gcacagccagcctagatcaactattaTGATGATCCCCAAAAGAAACAAACAAA) -3 '(the sequence is shown as SEQ ID: 3) and CNGC3-1305-R (5) -gcccttgctcatcggttTCATCCATAGGAAAACTCAGG) -3' (the sequence is shown as SEQ ID: 4) are designed according to the AtCNGC3 sequence (AT 2G 46430) 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 tapping 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 (3) thermally shocking the ligation product to transform escherichia coli DH5 alpha, shake culturing overnight in an LB culture medium, 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-AtCNGC3. The nucleotide sequence of the AtCNGC3 cloned and obtained 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 over-expression structure pC1305-AtCNGC3 of the AtCNGC3 gene is transformed into an agrobacterium strain with strong infectivity to Arabidopsis thaliana, such as GV3101, transformants are screened on a YEP culture medium containing kanamycin and rifampicin, and the agrobacterium carrying the over-expression structure pC1305-AtCNGC3 of the AtCNGC3 gene is obtained through PCR and sequencing identification. For the next step of genetic transformation of Arabidopsis thaliana.

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

Introducing an AtCNGC3 gene overexpression structure pC1305-AtCNGC3 into arabidopsis thaliana by an agrobacterium flower dipping method to obtain an arabidopsis thaliana T with the transferred pC1305-AtCNGC3 ₀ And (4) generation. The specific operation steps are as follows:

(i) Preparation of Arabidopsis thaliana

Cutting off the apical bud of the arabidopsis thaliana after bolting for one week, removing apical dominance to promote the collateral meristem of the arabidopsis thaliana, cutting off the pod and the blossom of the arabidopsis thaliana in the first two days of the agrobacterium transformation of the arabidopsis thaliana, only leaving the bud, 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 antibiotics 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 OD ₆₀₀ =1.0-1.2, transferring to a 50ml centrifuge tube, centrifuging at room temperature and 5000rpm for 8min, discarding supernatant, and adding sucrose-MgCl containing surfactant ₂ The buffer was resuspended and gently mixed.

(iii) Transformation of

The arabidopsis buds are completely immersed in the agrobacterium-infected liquid for about 1min, and then 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 at about 10 days and new leaves began to grow. Transferring the positive seedlings into a separate culture pot, placing the culture pot into a culture chamber for conventional management, and finally harvesting to obtain T ₀ And (4) seeds.

4) Screening, identifying and obtaining of arabidopsis pure 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 the Arabidopsis seeds, and whether normal rooting and healthy seedling growth can be carried out 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 obtaining an arabidopsis pure line of the transgenic AtCNGC3 gene overexpression structure pC1305-AtCNGC3 which has the characteristics of offspring which are not separated any more and can be stably inherited.

The invention obtains two pC1305-AtCNGC3 arabidopsis pure synthetic lines, named CNGC3-OE-1 and CNGC3-OE-2, which can all grow into healthy seedlings normally on a hygromycin resistant plate, and the expression water average of the 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 lines

The AtCNGC3 gene overexpression Arabidopsis thaliana homozygous line obtained in the step 4) is taken as a material, and the resistance of the AtCNGC3 gene overexpression Arabidopsis thaliana homozygous line 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 Sclerotinia sclerotiorum resistance is determined, and a foundation is laid for establishing and obtaining Sclerotinia sclerotiorum resistant crops by using the AtCNGC3 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 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: arabidopsis plants growing for 4-5 weeks with consistent growth were selected for inoculation. Spray the leaf with 0.1-Then 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 24 h), and analyzing the lesion area by using ImageJ software.

The inoculation experiment was repeated three times. Statistical analysis of Student's t-test was performed on lesion area. The results show that the AtCNGC3 overexpressing (AtCNGC 3-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 101.7mm ² The lesion areas of the two over-expression plants are respectively only 75.8mm ² And 78.9mm ² Significantly smaller than control plants (fig. 3B). And the bacteria amount detection result shows that the sclerotinia sclerotiorum biomass in the AtCNGC3-OE leaf blade is obviously less than that of the wild type, and only about half of the wild type (C).

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 leads to remarkable enhancement of resistance of Arabidopsis to sclerotinia sclerotiorum, so that the AtCNGC3 plays a positive control role in 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 AtCNGC3 ²⁺ Channel activity detection assay

In order to reveal the disease resistance regulation and control action mechanism of the AtCNGC3, the invention constructs a fluorescence detection technology based on the Aequorin protein to detect whether the AtCNGC3 has Ca or not ²⁺ 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 (Atcngc 3-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 opened, 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 with flower which is completely spread and has a cross shape and yellow stamen is selected as the 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: and detecting the T-DNA insertion sequence of the Atcngc3 mutant by three-primer PCR amplification to determine whether the plant is successfully hybridized. And subculturing the positive seedlings until obtaining homozygous Atcngc3-aequorin plants.

The invention successfully obtains the Atcngc3-aequorin homozygous line through the 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 elicitors ²⁺ And (4) streaming. Ca ²⁺ The final concentration of CTZ in the flow detection system is 10 muM, the final concentration of AtPep1 is 200nM, and the final concentration of SsNLP1/2 is 1 muM. The results show that three immune elicitors of AtPep1, ssNLP1 and SsNLP2 stimulate Ca generated by Atcngc3-aequorin plants ²⁺ The flow rate is obviously lower than that of Col-0-aequorin plants. Indicating that AtCNGC3 has Ca ²⁺ Channel activity and up-regulation of immunostimulant-induced Ca ²⁺ And (4) streaming. Revealing that AtCNGC3 regulates Ca ²⁺ 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 of the sclerotinia sclerotiorum, which is elucidated by the invention, a technical system for constructing the overexpression transgenic rape of the gene and creating and obtaining a new sclerotinia sclerotiorum resistance rape material by adopting a genetic engineering technology is established. The method mainly comprises the following steps:

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

The agrobacterium transformed with the over-expression structure pC1305-AtCNGC3 of the AtCNGC3 gene is obtained by the method 2) in the embodiment 1. And (4) carrying out the next step of genetic transformation of rape.

3) Creation and acquisition of AtCNGC3 gene-transferred over-expression structure pC1305-AtCNGC3 rape

Introducing the overexpression structure pC1305-AtCNGC3 of the AtCNGC3 gene into the rape by an agrobacterium-mediated method to obtain the rape T transformed with pC1305-AtCNGC3 ₀ And (4) generation. The concrete operation stepsThe method comprises the following steps:

seed cleaning and germination: 75% ethanol for 30-60s, and 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 2-3d;

agrobacterium infection and co-cultivation: 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;

bacteria removal (screening): inoculating the explants subjected to co-culture on a sterile culture medium, and performing illumination culture at 23 ℃ for 6 days;

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: and 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 a 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 obtaining the rape homozygous line of the transgenic AtCNGC3 gene overexpression structure pC1305-AtCNGC3 which has the progeny character which is not separated any more and can be stably inherited.

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

And (3) inoculating Sclerotinia sclerotiorum (sclerotiorum) to detect and analyze the resistance of the AtCNGC3 gene overexpression rape homozygous 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. Spray the leaf with 0.1-Then Tween 20. And (3) punching a hypha block with the edge of a colony being 3-5mm inwards 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 leaf for moisturizing, culturing the leaf in a greenhouse at 23 ℃, taking a picture for recording after a proper time (about 24 hours), and analyzing the lesion area by ImageJ software.

Through the inoculation analysis, the resistance regulation and control effect of 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 that the Arabidopsis thaliana calcium channel gene AtCNGC3 plays a positive role in the regulation of sclerotinia sclerotiorum resistance and that the gene regulates Ca by ²⁺ The flow regulates the mechanism of action of disease resistance. More importantly, the invention provides an application way and an application technology of AtCNGC3 in the creation of sclerotinia sclerotiorum resistant crop germplasm, and has important significance for green prevention and control of the sclerotinia sclerotiorum of crops.

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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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

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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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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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<212> DNA

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Claims

1. Arabidopsis thaliana calcium ion channel geneAtCNGC3Use in the control of sclerotinia sclerotiorum, characterized in that, the geneAtCNGC3The nucleotide sequence of (a) is as shown in SEQ ID:1, and the sequence of the encoded protein is shown as SEQ ID:2, by creating overexpressionAtCNGC3The transgenic Arabidopsis thaliana of (1) is used for obtaining the Arabidopsis thaliana material with increased sclerotinia sclerotiorum resistance.

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

（1）AtCNGC3construction and acquisition of Gene overexpression constructs

Will be provided withAtCNGC3Cloning 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 ofAtCNGC3Acquisition of Agrobacterium with Gene overexpression Structure

Will be constructedAtCNGC3The gene overexpression structure is used for transforming agrobacterium strains with strong infection capacity to arabidopsis thaliana by an electric shock method;

(3) OverexpressionAtCNGC3Transgenic Arabidopsis creation and acquisition

Through the flower dipping method of agrobacteriumAtCNGC3Introduction of the Gene overexpression Structure into Arabidopsis thaliana, obtaining the transformantsAtCNGC3Of gene over-expression structuresArabidopsis thaliana;

(4) OverexpressionAtCNGC3Obtaining of transgenic Arabidopsis homozygous lines

Respectively with antibiotic resistance andAtCNGC3the 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 moreAtCNGC3The transgenic Arabidopsis homozygous line of (1);

(5) Overexpression of Sclerotinia sclerotiorum resistanceAtCNGC3Screening, identifying and obtaining of transgenic arabidopsis homozygous lines

To overexpressAtCNGC3The transgenic arabidopsis pure line is a material, the resistance to sclerotinia sclerotiorum is detected and analyzed, and the transgenic line with enhanced disease resistance is obtainedAtCNGC3The gene arabidopsis thaliana.