CN117210461A

CN117210461A - Botrytis cinerea acetolactate synthase gene ALS and RNA interference and application thereof

Info

Publication number: CN117210461A
Application number: CN202311138342.5A
Authority: CN
Inventors: 余心怡; 渠慎春; 林欣欣; 曹丽芳; 周停停; 胡凯旭; 李芳竹
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2023-05-12
Filing date: 2023-09-05
Publication date: 2023-12-12

Abstract

The invention discloses an acetyllactic acid synthase gene ALS of a viticola and RNA interference and application thereof. The application of the substances inhibiting the expression of acetolactate synthase gene ALS shown in SEQ ID NO.1 in preventing and controlling the plasmodiophora viticola diseases. The RNAi fungus expression vector is used for verifying that the silencing of the gene affects the pathogenicity of the viticola, and can be used as an effective target for disease prevention and control. The agrostis viticola acetolactate synthase gene ALS and the RNA interference means thereof can be applied to plant disease resistance improvement or development of biological antibacterial agents to realize control of agrostis viticola diseases.

Description

Botrytis cinerea acetolactate synthase gene ALS and RNA interference and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, relates to a key gene of an agrostis and application thereof, in particular to an acetolactate synthase gene ALS of the agrostis and an RNA interference means thereof, and also relates to application of RNA interference of the acetolactate synthase gene ALS in prevention and control of agrostis diseases of the agrostis.

Background

The botrytis cinerea Botryosphaeria dothidea belongs to Ascomycota (Ascomycota), and apple ring rot (Physalospora piricola Nose) caused by botrytis cinerea (Botryosphaeria) is one of the most destructive apple fungus diseases in the world, so that not only is fruit branches damaged, but also branches are ulcerated, the quality of fruits is reduced, and in extreme cases, death of hosts is caused, and serious influence is caused to main apple producing areas in the world such as China, japan, korea, the United states, australia and south Africa. The pathogenic bacteria are also the most common and important pathogenic fungi causing canker and wilt of woody plants, can infect plant species from 24 different genera, and in addition to apples, jeopardize the safe production of a variety of fruit trees and economic tree species such as pears, peaches, grapes, mangos, olives, poplars, eucalyptus, and the like. Infection of the plants by the viticola comprises a period of endophytic latency, common quarantine measures are often escaped, disease monitoring of apple ring rot is time-consuming and labor-consuming, and the obtained effect is still poor. On the other hand, although the conventional disease control measures depending on chemical agents can play a certain role in controlling apple ring rot, the long-term use of a large amount of chemical bactericides not only increases the production cost, but also brings great pressure to environmental safety, and also causes the problem of fungal drug resistance. Therefore, the development of a green prevention and control strategy for the Puccinia viticola is always a problem to be solved in apple production.

Molecular breeding can break through the limit between seeds, shortens the breeding period, improves the breeding efficiency, and is the most rapidly developed crop germplasm optimization means at present. In the last few decades, substantial progress has been made in the study of the molecular mechanism of apple germplasm anti-ring disease, isolating and identifying many defense-related genes. The genes are transferred into plants through genetic engineering, so that the disease resistance of transgenic plants can be enhanced. However, apples are used as woody plants, the genome is highly heterozygous, the genetic background is very complex, the introduced exogenous genes possibly cannot play an expected immune effect, and even other agronomic characters of the apples are changed, so that the yield and quality of fruits are adversely affected.

RNA interference (RNAi) is a phenomenon of gene silencing induced by non-coding small RNAs (sRNA), which act on target mRNA with sequence complementarity to block expression of homologous mRNA in a direct cleavage or translational inhibition manner. RNAi pathways are well conserved and exist in almost all eukaryotes, and plants and fungi can induce gene silencing by endogenous sRNA. sRNA can shuttle between closely contacted hosts and parasites in the form of naked molecules, protein complexes, or vesicle transport, inducing gene silencing in the recipient cells. Cross-border RNAi based on sRNA bidirectional trafficking is a common mechanism for cross-border gene expression regulation during plant-pathogen interactions.

RNAi carrier of specific target fungus key gene is designed and fungus cell is introduced to directionally interfere the expression of target gene, and pathogen infection is repressed through cross-range regulation and control to raise the disease resistance of host. The prevention and control strategy based on the cross-boundary RNAi can effectively control plant diseases which are difficult to solve by conventional cultivation management measures, and is particularly suitable for latent fungus diseases. The method replaces the traditional pesticide control method, can reduce the influence of chemical drugs on the environment, simultaneously avoids the problem of pathogenic bacteria drug resistance, and is a feasible and environment-friendly disease control means with wide application prospect. Effective practice has been achieved in many different fungal pathological systems, successfully applied to the control of wheat leaf rust and scab, cotton verticillium wilt and powdery mildew and gray mold.

Although agricultural disease control techniques utilizing cross-border RNAi are known in the art, the use of such techniques for controlling fungal diseases of woody crops has not been reported. Meanwhile, a key factor affecting the effectiveness of the technology is to select proper target genes of fungi, namely genes essential for the growth, proliferation and virulence of the fungi, and the functional deficiency of the target genes destroys the pathogenicity of the fungi; the sRNA interference fragment aiming at the fungal target gene is designed, so that the specific silencing of the target gene can be triggered in the fungal body with high efficiency; the intake of exogenous sRNA by pathogenic fungi is induced by a proper means. Preliminary analysis of the viticola isolated from apple ring rot showed that its genome was enriched with a large number of genes encoding potentially pathogenic proteins. However, the function and mechanism of action of only a few genes are currently being further studied. As one of the most economically influencing pathogenic fungi, researchers have limited knowledge of the growth regulation and pathogenesis of Botrytis cinerea.

Acetolactate synthase (acetohydroxyacid synthase, AHAS, e.g. c.2.2.1.6, also abbreviated as ALS) is the first key enzyme in the biosynthesis pathway of valine, leucine and isoleucine in bacteria, fungi and higher plants, essential in the growth, development and virulence of phytopathogenic fungi. Acetolactate synthase inhibitors such as Sulfonylurea (SU) compounds such as chlorsulfuron, tribenuron-methyl and derivatives thereof change the catalytic activity of ALS protein, and can effectively inhibit the development of plant pathogenic fungi when used as antifungal agents.

Deletion of the ILVx gene encoding ALS subunit results in fungal BCAA auxotrophs, fusarium graminearum f.graminearum exhibiting inhibition of aerial hypha growth and red pigmentation, aspergillus oryzae m.oryzae conidiogenesis morphological defects, microsclerotium formation destruction by verticillium dahliae v.dahliae, and severe effects on fungal pathogenicity.

ALS catalyzes the life process of branched-chain amino acid synthesis, which is not existed in mammals, and ALS gene is used as a target spot for resisting harmful organisms such as weeds, pathogenic bacteria and the like, and has biological safety for human bodies. Therefore, the acetolactate synthase gene is used as an ideal target point of a novel control strategy for the botrytis cinerea diseases based on cross-border RNAi.

Disclosure of Invention

The invention aims to provide an acetolactate synthase gene ALS which influences the pathogenicity of the plasmodium viticola, and the acetolactate synthase gene ALS can be used as an effective target for preventing and controlling the plasmodium viticola diseases.

It is another object of the present invention to provide an RNA interference means against the Als gene of said Portland bacteria.

The third purpose of the invention is to apply the RNA interference of the agrostis viticola acetolactate synthase gene ALS to plant resistance improvement and biological antibacterial agents to realize the prevention and control of agrostis viticola diseases.

To achieve the above object, the general technical scheme of the present invention is as follows:

the invention firstly provides an acetyllactic acid synthase gene ALS of the Botrytis cinerea, and the nucleotide sequence of the acetyllactic acid synthase gene ALS is shown as SEQ ID NO. 1; the protein sequence coded by the gene is shown as SEQ ID NO. 2.

The acetolactate synthase gene ALS of the invention affects pathogenicity of the Portal cavity bacteria. The invention constructs RNAi fungus expression vector aiming at ALS genes, and silences the acetolactate synthase gene ALS of the highly pathogenic Botrytis strain LW347. Transformed strains were inoculated on Potato Dextrose Agar (PDA) plates and after 5 days of culture, ALS-silenced strains DeltaALS-hyg showed a significant growth defect compared to the empty vector-carrying control strain GFP-hyg. According to the invention, apple leaves are infected by two transformed strains, and after 48 hours of inoculation, the host lesion size and pathogenic bacteria biological quantity measurement result show that the colonization, propagation capacity and virulence of the delta ALS-hyg strain are obviously lower than those of GFP-hyg control strains, and the primary demonstration that the acetolactate synthase gene ALS is related to the pathogenicity of the viticola.

The application of the acetolactate synthase gene ALS shown in SEQ ID NO.1 as a target point in the preparation of a preparation for preventing and controlling the plasmopara viticola disease designs RNAi substances by taking the acetolactate synthase gene ALS as the target point so as to inhibit the plasmopara viticola.

The application of the substances inhibiting the expression of acetolactate synthase gene ALS shown in SEQ ID NO.1 in preventing and controlling the plasmodiophora viticola diseases.

Preferably, the substance inhibiting the expression of acetolactate synthase gene ALS shown in SEQ ID NO.1 is selected from miRNA or dsRNA for the acetolactate synthase gene ALS.

Preferably, the miRNA against the acetolactate synthase gene ALS is selected from any one of the mirnas shown in SEQ ID No.3 or SEQ ID No. 4; the dsRNA interference fragment aiming at the acetolactate synthase gene ALS is selected from dsRNA shown in any one of SEQ ID No. 5-7.

The application of a substance for inhibiting the expression of acetolactate synthase gene ALS shown in SEQ ID NO.1 in preparing a preparation for preventing and controlling the plasmodiophora viticola disease.

A preparation for controlling an Alternaria viticola disease, which comprises the dsRNA interference fragment for the acetolactate synthase gene ALS according to claim 7.

As a preferred embodiment of the present invention, the formulation is a dsRNA solution having a concentration of 10 ng/. Mu.L or more.

The invention adopts PEG mediated protoplast transformation technology, and respectively introduces miRNA/miRNA or dsRNA of 2 artificial miALS or 3 dsALS obtained by in vitro transcription of T7 RNA polymerase into an Botrytis cinerea strain LW347. Inoculating the transformed strain to a Potato Dextrose Agar (PDA) plate, and after culturing for 5 days, compared with a simulated transformed control, the growth of the Portland bacteria transformed with different RNA interference fragments is inhibited to different degrees; the transformed strains into which dsALS-1 (SEQ ID No. 5) and dsALS-2 (SEQ ID No. 6) were introduced, respectively, exhibited significant growth defects, indicating that the RNA interference effects of dsALS-1 and dsALS-2 on the ALS genes were most pronounced.

The RNA interference means of the acetolactate synthase ALS gene can be applied to prevention and control of the plasmodiophora viticola diseases, and specifically comprises the following steps: (1) Expressing the ALS gene RNA interference fragment in a host to improve the disease resistance of the plant to the Botrytis cinerea; (2) And (3) taking the ALS gene RNA interference fragment as an active substance to prepare the biological antibacterial agent of the Botrytis cinerea.

The RNA interference means of the acetolactate synthase ALS gene can be applied to improving the disease resistance of plants to the Botrytis cinerea, and comprises the following steps: (1) Constructing a plant expression vector containing the ALS gene RNA interference fragment; (2) Transforming the plant expression vector into a plant or plant cell; (3) And screening to obtain the transgenic plant with enhanced resistance of the viticola.

The transformation protocol of the present invention, as well as the protocol for introducing the ALS gene RNA interference fragment into a plant, may vary depending on the type of plant or plant cell used for transformation.

Plant expression vectors used to introduce the RNA interference fragments into plant cells include: hpRNA interference vectors and virus-mediated gene silencing (Virus Induced Gene Silencing, VIGS) vectors. Methods for constructing plant expression vectors containing the ALS gene RNA interference fragments are well known to those skilled in the art.

Suitable methods for introducing the plant expression vector into a plant cell include: microinjection, electroporation, agrobacterium-mediated transformation, direct gene transfer, and the like.

The plant of the present invention comprises: the host plant of the Puccinia is preferably any one or more of horticultural and commercial crops including, but not limited to, apple, pear, peach, bayberry, grape, mango, chestnut, walnut, pistachio, eucalyptus, poplar, alder or myrtle.

According to the phenotypic change of the transformed strain of the Botrytis cinerea, two ALS gene RNA interference fragments with nucleotide sequences shown as SEQ ID No.5 and SEQ ID No.6 are selected and used for constructing a VIGS vector. The two DNA fragments are respectively connected to a tobacco brittle virus (Tobacco rattle virus, TRV) pTRV2 vector to form a TRV-VIGS vector aiming at the ALS gene, agrobacterium is introduced, the TRV-VIGS vector is matched with pTRV1 for apple infiltration infection, and finally TRV positive apple TRV-dsALS-1 (expressing RNA interference fragment dsALS-1 with the nucleotide sequence shown as SEQ ID No. 5) and TRV-dsALS-2 (expressing RNA interference fragment dsALS-2 with the nucleotide sequence shown as SEQ ID No. 6) for expressing the RNA interference fragment of the ALS gene are obtained. The invention further carries out resistance evaluation of the grape vine chamber bacteria on the TRV apples expressing the ALS gene RNA interference fragment. The results of the host symptom analysis showed that the sensitivity of TRV-dsALS-1 and TRV-dsALS-2 apples to infection was significantly lower than that of the control plant (TRV-00) permeated with TRV empty, and the lesion areas were reduced by 43.9% and 61.2%, respectively. The DNA of the leaf with the inoculation point being the circle center diameter of 2cm is extracted for fungus biomass measurement, and the fungus biomass of two apples carrying RNA interference fragments of ALS genes is obviously reduced, which is only 66.7 and 36.8 percent of TRV-00 control plants. The results of the expression level analysis show that the expression levels of the ALS genes of the agrostis in the inoculated parts of the TRV-dsALS-2 and the TRV-dsALS-3 apples are obviously reduced, wherein the expression level of the ALS genes in the TRV-dsALS-2 group is reduced to 40.6% of that of the TRV-00 control. The analysis of host symptoms, the determination of fungal biomass and the analysis of ALS gene expression quantity are combined to obtain that the TRV-dsALS-2 (the nucleotide sequence of the RNA interference fragment is shown as SEQ ID No. 6) apple has stronger resistance to the infection of the Botrytis cinerea, which indicates that the dsALS-2 (the nucleotide sequence of the RNA interference fragment is shown as SEQ ID No. 6) is expressed in plants as the RNA interference fragment of the ALS gene of the Botrytis cinerea, so that better interference effect can be achieved, and the disease resistance of plants can be effectively improved.

The RNA interference means of acetolactate synthase ALS gene of the invention can also be applied to the biological antibacterial agent of the Botrytis cinerea, comprising the following steps: (1) synthesizing dsRNA of the ALS gene RNA interference fragment; (2) preparing an antibacterial agent using the dsRNA as an active substance; (3) Spraying an antimicrobial agent containing the ALS gene RNA interference fragment dsRNA on plants to inhibit the infection of the plasmodium viticola.

Methods for synthesizing the ALS gene interference fragment dsRNA of the present invention are well known to those skilled in the art and include: in vitro transcription, microbial fermentation, and plant expression.

The invention synthesizes the ALS gene RNA interference fragment dsALS-1 or dsALS-2 (the nucleotide sequence is shown as SEQ ID No.5 or SEQ ID No. 6) into dsRNA by adopting an in vitro transcription technology based on T7 RNA synthetase, and dilutes the dsALS gene RNA interference fragment dsALS-1 or dsALS-2 into 50 ng/. Mu.L by using ultrapure water without RNase. The dsRNA solution is coated on a PDA flat plate and is used for culturing the Botrytis cinerea after being completely absorbed. After 5 days of inoculation on PDA plates containing both dsRNAs, the R.viticola showed significant growth defects compared to controls inoculated on ultra pure water (RNase-) treated PDA plates. Fluorescent quantitative PCR (RT-qPCR) analysis shows that the expression level of fungus ALS genes is obviously reduced, and the portulaca oleracea can absorb exogenous dsRNA in the environment and induce the silencing of fungus target genes.

The invention applies the dsRNA solution (50 ng/. Mu.L) of the RNA interference fragment (with the nucleotide sequence of SEQ ID No.5 or SEQ ID No. 6) of the ALS gene to the surface of apple leaves, and inoculates the Botrytis cinerea strain LW347 to the application position after air drying. After 48 hours of inoculation, the symptoms of the inoculated part of the dsRNA solution of the RNA interference fragments of the two ALS genes are obviously lighter than those of the control part of ultra pure water (RNase-) application, the area of the disease spots is reduced to 47.5% -69.4%, the biomass of pathogenic bacteria is obviously reduced, the inoculated point is used as the center of a circle, the biomass of fungi in blades with the peripheral diameter of 2cm is also obviously reduced to 53.8% -76.0% of the control part, and the RNA interference fragments of the ALS genes can be used as antibacterial active substances to inhibit the infection of the viticola on plants.

The invention further uses ultrapure water (RNase-) to dilute the dsRNA solution of the ALS gene RNA interference fragment with the nucleotide sequence of SEQ ID No.6 to 25, 10, 5 and 1 ng/mu L, the dsRNA solution is respectively applied to the surfaces of apple leaves, and after the apple leaves are air-dried, the apple leaves are inoculated with an agrobacteria strain LW347. The results of fungus bioassay with the diameter of 2cm at the periphery of the inoculation point show that 10 ng/. Mu.L of dsASL-2 solution has a certain antibacterial effect, but the dsRNA solution with high concentration can play a better role in protecting plants.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the invention provides an acetolactate synthase gene ALS, which utilizes RNAi fungal expression vectors to verify that silencing of the gene affects pathogenicity of the viticola and can be used as an effective target for disease prevention and control. Two engineering miRNAs and three dsRNA interference fragments aiming at the ALS genes are designed and obtained, and the interference fragments (the nucleotide sequences of the dsRNAs are shown as SEQ ID No.5 and SEQ ID No. 6) which can play a remarkable RNA interference effect are screened out by directly introducing the interference fragments into fungi. The RNA interference fragment with the nucleotide sequence of SEQ ID No.5 or SEQ ID No.6 is further introduced into apples by using a TRV-mediated VIGS technology, so that an ALS gene interference plant is obtained. The RNA interference fragment with the best interference effect is determined by combining host symptom identification, pathogenic bacteria biomass measurement and target gene expression level analysis, and the nucleotide sequence of the RNA interference fragment is shown as SEQ ID No. 6. The ALS gene RNA interference fragment is further prepared into an antibacterial agent, and the effect of resisting the viticola can be achieved when the concentration of dsRNA solution is 10 ng/mu L. The agrostis viticola acetolactate synthase gene ALS and the RNA interference means thereof can be applied to plant disease resistance improvement or development of biological antibacterial agents to realize control of agrostis viticola diseases.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Drawings

FIG. 1 is a schematic diagram of an RNAi fungal expression vector of the ALS gene.

FIG. 2 is an identification of ALS silencing strains. RT-PCR verification of PDK intron and hyg; identification of the expression level of ALS genes.

FIG. 3 is a phenotypic identification of ALS gene silencing Puccinia.

FIG. 4 is a pathogenicity analysis of ALS gene-silenced Puccinia. Wherein, diagram a: evaluation of host symptoms; graph B: and (5) measuring the biomass of pathogenic bacteria.

FIG. 5 is a phenotypic identification of Portland bacteria strain LW347 incorporating different RNA interference fragments of the ALS gene.

FIG. 6 is an analysis of ALS gene expression levels of Rabdosia sp with different RNA interference fragments introduced.

FIG. 7 is a schematic representation of pTRV2 vector for TRV-VIGS.

FIG. 8 is an RT-PCR identification of TRV positive apple plants. Wherein M: marker, WT: wild type.

FIG. 9 is an evaluation of disease resistance of TRV apples expressing an ALS gene interference fragment. Wherein, diagram a: counting the area of the host disease spots; graph B: measuring the biomass of pathogenic bacteria; graph C: analysis of fungal ALS gene expression levels.

FIG. 10 is an identification of uptake of exogenous dsRNA in the environment by Portland bacteria strain LW347. Wherein, diagram a: fungal phenotype in PDA plates containing ALS gene RNA interference fragments; graph B: ALS gene expression level analysis.

FIG. 11 is an evaluation of the antibacterial effect of ALS gene RNA interference fragment on apple leaves. Wherein, diagram a: identifying host symptoms; graph B: counting the size of the lesion; graph C: measuring the biomass of pathogenic bacteria; graph D: analysis of fungal ALS gene expression levels.

FIG. 12 is an evaluation of antibacterial effect of dsRNA solutions of ALS gene RNA interference fragments at different concentrations. Wherein, diagram a: counting the size of the host lesion; graph B: and (5) measuring the biomass of pathogenic bacteria.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. It should be understood that the embodiments described are exemplary only and should not be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.

Example 1 identification of the Als Gene of Brevibacterium (preparation example 1)

Isolation and cloning of ALS Gene

1) The Botrytis cinerea strain LW347 (isolated from apple ring rot disease strain, offered by Zhou Zengjiang of Zhengzhou fruit tree institute of China academy of sciences) was inoculated on a PDA plate, and after dark culture at 24℃for 5 days, mycelia were scraped and total RNA was extracted. Total plant RNA extracted from Trimerella TSINGKE extraction kit (see manufacturer's instructions for specific procedures);

2) Synthesis of first Strand cDNA

First strand cDNA was synthesized using PrimeScript reverse transcription kit (Takara) using total RNA of Botrytis cinerea as a template. Genomic DNA was first removed and reverse transcribed using the RT Primer Mix provided in the kit according to the manufacturer's instructions.

3) Amplification of full-Length cDNA of ALS Gene

Acetolactate synthase ALS in the present invention is isolated from NCBI Genebank (National Center for Biotechnology Information,https://www.ncbi.nlm.nih.gov/) Published KAF4307733 corresponds. The tBLASTn program is used for comparing the amino acid sequence and the genome sequence information of the Botrytis cinereahttps:// www.ncbi.nlm.nih.gov/assembly/GCA_011503125) Sequence information of ALS gene was obtained. According to the sequence information, a specific primer is designed, and the whole cDNA of the ALS gene is obtained through conventional PCR amplification. The primers used were as follows:

ALS F：ATGCAGCCTCACACAGTGG

ALS R：TCATGCCACGATAGCCTCCA

the nucleotide sequence of 1-1809 base in ALS gene full-length cDNA as shown in SEQ ID NO.1 (total length of sequence is 1809 bp) is obtained by Sanger sequencing, and the gene codes for the amino acid sequence as shown in SEQ ID NO.2 (total length is 602 nt). ALS gene full-length cDNA may be synthesized by a biological company.

2. Construction of an ALS Gene silencing Strain

Designing a gene specific primer (delta ALS F: GGTACCAAGACATCCCCAACC; delta ALS R: GGTTACGACGAGCGGGTG) by using a first strand cDNA of the Botrytis cinerea as a template, amplifying to obtain an intermediate fragment (308 bp) of the ALS gene of the Botrytis cinerea by using a conventional PCR technology, connecting the intermediate fragments of sense and antisense to two ends of PDK intron by using an overlap PCR technology to obtain a delta ALS fragment, and constructing the delta ALS fragment into a fungus expression vector pBHT2-GFP to replace GFP genes to obtain an RNAi fungus expression vector pBHT 2-delta ALS of the ALS genes (figure 1).

1) Extracting the Botrytis cinerea protoplast. (1) 6 pieces of about 3X 3mm were picked from the edge of the Portland fungi LW347 dark cultured at 24℃for 5 days on a PDA plate ² Is inoculated into 100mL of PDB liquid culture medium and is cultured for 42 hours at 24 ℃ and 60 rpm. (2) The medium was removed by centrifugation at 3000g for 10min at normal temperature, the mycelia were rinsed 3 times with 0.7M NaCl and the water was blotted off with sterile filter paper. (3) 1g of mycelium is weighed and 10mL of crashing enzyme (3%driselase,0.7M NaCl) is added and the enzymolysis is carried out at 28 ℃ and 80rpm for 3.5 hours. (4) Filtering the enzymolysis solution with three layers of mirror cleaning paper, centrifuging 3000g of the filtrate at 4deg.C for 10min, and collecting protoplast. (5) STC (1.2M sorbitol,0.01M Tris-HCl pH 7.5,0.05M CaCl) ₂ ) The pellet was washed twice, resuspended in STC and diluted to 1X106/mL and stored on ice.

2) Genetic transformation of Botrytis (transformation of the fungus described below is only used to verify the function of the ALS gene, the transformed strain described below is not a algebraic biological material). (1) The pBHT 2-DeltaALS recombinant plasmid and the pBHT2-GFP vector were transformed into Agrobacterium AGL1, respectively. (2) Fresh Agrobacterium in 500. Mu.L LB liquid medium (50. Mu.g/mL kanamycin) was taken and added to the induction liquid medium (IM, MM (K) ₂ HPO ₄ 2.05g/mL,KH ₂ PO ₄ 1.45g/mL,NaCl 0.15g/mL,MgSO4.7H2O 0.50g/mL,CaCl ₂ .6H ₂ O 0.1g/mL,FeSO ₄ .7H ₂ O 0.0025g/mL,(NH4) ₂ SO ₄ 0.5g/mL),40mM 2-(N-morpholino)ethanesulfonic acid(MES),pH 5.3；10mM glucose,0.5% (w/v) glycidol, 200. Mu.M Acetostingo (AS)), cultured at 28℃for 5-6 hours to 0D6000.5-0.6. (3) mu.L of recovery medium (Yeast extract Peptone Sucrose, YPS, eye extract 3.5g/L, peptone 5g/L,1M suspension) was added to 100. Mu.L of Botrytis cinerea protoplast, and the mixture was incubated at 25℃for 2 hours. (4) 100. Mu.L of IM containing recombinant Agrobacterium was added to 200. Mu.L of the culture-recovered Portland bacteria protoplast, mixed and spread evenly on a glass paper-covered induction plate (AIM, MM,40mM MES,pH 5.3,5mM glucose,0.5% (w/v) glycitol, 200. Mu.M Acetokringine (AS), agar 1.5%), and cultured at 22℃for 48h. (5) Cellophane on AIM plates was cut into strips 0.5cm wide, transferred to screening plates (PDA, hygromycin B8 mg/L, cefotaxin 60mg/L, streptomycin60 mg/L) and incubated dark at 28℃for 7 days. (6) The new mycelia were picked and transferred to PDA plates containing 10mg/L hygromycin B and incubated at 28℃for 5 days.

3) Identification of positive strains. Genomic DNA of the transformed strain was extracted using TIANGEN plant genomic DNA extraction kit according to the manufacturer's instructions, and the results of RT-PCR identification of the transgenic positive strain are shown in FIG. 2A, using the following primers:

PDKintron F GCAATGAAAGACGGTGAGCTG

PDKintron R GGCATGATGAACCTGAATCGC

hyg F CGAGAGCCTGACCTATTGCAT

hyg R TCCGTCAGGACATTGTGGAG

total RNA of the transformed strain was extracted and reverse transcribed into first strand cDNA, and the expression level of ALS gene of the transgenic positive strain was identified by RT-qPCR, and the result is shown in FIG. 2B. The primers used were as follows:

ALS-q F CGATGTGGTGACGAATCAGGT

ALS-q R ACCTGTGATGCTGCATACGAA

3. phenotypic identification of an ALS silencing strain of Portland bacteria

1) And (5) evaluating fungus growth. The transformed strain was inoculated on a PDA plate, and after 5 days of dark culture at 24℃the colony morphology was observed. The Δals-hyg strain that silenced the ALS gene exhibited a significant defect in growth compared to the GFP-hyg control strain, indicating that ALS gene silencing affected the growth of portulaca viticola (fig. 3).

2) Analysis of fungal pathogenicity. Taking apple leaves at 3-4 leaves of current annual branch, and winding the leaves with wet absorbent cotton to preserve moisture. A small hole is pricked on the surface of a leaf by a needle head, a transformed strain is inoculated on a wound, the front surface of the leaf is upwards at 25 ℃ (moisturizing culture (16 h illumination/8 h darkness)), after inoculation is carried out for 48 hours, leaf symptoms are recorded by photographing, and the size of the disease spots is measured, a leaf disc with the inoculation point as the circle center diameter of 2cm is taken, the biomass of the disease spots is measured (see Yu et al, malus hupehensis miR168 targets to ARGONAUTE1 and contributes to the resistance against Botryosphaeria dothidea infection by altering defense response.plant and Cell Physiology,2017,58 (9): 1541-1557.), and the result shows that the symptom of an ALS gene silencing strain inoculation position on the apple leaf is lighter, the disease spot area is about 37.4 percent of the inoculation position of a control strain, and the biomass of the disease spots is obviously reduced and only is 31.5 percent of the control strain, so that the ALS gene plays an important role in pathogenicity of the grape-chamber bacteria (figure 4).

Example 2 screening of RNA interference fragment of Alternaria viticola ALS Gene (preparation example 2)

1) RNA interference fragment design. Based on the sequence information shown in SEQ ID No.1, two engineered miRNAs and 3 dsRNA interference fragments for the Purpureae ALS gene were designed, namely, miALS-1 and miALS-2 (SEQ ID No. 3-4) and dsALS-1, dsALS-2 and dsALS-3 (SEQ ID No. 5-7) (Table 1). Two 21bp miallSDNA fragments were obtained by Shanghai strapdown synthesis, and three dsALSs were derived from different segments of ALS gene cDNA, obtained by conventional PCR means.

TABLE 1 RNA interference fragments of Alternaria viticola ALS genes

2) Genetic transformation of the Botrytis cinerea. The RNA interference fragment was introduced into the botrytis strain LW347 using PEG-mediated protoplast transformation technique. (1) The ALS gene interference fragment was transcribed in vitro to miRNA/miRNA or dsRNA using the nupraise Vazyme T7 RNAi Transcription Kit kit, see manufacturer's instructions for specific procedures. (2) The Portugal protoplasts were extracted as described in example 1. (3) 1mL of protoplast was added with 10. Mu.g of the above miRNA/miRNA or dsRNA and 25. Mu.g of pBHT2-GFP plasmid, gently mixed and ice-bathed for 30min. (4) 1mL of PTC (40%PEG4000,0.1MCaCl2,0.1M Tris-HCl pH=8.0) was added dropwise and mixed well. (5) Incubation was performed at 30℃for 30min, centrifugation was performed at 2000g for 8min, and the pellet was resuspended in 3mL RB (1.2M sorbitol,1%dextrose,100. Mu.g/mL Kanamycin) and incubated at 26℃for 12h. (6) 20mL RB agar (RB, 1%agar,hygromycin B8mg/L) was added, gently mixed, plated at 10 mL/plate, and incubated at 26℃for 24 hours in the dark. (7) 15mL of PDA containing 10mg/Lhygromycin B was plated on RB plates and incubated at 26℃until colonies broke through the upper PDA.

3) And (5) identifying fungus phenotype. The transformed strain was inoculated on a PDA plate, and after dark culture at 24℃for 5 days, colony morphology was observed. The different RNA interference fragments transformed D.vitis showed different degrees of growth deficiency compared to the mock transformed control, wherein the transformed strains introduced with dsALS-2 (SEQ ID No. 6) and dsALS-3 (SEQ ID No. 7) showed more significant phenotypic changes (FIG. 5).

4) And (5) analyzing the expression level of the target gene. The hyphae of the transformed strain were scraped, total RNA extracted and reverse transcribed into first strand cDNA as described in example 1. The expression level of ALS gene is detected by using a real-time fluorescent quantitative PCR (RT-qPCR) method and using staphylococcus action gene as an internal reference gene. RT-qPCR was performed using the Quantum studio 6 system, and the optimized reaction procedure was: pre-denaturation at 95 ℃ for 30s;95℃for 5s,60℃for 34s,40 cycles. The amplification efficiency and primer specificity of the PCR reaction were analyzed based on the amplification curve and the melting curve. The results were analyzed using the 2-DeltaCt method. The primers used were as follows:

ALS-q F CGATGTGGTGACGAATCAGGT

ALS-q R ACCTGTGATGCTGCATACGAA

Actin-q F GCGTGAAATCGTTCGTGACAT

Actin-q R ATGGAGTTGAAGGTGGTGACG

the expression levels of ALS genes of the Portland bacteria introduced with different RNA interference fragments were significantly reduced compared to the control strain, wherein the expression levels of dsALS-1 and dsALS-2 were significantly changed more than those of the other strains (decreasing levels reached 80.5% and 71.8%, respectively), indicating that SEQ ID No.5 and SEQ ID No.6 gave a better interference effect on the ALS genes as RNA interference fragments (FIG. 6).

Example 3 expression of an ALS Gene RNA interference fragment in apple increases its resistance to Rabbit

The invention adopts TRV-mediated VIGS technology, and introduces preferred ALS gene RNA interference fragments dsALS-1 and dsALS-2 (nucleotide sequences are SEQ ID No.5 and SEQ ID No.6 respectively) into apples so as to improve the resistance of the grape vine chamber bacteria of plants (the transformation of apples is only used for verifying that the RNA interference of ALS genes can be applied to plant disease resistance improvement, and the transformed plants are not biological materials of substitution phase).

1) TRV penetration infestation of apples. (1) The TRV system vector is provided by the university of bloom Liu Yule teacher. ALS gene interfering fragments dsALS-1 and dsALS-2 having nucleotide sequences of SEQ ID No.5 and SEQ ID No.6, respectively, were ligated into the MCS region of pTRV2 plasmid (FIG. 7). After verification of DNA sequencing, the recombinant plasmid and the companion plasmid pTRV1 were transformed into agrobacterium EHA105, respectively, for infiltration infection of apples. (2) The apple aseptic seedlings are subjected to secondary culture in a plant tissue culture chamber (25 ℃ C., photoperiod 16h/8 h) by using an MS culture medium (0.3 mg/L IAA,0.2 mg/L6-BA, 0.1mg/L GA 3); rooting in 1/2MS culture medium (1.0 mg/LIAA,0.4mg/L IBA). The root seedlings are subjected to TRV vacuum infiltration before transplanting. The plants from which the medium was washed were immersed in an agrobacterial suspension carrying the TRV vector and evacuated for 90s at a pressure of 40kPa and repeated once. Washing the bacterial liquid with distilled water, and transplanting the plants in an illumination incubator. (3) Four weeks after transplanting, new leaves with the third leaf position fully developed are collected, and the TRV virus diffusion condition in plants is detected by using a semi-quantitative PCR (RT-PCR) technology, and the result is shown in figure 8. The primers used were as follows:

TRV1-rt F CCGAGGTAAAAGCCAAGTCTG

TRV1-rt R GAATCGTCTCCACTGACAACC

TRV2-rt F GTTACTCAAGGAAGCACGAT

TRV2-rt R AACTTCAGACACGGATCTAC

EF1-α-rt F ATTCAAGTATGCCTGGGTGC

EF1-α-rt R CAGTCAGCCTGTGATGTTCC

2) Disease resistance evaluation of TRV apples. RT-PCR results show that the viral RNA successfully diffuses in the TRV-infiltrated apple plants. Apple plants TRV-dsALS-1 and TRV-dsALS-2 express ALS gene interference fragments dsALS-1 and dsALS-2 respectively, and TRV-00 plants infiltrated with TRV empty are used as a control. After 48 hours of inoculation of the Botrytis cinerea strain LW347, the symptoms of the diseased leaves were evaluated and the biomass of the pathogenic bacteria was determined, as described in example 1; at the same time, total RNA at the inoculation site was extracted and the expression level of the pathogenic ALS gene was analyzed as described in example 2.

The lesion areas of TRV-dsALS-1 and TRV-dsALS-2 apples were reduced by 43.9% and 61.2% respectively compared with TRV-00 control plants, indicating a reduced susceptibility to infection by Botrytis cinerea; the fungal biomass at the inoculation site was also significantly lower than that of the control plants, 66.7% and 36.8% of TRV-00, respectively. In addition, the ALS gene RNA interference fragment expressed in apples can inhibit the expression of the target genes of invasive Botrytis cinerea, and the expression amount of the ALS genes at the infection sites of TRV-dsALS-1 and TRV-dsALS-2 fungi is about 62.4% and 40.6% of that of the control plants (FIG. 9). Among them, TRV-dsALS-2 apple (the nucleotide sequence of the RNA interference fragment is shown as SEQ ID No. 6) shows stronger resistance to Puccinia. The RNA interference fragment dsALS-2 (the nucleotide sequence is shown as SEQ ID No. 6) has a good interference effect on the ALS gene of the viticola, and the expression of the fragment in plants can effectively improve the disease resistance of the plants.

EXAMPLE 4ALS Gene RNA interference fragment as antibacterial active substance protection against infection with Aphanothece viticola (application example 2)

Preferred ALS gene RNA interference fragments dsALS-1 and dsALS-2 (nucleotide sequences SEQ ID No.5 and SEQ ID No.6 respectively) are prepared as antibacterial agents, and can protect apples from infection of Puccinia viticola. 1) And synthesizing dsRNA. The procedure of example 2 was followed to synthesize RNA interference fragments dsALS-1 and dsALS-2 (nucleotide sequences SEQ ID No.5 and SEQ ID No.6, respectively) into dsRNA.

2) Fungal uptake assay. (1) The synthesized dsRNA was diluted to 50 ng/. Mu.L in RNase-free ultrapure water and applied to PDA plates (1 mL/plate). After complete absorption, the Botrytis cinerea strain LW347 was inoculated thereon, and after dark culture at 24℃for 5 days, colony morphology was observed. The expression level of ALS gene in hyphae was detected by RT-qPCR method according to the method of example 2. The results showed that the plating of D.vitis on dsALS-1 and dsALS-2 treated PDA plates resulted in significant growth defects, and that the expression level of ALS gene was significantly lower than that of the control plated with ultrapure water (RNase-). The dsRNA in the environment was able to be taken up by the portugal bacteria and induced silencing of the target gene in the fungus (fig. 10).

3) dsRNA is used as an antimicrobial active. (1) 50 ng/. Mu.L of dsRNA solutions containing the RNA interfering fragments of ALS gene dsALS-1 or dsALS-2 were applied to the surfaces of apple leaves (100. Mu.L/site), respectively, using ultrapure water (RNase-) as a control. (2) After air-drying, the Botrytis cinerea strain LW347 was inoculated to the site of application as described in example 1, cultured in a moisture-retaining manner at 25℃for 48 hours, photographed, recorded and evaluated for development of leaf lesions, fungal biomass was measured at the site of inoculation, and the expression level of fungal ALS gene was examined as described in examples 2 and 3. The results showed that the inoculation spot area of the applied dsRNA solution was significantly smaller than the control inoculation spot (dsALS-1 and dsALS-2 reduced to 69.4% and 47.5%, respectively) and pathogenic biomass was 76.0% and 53.8% of the control site, respectively. The ALS gene expression level of the Portland bacteria inoculated to the dsRNA treatment site was significantly reduced as compared to the fungus inoculated to the ultrapure water treatment site (FIG. 11). (3) The dsALS-2 solution was further diluted to 25, 10, 5 and 1 ng/. Mu.L with ultrapure water (RNase-) and, after application to apple leaves, the Botrytis cinerea strain LW347 was inoculated, and the protective effect of dsRNA solutions of different concentrations was analyzed by the method described above. The results indicate that RNA interference fragments dsALS-1 or dsALS-2 can be used as antibacterial active substances, wherein 10 ng/. Mu.L of dsALS-2 can be sprayed on apple leaves to effectively inhibit the infection of the plasmodium viticola, and a higher concentration of dsRNA solution can play a better role in protecting plants (figure 12).

Claims

The application of the acetolactate synthase gene ALS shown in SEQ ID No.1 as a target point in the preparation of a preparation for preventing and controlling the plasmopara viticola disease is characterized in that the acetolactate synthase gene ALS is used as the target point to design RNAi substances so as to inhibit the plasmopara viticola.
2. The application of the substances inhibiting the expression of acetolactate synthase gene ALS shown in SEQ ID NO.1 in preventing and controlling the plasmodiophora viticola diseases.
3. The use according to claim 2, wherein said substance inhibiting the expression of the acetolactate synthase gene ALS represented by SEQ ID No.1 is selected from the group consisting of miRNA or dsRNA directed against said acetolactate synthase gene ALS.
4. Use according to claim 3, characterized in that said miRNA against said acetolactate synthase gene ALS is selected from any one of the mirnas shown in SEQ ID No.3 or SEQ ID No. 4; the dsRNA interference fragment aiming at the acetolactate synthase gene ALS is selected from dsRNA shown in any one of SEQ ID No. 5-7.
5. The application of a substance for inhibiting the expression of acetolactate synthase gene ALS shown in SEQ ID NO.1 in preparing a preparation for preventing and controlling the plasmodiophora viticola disease.
6. The use according to claim 5, wherein said substance inhibiting the expression of the acetolactate synthase gene ALS represented by SEQ ID No.1 is selected from the group consisting of miRNA or dsRNA directed against said acetolactate synthase gene ALS.
7. The use according to claim 6, characterized in that the miRNA against the acetolactate synthase gene ALS is selected from any one of the mirnas shown in SEQ ID No.3 or SEQ ID No. 4; the dsRNA interference fragment aiming at the acetolactate synthase gene ALS is selected from dsRNA shown in any one of SEQ ID No. 5-7.
8. A preparation for controlling an Alternaria viticola disease, which comprises the dsRNA interference fragment for the acetolactate synthase gene ALS according to claim 7.
9. The formulation of claim 8, wherein the formulation is a dsRNA solution at a concentration of 10ng/μl or greater.