CN114316007A - Disease-resistant functional application of rape secretory peptide gene BnRALF10 - Google Patents

Abstract

The invention provides an application of a rape secretory peptide gene BnRALF10 in disease resistance, which is an application of a rape (Brassica napus) secretory peptide gene BnRALF10 in sclerotinia sclerotiorum disease prevention and control, and an application of obtaining a rape material with changed disease resistance by creating transgenic rape. The invention constructs the overexpression and RNAi transgenic rape of the gene, discloses the strong positive regulation and control function of the gene to the sclerotinia sclerotiorum resistance of rape for the first time, and provides the application of obtaining the rape material with obviously increased sclerotinia sclerotiorum resistance by creating BnRALF10 gene overexpression rape and the application of obtaining the rape material with obviously reduced sclerotinia sclerotiorum resistance by creating BnRALF10-RNAi rape. The BnRALF10 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 sclerotinia sclerotiorum.

Description

Disease-resistant functional application of rape secretory peptide gene BnRALF10

Technical Field

The invention belongs to the field of plant disease resistance biotechnology, relates to the application of a disease resistance function of a rape secretory peptide gene BnRALF10, and particularly relates to the application of a disease resistance regulation function of a rape secretory peptide gene BnRALF10 in sclerotinia sclerotiorum disease prevention and control.

Background

1. Plant disease-resistant genetics regulation and control technology

Plant disease resistance is a genetic trait of host plants to resist pathogen infection and damage, and is controlled by genes. Activation of disease-resistant signaling by plant receptors through recognition of pathogen ligands is the first barrier for plants to defend against pathogens. The exciton ligand, which is located outside the cell membrane, is the key to open the plant defence response. Therefore, these promoter genes play a crucial role in conferring disease resistance to plants. In order to accurately sense the danger signals from pathogens, plants have evolved large amounts of secreted peptides that act as immune stimulators such as lesion-associated molecular patterns (DAMPs). Rapid alkalizing factors (RALFs) are a class of DAMPs, and play an important role in stimulating and regulating plant immunity of various pathogens. Therefore, the generation of crop germplasm with enhanced disease resistance by using the RALF gene has the advantages of good resistance regulation effect, broad-spectrum regulation effect on resistance of different pathogens and the like.

2. Sclerotinia preventing and controlling technology

Plant sclerotinioses are caused by infection with Sclerotinia sclerotiorum (sclerotirotirus). Sclerotinia sclerotiorum is a dead body 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 a major approach due to the lack of deep understanding of the immune resistance regulatory mechanism of the bacterium and the lack of stable, highly resistant varieties. However, chemical control is easy to pollute the environment, soil property is reduced, pathogenic bacteria also have potential drug resistance, improper medication management can pose potential threat to future crop yield, and the optimal application period is difficult to predict. The identification of important sclerotinia rot resistance regulating gene, the creation and utilization of disease-resistant variety, is important for the green prevention and control of sclerotinia rot.

3. Plant disease-resistant breeding technology

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

Disclosure of Invention

The invention aims to provide the application of the disease resistance function of rape secretory peptide gene BnRALF10, wherein the function refers to the excitation regulation function of rape (Brassica napus) secretory peptide gene BnRALF10 on disease resistance, and the application can be applied to the creation of germplasm of antibacterial nuclear disease (pathogen: Sclerotinia sclerotiorum).

The application is the application of obtaining the rape material with changed disease resistance by creating transgenic rape (Brassica napus). The application of the rape material for enhancing the sclerotinia sclerotiorum resistance is obtained by creating the transgenic rape of which the overexpression of BnRALF10 is realized. Or the BnRALF10-RNAi transgenic rape is created to obtain the rape material with reduced sclerotinia sclerotiorum resistance.

The invention uses double 11 varieties cDNA in rape as a template, obtains rape gene BnRALF10 through PCR cloning, and the nucleotide sequence is shown as SEQ ID: 1, the Open Reading Frame (ORF) of the gene has the length of 360bp, the coded protein consists of 119 amino acids, and the sequence of the coded protein is shown as SEQ ID: 2, respectively. The BnRALF10 protein is obtained by cutting and processing a precursor protein and contains functional motifs such as RRIL and YISY. The nucleotide and protein sequences of the cloned BnRALF10 are respectively identical to the LOC106379883 nucleotide and XP _013675189.1 protein sequences of the rape seed ZS11 in the NCBI database.

Prior to the present invention, the function of the gene was not reported in any public. The invention firstly clarifies the regulation and control function and mechanism of the gene on the sclerotinia rot resistance of rape by constructing the overexpression of the gene, RNAi transgenic rape and the disease resistance analysis thereof. Results show that compared with non-transgenic wild type control rape plants, the overexpression transgenic rape plants are obviously more resistant to sclerotinia sclerotiorum, and RNAi transgenic plants are obviously more sensitive to sclerotinia sclerotiorum, which indicates that BnRALF10 strongly and positively regulates the resistance of rape to sclerotinia sclerotiorum.

Based on the function of the BnRALF10 gene explained in the invention, the application of the invention aims to provide the application of the rape BnRALF10 gene in obtaining a rape material with changed disease resistance by creating transgenic rape, and comprises (1) the application in obtaining a rape material with enhanced resistance to sclerotinia sclerotiorum by creating transgenic rape with over-expression BnRALF10 (see the description in example 1 specifically); and (2) application of the transgenic rape with BnRALF10-RNAi creation to obtain rape material with reduced resistance to sclerotinia sclerotiorum (see the description in example 2).

1. The application of the rape BnRALF10 gene in preparing transgenic rape with over-expressed BnRALF10 to obtain rape material with enhanced resistance to sclerotinia rot. The method is realized by the following steps:

(1) construction and acquisition of BnRALF10 gene overexpression structure

Cloning the BnRALF10 gene Open Reading Frame (ORF) into a plant expression vector, so that the expression is driven by a strong promoter;

(2) acquisition of agrobacterium for transforming BnRALF10 gene overexpression structure

Transforming the constructed BnRALF10 overexpression structure into an agrobacterium strain with strong infection capacity on rape by an electric shock transformation method;

(3) creation and acquisition of transgenic rape with overexpression of BnRALF10

Introducing the BnRALF10 gene overexpression structure into the rape by an agrobacterium-mediated method to obtain the rape with a BnRALF10 gene overexpression structure;

(4) acquisition of transgenic rape homozygous line of overexpression BnRALF10

Respectively taking antibiotic resistance and BnRALF10 gene expression as detection indexes, detecting the character separation condition of transgenic plant progeny, and obtaining a transgenic rape homozygous line of overexpression BnRALF10, wherein the progeny character is not separated any more and can be stably inherited;

(5) screening, identifying and obtaining transgenic rape homozygous lines of overexpression BnRALF10 with enhanced sclerotinia disease resistance

The transgenic rape homozygous line of overexpression BnRALF10 is used as a material, the resistance to sclerotinia sclerotiorum is detected and analyzed, and the transgenic rape BnRALF10 with enhanced disease resistance is obtained.

2. The application of rape BnRALF10 gene in preparing BnRALF10-RNAi transgenic rape to obtain rape material with reduced sclerotinia sclerotiorum resistance. The method is realized by the following steps:

(1) construction and acquisition of RNAi structure of BnRALF10 gene

Respectively and sequentially cloning the forward interference fragment and the reverse interference fragment of the BnRALF10 gene into an RNAi vector (pART-RNAi) vector by a homologous recombination method to obtain an RNAi structure pART-RNAi-BnRALF10 of BnFER 1;

(2) acquisition of Agrobacterium transformed with BnRALF10 Gene RNAi Structure

B, transforming the BnRALF10 gene RNAi structure (pART-RNAi-BnRALF10) into an agrobacterium strain with strong infection capacity to rape by methods such as heat shock;

(3) creation and acquisition of BnRALF10 gene transferred RNAi structure rape

Introducing the RNAi structure of the BnRALF10 gene into the rape by an agrobacterium-mediated method to obtain the rape with the RNAi structure of the BnRALF10 gene;

(4) acquisition of BnRALF10 gene-transferred RNAi structure rape homozygous line

Respectively taking antibiotic resistance and BnRALF10 gene expression as detection indexes, detecting the character separation condition of transgenic plant progeny, and obtaining a rape homozygous line of a BnRALF10 gene RNAi structure, wherein the progeny character of the rape homozygous line is not separated any more and can be stably inherited;

(5) screening, identifying and obtaining of BnRALF10 gene-transformed RNAi structure rape homozygous lines with reduced sclerotinia sclerotiorum resistance

The BnRALF10 gene RNAi structure (pART-RNAi-BnRALF10) transferred rape homozygospore system is used as a material to detect and analyze resistance to sclerotinia sclerotiorum, and BnRALF10 gene RNAi rape with reduced resistance to sclerotinia sclerotiorum is obtained.

The invention has the advantages that: (1) the BnRALF10 gene provided by the invention is a high-quality antibacterial nuclear disease regulation gene resource, and the disease-resistant material obtained by using the gene has the advantages of strong disease resistance and the like. Rape sclerotiniose resistance is quantitative trait resistance, controlled by multiple genes. In general, a single gene regulates this resistance to a lesser extent. Therefore, the sclerotinia sclerotiorum resistant materials of the rapes are all deficient all over the world, and stable high-resistant materials are not available. The BnRALF10 gene is a plant secretory peptide gene, is used as a DAMP immune exciton gene, plays a role of a starter of immune signal transduction, has good resistance regulation and control effects, and is a high-quality disease-resistant regulation and control gene resource. The method for creating disease-resistant varieties and germplasm by using RALF is an economic, effective and safe way for green disease control. Therefore, the BnRALF10 gene is a brand new gene resource suitable for creating and breeding new materials and new varieties of sclerotinia sclerotiorum resistant rapes. (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 immune-stimulant gene BnRALF10, adopts a genetic engineering method to create and culture a rape material with high resistance to sclerotinia rot, and has the characteristics of short period, rapid breeding and the like.

Drawings

FIG. 1 provides evidence of various transgenic rape plants obtained in the present patent, showing the results of the expression detection of BnRALF10 gene in the transgenic rape plants. The expression level of BnRALF10 gene in OE plant (A) over-expressing BnRALF10 gene and RNAi plant (B) silencing BnRALF10 gene is detected by real-time fluorescent quantitative PCR. Rape BnACTN 7 is selected as an internal reference gene, and the relative expression quantity of BnRALF10 gene in wild type ZS11 is set as 1. The experiment was repeated three times and the expression results were analyzed by one-way ANOVA and the data are expressed as mean. + -. SE. The significance of the differences is indicated by different numbers (, P < 0.01;, P < 0.005;, P < 0.001). The results show that the expression level of BnRALF10 in both over-expression (OE) lines 6 and 7 is significantly increased, which is 254.1 times and 85.7 times that of the control (A), while the expression level of BnRALF10 in RNAi lines 4 and 8 is significantly decreased, which is only 8.6% and 16.5% (B) of the control plants. The plants are shown to be true BnRALF10 gene overexpression plants and RNAi plants.

FIG. 2 provides evidence of the sclerotinia sclerotiorum resistance regulatory function of BnRALF10 gene, showing that BnRALF10 strongly positively regulates the resistance of Brassica napus to Sclerotinia sclerotiorum. The inoculation analysis of Sclerotinia sclerotiorum UF-1 is carried out on rape BnRALF10 transgenic plants. The graph shows the results of quantitative statistical analysis of phenotype (A) and lesion area 24h after inoculation (B). The inoculation experiment was repeated three times. One-way ANOVA analysis was performed on the lesion area and the data are expressed as mean. + -. SE. Significance of differences is indicated by different numbers (, P)<0.01；***，P<0.005). The results show that the over-expressed plants show a more disease resistant phenotype than the wild type plants ZS11(WT), whereas the RNAi plants are significantly more susceptible than the control (a). The result of quantitative analysis of lesion area shows that the lesion area of WT plants is 152mm²The average lesion areas of BnRALF10 over-expression plants-6 and-7 are only 26mm respectively²、65mm²Significantly less than control; the lesion areas of RNAi plants-2 and-8 respectively reach 264mm on average²、243mm²Significantly greater than control (B). These results indicate that BnRALF10 strongly positively regulates resistance of oilseed rape to sclerotinia.

Detailed Description

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

Example 1

The invention clones a rape immune exciton gene BnRALF10, clarifies the sclerotinia rot resistance regulation function of the gene by constructing the overexpression transgenic rape for the first time, and reveals that the gene has strong positive regulation function on sclerotinia rot resistance of rape. Because the overexpression of the BnRALF10 gene leads the remarkable enhancement of sclerotinia sclerotiorum resistance of rape, a new sclerotinia sclerotiorum resisting rape material can be created and obtained by constructing the overexpression transgenic rape of the gene and adopting a genetic engineering technology. The method mainly comprises the following steps:

1) cloning of rape BnRALF10 gene and construction of overexpression structure

The rape BnRALF10 gene provided by the invention is obtained by cloning through the following steps. Firstly, according to the sequence of BnRALF10 in the rape genome database, a primer BnRALF10-F (5'-ggacagcccagatcaactagtatgaactctcgcgccatct-3', the italic part is SpeI enzyme cutting site) (the sequence is shown in SEQ ID: 3) and a primer BnRALF10-R (5'-gcccttgctcaccatggatccacgcctgcatctcgtgatggt-3', the italic part is BamHI enzyme cutting site) (the sequence is shown in SEQ ID: 4) are designed. Extracting total RNA of double 11 leaves in a rape variety by using a TRIzol reagent, performing Reverse transcription to synthesize first-strand cDNA by using an RT-PCR (Reverse transcription-polymerase chain reaction) method, performing PCR amplification by using the cDNA as a template to obtain a BnRALF10 CDS fragment, performing gel cutting, purification and recovery of a PCR product after electrophoresis of 1.5% agarose gel, connecting a pCAMBIA1305-GFP vector, thermally shocking and transforming escherichia coli DH5 alpha, performing shake culture overnight in an LB (LB) culture medium, extracting plasmids, respectively checking whether the extracted plasmids contain BnRALF10-GFP fusion genes by using a SpeI and BamHI enzyme digestion method and a PCR method using BnRALF10-F and GFP-R as primer pairs, and finally sending the plasmids to a company for sequencing verification, thereby successfully cloning and obtaining a BnRALF10 CDS gene full-length sequence and an overexpression structure. The nucleotide sequence of BnRALF10 is shown in SEQ ID: 1, the Open Reading Frame (ORF) of the gene has the length of 360bp, the coded protein consists of 119 amino acids, and the sequence of the coded protein is shown as SEQ ID: 2, respectively. The gene coding product contains functional motifs such as RRXL and YISY. The nucleotide and protein sequences of the cloned BnRALF10 are respectively identical to the LOC106379883 nucleotide and XP _013675189.1 protein sequences of the rape seed ZS11 in the NCBI database. Prior to the present invention, the function of this gene was not reported in any public. The overexpression vector pCAMBIA-1305GFP uses a CaMV 35S promoter to drive the expression of a target gene and simultaneously carries a GFP label, thereby being convenient for molecular identification and gene function research.

2) Acquisition of Agrobacterium transformed with BnRALF10 gene overexpression Structure pCAMBIA-1305GFP-BnRALF10

The BnRALF10 gene overexpression structure pCAMBIA-1305GFP-BnRALF10 is transformed into an agrobacterium strain with strong infectivity on rape, such as EHAl05, by a method of heat shock and the like, a transformant is screened on an LB solid culture medium containing kanamycin, and agrobacterium carrying the BnRALF10 gene overexpression structure pCAMBIA-1305GFP-BnRALF10 is obtained by double enzyme digestion, PCR and sequencing identification of SpeI and BamHI. Used for the next step of rape genetic transformation.

3) Creation and acquisition of BnRALF10 gene-transferred overexpression structure pCAMBIA-1305GFP-BnRALF10 rape

Introducing BnRALF10 gene overexpression structure pCAMBIA-1305GFP-BnRALF10 into rape by agrobacterium-mediated method, and finally obtaining rape T transformed with pCAMBIA-1305GFP-BnRALF10₀And (4) generation. The specific operation steps are as follows:

(i) induction of canola callus

Disinfecting and cleaning the surface of the embryo: taking the well-matured ZS11 rape seeds, and soaking the seeds for 15min by using sterile water; sterilizing with 75% ethanol for 60s, sterilizing with sodium hypochlorite solution (effective chlorine of 2%) for 15min, washing with sterile water for 4 times, and inoculating on 1/2MS +1 mg/L6-BA solid culture medium. About 30 seeds per dish. Sealing with preservative film, and culturing in incubator at 28 deg.C.

Preparing an explant: after 7-10 days of culture, the hypocotyl is taken out and placed in a pre-culture medium for 2D (MS +1 mg/L2, 4-D +1 mg/L6-BA pH 5.8), and the callus at this time is most suitable for agrobacterium infection.

(ii) Agrobacterium infection of canola callus

And (3) culturing agrobacterium: the glycerol strain of Agrobacterium tumefaciens EHA105 carrying a BnRALF10 gene overexpression structure pCAMBIA-1305GFP-BnRALF10 is streaked on an LB solid culture medium (50mg/L kanamycin +50mg/L rifampicin), after two days of culture at 28 ℃, a single clone is picked and inoculated into 5ml of LB liquid culture medium (containing 50mg/L kanamycin +50mg/L rifampicin), and the mixture is subjected to constant-temperature shaking culture at 28 ℃ and 220rpm for overnight culture until OD is reached₆₀₀When the concentration was about 0.6, the cells were collected by centrifugation at 5000rpm for 10min, and the collected cells were resuspended to OD with MS liquid medium₆₀₀When the yield is 0.4, the strain can be used for rape transformation.

Agrobacterium infection and co-culture: placing the pre-cultured healthy bright yellow callus in the resuspended Agrobacterium liquid, gently shaking for 15min, blotting the explant surface liquid with sterile filter paper, and culturing in the dark for 3 days on a co-culture medium (MS +0.15mg/L NAA +3 mg/L6-BA +5mg/L AgNO 3).

Bud induction and differentiation: after co-culture, explants were transferred to shoot inducing differentiation medium (MS +0.15mg/L NAA +3 mg/L6-BA +5mg/L AgNO3+25mg/L Hyg +300mg/L Timentin pH 5.8) to induce sprouting with medium replacement every two weeks.

(iii) Rooting and resistant plant screening

When the resistant bud grows to 2cm, cutting the bud and transferring the bud into a rooting culture medium (MS +0.15mg/L NAA +300mg/L Timentin +25mg/L Hyg pH 5.8), when a complete plantlet is formed, cutting partial leaves, and carrying out PCR amplification to detect a positive plant.

4) Screening, identifying and obtaining rape homozygous lines of BnRALF10 gene overexpression structure pCAMBIA-1305GFP-BnRALF10

And (3) detecting the character separation condition of the transgenic plant progeny by respectively using hygromycin resistance and BnRALF10 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 BnRALF10 gene overexpression structure pCAMBIA-1305GFP-BnRALF10, the progeny characters of which are not separated any more and can be stably inherited.

The invention obtains two BnRALF10 overexpression rape homozygous lines, OE-line 6 and OE-line 7, which can all grow into healthy plantlets on a hygromycin resistant plate, and the pCAMBIA-1305GFP-BnRALF10 gene expression level is obviously higher than that of an empty vector, and is 254.1 times and 85.7 times of that of the control (figure 1A). The plants are shown to be true BnRALF10 gene over-expression plants.

5) Disease resistance detection analysis of BnRALF10 gene overexpression rape homozygous line

The BnRALF10 gene overexpression rape homozygote obtained in the step 4) is taken as a material, and Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is inoculated to detect and analyze the resistance of the BnRALF10 gene to rape sclerotiniose, so that the regulation and control function of the BnRALF10 gene to rape sclerotiniose resistance is determined, and whether a new sclerotiniose resistant rape material can be created and obtained by constructing overexpression transgenic rape of the gene and adopting a genetic engineering technology.

Activation culture of sclerotinia sclerotiorum: the method comprises the steps of selecting plump and pollution-free sclerotinia sclerotiorum sclerotia, cutting the sclerotinia sclerotiorum into two halves by using 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 5mm to punch mycelium blocks with the edges of bacterial colonies facing inwards by 3-5mm, inoculating the hypha blocks with one side facing downwards to a new PDA solid plate, and culturing at 23 ℃ in the dark place for about 48 hours to perform inoculation.

Inoculation of sclerotinia sclerotiorum: and selecting the rape plants with consistent growth vigor for inoculation. And (3) taking hypha blocks with the edges of colonies inward facing 3-5mm by using a puncher with the diameter of 5mm, inoculating the hypha blocks to fully developed leaves with the hypha surfaces facing downward, symmetrically inoculating two hypha blocks to the left half and the right half of each leaf respectively, covering a film on each leaf for moisturizing, culturing in a greenhouse at 23 ℃, photographing and recording after a proper time (about 36 hours), and analyzing the lesion area by using ImageJ software.

The inoculation experiment was repeated three times. One-way ANOVA analysis was performed on the lesion area and the data are expressed as mean. + -. SE. The results show that BnRALF10 overexpressing plants were significantly more disease resistant than wild type plants (WT) (fig. 2A). The result of quantitative analysis of lesion area shows that the lesion area of WT plants is 152mm²The average lesion areas of BnRALF10 over-expression plants-6 and-7 are only 26mm respectively²、65mm²Significantly less than the control (fig. 2B).

These results indicate that BnRALF10 overexpressing plants are significantly more resistant to sclerotinia than wild type plants. Overexpression of the BnRALF10 gene leads to remarkable enhancement of sclerotinia sclerotiorum resistance of rape, so that BnRALF10 plays a strong positive regulation role in sclerotinia sclerotiorum resistance of rape. The result also proves that a new antibacterial sclerotinia rot rape material can be created and obtained by constructing the overexpression transgenic rape of BnRALF10 and adopting a genetic engineering technology.

Example 2

The embodiment 1 of the invention clarifies the positive regulation function of rape gene BnRALF10 on sclerotinia sclerotiorum resistance by constructing the over-expression plant of BnRALF10 gene. This example illustrates the RNAi rapeseed construction technology of BnRALF10 gene, and further proves the positive regulation function of BnRALF10 on sclerotinia sclerotiorum resistance by this material. This example provides a technique for creating a new material for obtaining rape with reduced resistance to sclerotinia sclerotiorum by constructing RNAi rape with BnRALF10 gene, and the material can be used for gene function and mechanism of action analysis. The method mainly comprises the following steps:

(1) construction and acquisition of RNAi structure of BnRALF10 gene

The sequence of the cloned BnRALF10 is used for designing forward interference fragment primers BnRALF10-F2 (5'-atttggagaggacacgctcgagatgaactctcgcgccatctac-3', the italic part is Xho I restriction site) (the sequence is shown as SEQ ID: 5), and BnRALF10-R2 (5'-accaagctggggtaccgaattcacgctggcagttgtaatacg-3', the italic part is Eco RI restriction site) (the sequence is shown as SEQ ID: 6). Taking ZS11 cDNA as a template and BnRALF10-F2/BnRALF10-R2 as a primer pair, obtaining a 300bp BnRALF10 forward interference fragment through PCR amplification, and then obtaining an intermediate RNAi vector pART-RNAi-BnRALF10(+) with the 300bp BnRALF10 forward interference fragment subcloned through the steps of Xho I and Eco RI double digestion, connection, transformation, streptomycin culture medium plate screening, enzyme digestion, PCR identification and the like. Then, the sequence of BnRALF10 is used to design a reverse interference fragment primer BnRALF10-F3 (5'-tgggttcgaaatcgataagcttacgctggcagttgtaatacg-3', the italic part is Hind III restriction site) (the sequence is shown in SEQ ID: 7), and BnRALF10-R3 (5'-ctcattaaagcaggactctagaatgaactctcgcgccatctac-3', the italic part is Xba I restriction site) (the sequence is shown in SEQ ID: 8). Taking ZS11 cDNA as a template and BnRALF10-F3/BnRALF10-R3 as a primer pair, obtaining a 300bp BnRALF10 reverse interference fragment through PCR amplification, and then obtaining an RNAi vector pART-RNAi-BnRALF10 with forward and reverse interference fragments of 300bp BnRALF10 subcloned through steps of HindIII/Xba I double enzyme digestion, connection, transformation, streptomycin culture medium plate screening, enzyme digestion, PCR, sequencing identification and the like.

(2) Acquisition of Agrobacterium transformed with BnRALF10 Gene RNAi Structure

The RNAi vector structure pART-RNAi-BnRALF10 of the BnRALF10 gene is transformed into an agrobacterium strain with strong infection capacity to rape, such as EHAl05, a transformant is screened on a YEP culture medium containing 50mg/L streptomycin and 50mg/L spectinomycin, and agrobacterium carrying the RNAi structure pART-RNAi-BnRALF10 of the BnRALF10 gene is obtained through Hind III and Xba I double enzyme digestion, PCR and sequencing identification. Used for the next step of rape genetic transformation.

(3) Creation and acquisition of BnRALF10 gene transferred RNAi structure rape

Introducing the RNAi structure pART-RNAi-BnRALF10 of the BnRALF104 gene into rape by an agrobacterium-mediated method, and finally obtaining the rape T transformed with pART-CAM-RNAi-BnRALF10₀And (4) generation. The specific procedures were as described in 4) of example 1.

(4) Acquisition of BnRALF10 gene-transferred RNAi structure rape homozygous line

And respectively detecting the character separation condition of the transgenic plant progeny by taking the antibiotic resistance and the BnRALF10 gene expression as detection indexes. Kanamycin resistance screening is carried out on a kanamycin-containing plate aiming at rape seeds, and whether healthy seedlings can normally grow on the kanamycin-resistant plate or not is observed. The gene expression is detected by methods such as real-time fluorescence quantitative PCR and the like. Obtaining the rape homozygous line of the BnRALF10 gene RNAi structure pART-RNAi-BnRALF10, the progeny character of which is not separated any more and can be stably inherited. These rape homozygous lines can all grow into healthy seedlings normally on kanamycin-resistant plates, and the expression level of BnRALF10 gene is obviously lower than that of wild plants.

The invention obtains two BnRALF10-RNAi rape homozygous lines, RNAi-line 4 and line 8, which can be normally grown into healthy seedlings on a kanamycin-resistant plate, wherein the expression level of the BnRALF10 gene is obviously lower than that of a wild plant, and only 8.6 percent and 16.5 percent of the wild plant are respectively obtained (figure 1B). The two strains are shown to be true BnRALF10 gene RNAi strains.

(5) Screening, identifying and obtaining BnRALF10 gene-transformed RNAi structure rape homozygous lines for sclerotinia sclerotiorum

Two rape homozygous lines which are transformed into BnRALF10 gene RNAi structures (pART-RNAi-BnRALF10) are taken as materials to detect and analyze the resistance to sclerotinia sclerotiorum. The inoculation method and evaluation of disease resistance were as described in 5) of example 1).

The result of sclerotinia inoculation analysis of the BnRALF10-RNAi plant constructed by the invention shows that the BnRALF10-RNAi plant is obviously more susceptible than the wild-type plant (WT) (FIG. 2A). The result of quantitative analysis of lesion area shows that the lesion area of WT plants is 152mm²The lesion areas of BnRALF10 RNAi plants-4 and-8 respectively reach 264mm on average²、243mm²(FIG. 2B). The BnRALF10-RNAi plant is shown to have significantly lower resistance to sclerotinia sclerotiorum than the wild type plant. Inhibition of the expression of the BnRALF10 gene resulted in a significant reduction in resistance of canola to sclerotinia sclerotiorum. The invention further proves the conclusion that BnRALF10 gene regulates resistance to sclerotinia by constructing BnRALF10-RNAi rape on one hand, and successfully creates and obtains a novel rape material with obviously reduced sclerotinia resistance by constructing BnRALF10-RNAi rape on the other hand.

In summary, the results of the invention combined with fig. 1-2 reveal for the first time that the rape secretory peptide promoter gene BnRALF10 plays a strong positive regulatory role in resistance to sclerotinia rot of rape. The invention provides an application approach and an application technology of BnRALF10 in creating germplasm of an antibacterium nucleopathy crop, provides an embodiment and successfully obtains a rape with high antibacterium nucleopathy.

Sequence listing

<110> Zhejiang university

<120> application of disease resistance function of rape secretory peptide gene BnRALF10

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 360

<212> DNA

<213> rape (Brassica napus)

<400> 1

atgaactctc gcgccatcta cgccgtcgtc gcgattctcg ccatcgtaat ctcagccgtc 60

gacgcaagcg gcttcggttt cggacactcg cttgatttcg taaggaccgg atcttctact 120

ctcttctctg gatgcgaagg atcgatcgct gagtgcatcg ccgaggaaga ggagatggag 180

ttcgattcgg atatcagcag gcgcatttta gcgcagaaga agtacgttag ctacggtgcg 240

atgaggaaga acagtgtgcc ttgctcgcgc cgcggagctt cgtattacaa ctgccagcgt 300

ggcgctcagg cgaatcctta ccgccgtgga tgcagcacca tcacgagatg caggcgttga 360

<210> 2

<211> 119

<212> PRT

<213> rape (Brassica napus)

<400> 2

Met Asn Ser Arg Ala Ile Tyr Ala Val Val Ala Ile Leu Ala Ile Val

1 5 10 15

Ile Ser Ala Val Asp Ala Ser Gly Phe Gly Phe Gly His Ser Leu Asp

20 25 30

Phe Val Arg Thr Gly Ser Ser Thr Leu Phe Ser Gly Cys Glu Gly Ser

35 40 45

Ile Ala Glu Cys Ile Ala Glu Glu Glu Glu Met Glu Phe Asp Ser Asp

50 55 60

Ile Ser Arg Arg Ile Leu Ala Gln Lys Lys Tyr Val Ser Tyr Gly Ala

65 70 75 80

Met Arg Lys Asn Ser Val Pro Cys Ser Arg Arg Gly Ala Ser Tyr Tyr

85 90 95

Asn Cys Gln Arg Gly Ala Gln Ala Asn Pro Tyr Arg Arg Gly Cys Ser

100 105 110

Thr Ile Thr Arg Cys Arg Arg

115

<210> 3

<211> 40

<212> DNA

<213> Artificial sequence (Unknow)

<400> 3

ggacagccca gatcaactag tatgaactct cgcgccatct 40

<210> 4

<211> 42

<212> DNA

<213> Artificial sequence (Unknow)

<400> 4

gcccttgctc accatggatc cacgcctgca tctcgtgatg gt 42

<210> 5

<211> 43

<212> DNA

<213> Artificial sequence (Unknow)

<400> 5

atttggagag gacacgctcg agatgaactc tcgcgccatc tac 43

<210> 6

<211> 42

<212> DNA

<213> Artificial sequence (Unknow)

<400> 6

accaagctgg ggtaccgaat tcacgctggc agttgtaata cg 42

<210> 7

<211> 42

<212> DNA

<213> Artificial sequence (Unknow)

<400> 7

tgggttcgaa atcgataagc ttacgctggc agttgtaata cg 42

<210> 8

<211> 43

<212> DNA

<213> Artificial sequence (Unknow)

<400> 8

ctcattaaag caggactcta gaatgaactc tcgcgccatc tac 43

Claims (5)

1. The disease-resistant function application of a rape secretory peptide gene BnRALF10 is characterized in that the function refers to the function of stimulating and regulating disease resistance of a rape (Brassica napus) secretory peptide gene BnRALF10, the application is the application of obtaining a rape material with changed disease resistance by creating transgenic rape, and the nucleotide sequence of the gene is shown as SEQ ID: 1, and the sequence of the encoded protein is shown as SEQ ID: 2, respectively.

2. Use according to claim 1, characterized in that in the acquisition of sclerotinia disease resistance-enhancing rape material by creating transgenic rape overexpressing BnRALF 10.

3. Use according to claim 1, characterized in that in the obtaining of rape material with reduced resistance to sclerotinia sclerotiorum by creating BnRALF10-RNAi transgenic rape.

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

(1) construction and acquisition of BnRALF10 gene overexpression structure

The BnRALF10 gene Open Reading Frame (ORF) is cloned into a plant expression vector and driven by a strong promoter to express,

(2) acquisition of agrobacterium for transforming BnRALF10 gene overexpression structure

The constructed BnRALF10 gene overexpression structure is transformed into an agrobacterium strain with strong infection capability to rape by methods such as heat shock,

(3) creation and acquisition of transgenic rape with overexpression of BnRALF10

Introducing the BnRALF10 gene overexpression structure into rape by an agrobacterium-mediated method to obtain the rape with a BnRALF10 gene overexpression structure,

(4) acquisition of transgenic rape homozygous line of overexpression BnRALF10

Respectively using antibiotic resistance and BnRALF10 gene expression as detection indexes to detect the character separation condition of transgenic plant progeny so as to obtain transgenic rape homozygous line which can stably inherit and does not separate progeny character and overexpress BnRALF10,

(5) screening, identifying and obtaining transgenic rape homozygous lines of overexpression BnRALF10 with enhanced sclerotinia disease resistance

The transgenic rape homozygous system of the overexpression BnRALF10 is used as a material, the resistance to sclerotinia rot of rape is detected and analyzed, and the transgenic rape BnRALF10 with enhanced sclerotinia rot resistance is obtained.

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

(1) construction and acquisition of RNAi structure of BnRALF10 gene

The forward interference fragment and the reverse interference fragment of the BnRALF10 gene are respectively and sequentially cloned into an RNAi vector (pART-RNAi) vector through a homologous recombination method, and the RNAi structure pART-RNAi-BnRALF10 of the BnFER1 is obtained.

(2) Acquisition of Agrobacterium transformed with BnRALF10 Gene RNAi Structure

The BnRALF10 gene RNAi structure (pART-RNAi-BnRALF10) is transformed into an agrobacterium strain with strong infection capacity to rape by methods such as heat shock.

(3) Creation and acquisition of BnRALF10 gene transferred RNAi structure rape

The RNAi structure of the BnRALF10 gene is introduced into the rape by an agrobacterium-mediated method, and the rape with the BnRALF10 gene RNAi structure is obtained.

(4) Acquisition of BnRALF10 gene-transferred RNAi structure rape homozygous line

Antibiotic resistance and BnRALF10 gene expression are respectively used as detection indexes to detect the character separation condition of transgenic plant progeny, and obtain the rape homozygous line of the BnRALF10 gene RNAi structure, the progeny character of which is not separated any more and can be stably inherited.

(5) Screening, identifying and obtaining of BnRALF10 gene-transformed RNAi structure rape homozygous lines with reduced sclerotinia sclerotiorum resistance

The BnRALF10 gene RNAi structure (pART-RNAi-BnRALF10) transferred rape homozygospore system is used as a material to detect and analyze resistance to sclerotinia sclerotiorum, and BnRALF10 gene RNAi rape with reduced resistance to sclerotinia sclerotiorum is obtained.

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