CN109913492B

CN109913492B - Method for inhibiting geminivirus infection through synergistic effect of arabidopsis PEPR2 protein and Atpep1 small peptide

Info

Publication number: CN109913492B
Application number: CN201910228312.0A
Authority: CN
Inventors: 赖建彬; 阳成伟; 曾润秀; 刘晓诗
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2019-03-25
Filing date: 2019-03-25
Publication date: 2021-05-18
Anticipated expiration: 2039-03-25
Also published as: CN109913492A

Abstract

The invention discloses a method for inhibiting geminivirus infection by the synergistic effect of arabidopsis PEPR2 protein and Atpep1 small peptide, in particular to the field of methods for inhibiting geminivirus infection, which specifically comprises the following steps: step one, selecting a plant with geminivirus; step two, the PEPR2 is over-expressed in the plant, and meanwhile, AtPep1 small peptide is applied exogenously to synergistically activate an antiviral mechanism. The invention utilizes the method of the synergistic effect of the plant-derived gene and the small peptide to improve the resistance of the plant to the geminivirus and solve the problem that the geminivirus causes serious harm to the production of crops in China, under the action of the AtPep1 small peptide, the PEPR2 has the regulation and control effect on the C4 protein coded by the BSCTV, and a specific controllable plant disease-resistant method is developed, because the C4 is a pathogenic factor with a conserved sequence in various geminiviruses, the technology can be applied to other geminiviruses to improve the antiviral capability of crops in the future.

Description

Method for inhibiting geminivirus infection through synergistic effect of arabidopsis PEPR2 protein and Atpep1 small peptide

Technical Field

The invention relates to the technical field of methods for inhibiting geminivirus infection, in particular to a method for inhibiting geminivirus infection through the synergistic effect of an arabidopsis PEPR2 protein and an Atpep1 small peptide.

Background

Geminiviruses (geminiviruses) are a class of plant DNA viruses with a broad host range, from which many important crops are threatened. The geminivirus is found in grain and economic crops in several provinces of China successively, which causes harm to agricultural production. Therefore, the deep research on the interaction relationship between the geminivirus and the plants has important significance for preventing and treating diseases, improving the crop yield and ensuring the development of agricultural production.

Geminiviruses have a single-stranded DNA genome, encoding only a few proteins, that complete their life cycle by infecting and interacting with host cells. After geminiviruses enter plant host cells through insect vectors, virus DNA is largely replicated in the nucleus, and then migrates to other parts of plants through intracellular and intercellular movement to form systemic infection, thereby generating diseases. The Beet Severe Curdy Top Virus (BSCTV) is a kind of geminivirus, can infect model plants, namely arabidopsis thaliana and nicotiana benthamiana, and is often used as a representative species for interaction research of plants and geminivirus.

Most geminiviruses encode a conserved C4 protein (called AC4, AL4 or L4 in some species), and C4 has been shown to be a major disease determinant in a variety of geminivirus studies. When the C4 gene of BSCTV is deleted, the virus pathogenicity is obviously reduced; when C4 of BSCTV is exogenously over-expressed, the cell division of the plant can be promoted, callus tissues are formed, and a phenotype similar to virus infection is generated. Existing studies indicate that the C4 protein may be involved in hormone signaling and cell cycle regulation in plants, thereby affecting the normal development of the host. However, the specific regulatory mechanism of the C4 protein in plant cells is not known, and methods for inhibiting viral infection by interfering with C4 function are lacking.

AtPeps small peptide-dependent signaling is an important pathway for plant activation of immune response, but current research on the pathway focuses on fungal and bacterial diseases, and no report is made on the function of the pathway in plant and virus interaction. AtPeps are a class of plant-encoded small peptides produced from the precursor proteins encoded by the eight PROPEP genes, and most studies have used AtPep1 as an example because of the close sequence proximity of these small peptides. AtPeps initiate downstream signal transduction mechanisms by recognizing receptor-like kinases PEPR1 and PEPR2 on the plasma membrane of plant cells. Previous studies have shown that binding of AtPep1 to PEPR1 or PEPR2 can result in the transport of these receptor kinases from the cytoplasmic membrane into the cell in endocytosed form. In the invention, the applicant firstly discovers that the PEPR2 protein of Arabidopsis can promote the endocytosis localization of the C4 protein of BSCTV so as to influence the function of the BSCTV in the determination of plant diseases and further inhibit the geminivirus infection (unpublished).

Prior art relating to the invention

The technical scheme of the prior art I is as follows:

the invention utilizes a method of combining PEPR2 overexpression and AtPep1 small peptide application to inhibit the infection of geminivirus BSCTV by influencing the positioning of a disease condition determining factor C4 in plant cells. Similar technology can be found in the invention patent of a new application of GDU3 gene (CN101775397B) (the specific scheme can be referred to the content of the patent), and the technology also utilizes the gene which is over-expressed and derived from arabidopsis thaliana to resist the infection of BSCTV. According to the invention, the resistance of plants to BSCTV is enhanced mainly by over-expressing the gene LSB1/GDU3 derived from arabidopsis thaliana, and the new application of the gene LSB1/GDU3 can be used for reducing agricultural loss caused by BSCTV.

The first prior art has the following defects:

the main problems of this technique are: the overexpression of the LSB1/GDU3 gene constitutively and continuously activates the disease-resistant pathway of the plant, and the continuous accumulation of disease-resistant signals causes the growth and development of the plant to be influenced under normal conditions; meanwhile, the virus resisting action mechanism of the method is unknown, and other unknown side effects can be caused in the application process. Compared with the technology, the virus resistant channel utilized by the invention needs to be activated when PEPR2 is simultaneously expressed and AtPep1 small peptide is applied, so that the resistance to BSCTV is generated, and therefore, the AtPep1 small peptide can be added under the condition of virus infection risk; meanwhile, the invention discovers a mechanism that the function of the AtPep1 and PEPR2 is influenced by jointly regulating and controlling the positioning of the disease determining factor C4 protein, and the mechanism is a more specific and controllable disease-resistant mode.

In addition, the existing technologies for effectively preventing and treating geminivirus are few, and mainly utilize overexpression or knockdown of plant genes to activate related signal paths so as to improve the resistance to the virus, and destroy the infection process through gene editing aiming at virus DNA; the overexpression and the knockdown of plant genes are mainly realized by constitutively activating a wide disease-resistant pathway such as salicylic acid and the like, or controlling the replication of viruses by influencing the plant cell cycle, so that the disease resistance is improved, and the influence on the growth and development of the plant can be generated; although the gene editing technology aiming at the virus DNA specifically destroys the structural integrity of the virus, the existing research shows that the technology may have off-target phenomenon at present and may influence the genome of the plant.

Disclosure of Invention

In order to overcome the defects of the prior art, the embodiment of the invention provides a method for inhibiting the infection of geminiviruses by the synergistic action of arabidopsis PEPR2 protein and Atpep1 small peptide, which improves the resistance of plants to the geminiviruses by utilizing the synergistic action of plant-derived genes and small peptide, is beneficial to agricultural safe production, solves the problem that the geminiviruses cause serious harm to the production of crops in China, develops a specific controllable plant disease-resistant method under the action of the Atpepr 1 small peptide and the regulation and control action of PEPR2 on C4 protein coded by BSCTV, because C4 is a pathogenic factor with a conserved sequence in various geminiviruses, the technology can be applied to other geminiviruses to improve the antiviral capability of future crops, solves the problems caused by the over-expression and plant gene knock-down method in the prior art to the growth and the possible off-target phenomenon, the influence on the genome of the plant itself.

In order to achieve the purpose, the invention provides the following technical scheme: a method for inhibiting geminivirus infection through synergistic effect of Arabidopsis PEPR2 protein and AtPep1 small peptide specifically comprises the following steps:

step one, the PEPR2 is over-expressed in the plant, and meanwhile, the AtPep1 small peptide is applied exogenously to synergistically activate an antiviral mechanism.

In a preferred embodiment, the PEPR2 gene and the PEPR2 protein are both derived from arabidopsis thaliana.

In a preferred embodiment, the AtPep1 is derived from the elicitor small peptide of arabidopsis thaliana.

In a preferred embodiment, the geminivirus in the first step may be particularly a beet severe aspergillovirus (BSCTV) encoding a pathogenic agent C4, i.e. BSCTV C4.

The invention has the technical effects and advantages that:

1. the invention utilizes the method of the synergistic effect of the plant-derived gene and the small peptide to improve the resistance of the plant to the geminivirus, is beneficial to the agricultural safety production and solves the problem that the geminivirus causes serious harm to the crop production in China;

2. the invention utilizes the regulation and control function of PEPR2 on C4 protein coded by BSCTV under the action of AtPep1 small peptide to develop a specific controllable plant disease-resistant method, because C4 is a pathogenic factor with a conserved sequence in a plurality of geminiviruses, the technology can be applied to other geminiviruses to improve the antiviral capability of future crops, and solves the problems that the plant growth and development are caused by using a method of over-expression and knocking down plant genes in the prior art, and the off-target phenomenon possibly exists to influence the genome of the plant;

3. the invention utilizes plant expression vectors of different types to carry out over-expression on the PEPR2 gene of arabidopsis thaliana, and simultaneously can utilize transgenic plants of arabidopsis thaliana or other host plants of pCang-PEPR2-MYC to improve the resistance of various plants to BSCTV and other geminiviruses by applying AtPep1 small peptide externally;

4. the invention proves that the combined use of the over-expression of PEPR2 and AtPep1 can affect the subcellular localization, pathogenic function and geminivirus infection of pathogenic factor C4 protein of geminivirus BSCTV from various aspects; since the single PEPR2 is overexpressed or the AtPep1 is applied, the virus infection cannot be effectively inhibited, the AtPep1 treatment can be carried out on the plant excessively expressing PEPR2 in a specific time and space, the antiviral capacity of the plant can be improved by applying the exogenous small peptide while the disease-resistant way of the plant is not continuously activated, the defects of other constitutive continuously activated disease-resistant way technologies are avoided, and meanwhile, the C4 protein activity is inhibited by specifically identifying the plant through the AtPep1-PEPR2, the mechanism is clearer, and the influence of other side effects is favorably avoided.

Drawings

FIG. 1 is a graph showing the results of the experiment of the synergistic effect of PEPR2 and AtPep1 in resisting viral infection in example 2 of the present invention.

FIG. 2 is a graph showing the experimental results of PEPR2 and AtPep1 affecting the C4 protein in the induction of plant callus in example 2 of the present invention.

FIG. 3 is a graph showing the results of experiments in which PEPR2 and AtPep1 synergistically affect the infestation of geminivirus BSCTV on plants in example 2 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

the method for inhibiting geminivirus infection through the synergistic effect of the arabidopsis PEPR2 protein and the Atpep1 small peptide shown in the figure 1-2 specifically comprises the following steps:

step one, selecting a plant with geminivirus;

step two, the PEPR2 is over-expressed in the plant, and meanwhile, AtPep1 small peptide is applied exogenously to synergistically activate an antiviral mechanism.

The PEPR2 gene is derived from arabidopsis thaliana, and the gene sequence of the PEPR2 gene at the 3267bp coding region is specifically TTTCAGTCTTGAGCTCTAATCTCAAATGAGGAATCTTGGGTTACTCGAAATTACTCTGCTTTGCTCTCTCTTTGTCTATTTCCGTATAGATTCTGTCTCTAGTTTAAACTCAGATGGTTTGGCTTTACTCTCGCTTCTCAAGCACTTTGATAAAGTCCCACTTGAAGTAGCTTCGACGTGGAAGGAGAACACATCTGAAACCACTCCATGTAATAATAACTGGTTTGGTGTCATTTGTGATCTTTCTGGTAATGTCGTCGAGACCCTTAATTTGTCTGCTTCTGGGCTTTCAGGCCAATTAGGTTCTGAAATTGGGGAGCTTAAGAGCTTGGTCACATTGGATCTCAGTCTTAACAGTTTCTCTGGTTTATTGCCTTCCACTTTAGGAAACTGTACTTCACTTGAGTATTTGGATTTGTCTAACAATGATTTTTCTGGAGAAGTTCCTGATATTTTTGGTAGCTTGCAGAATTTGACGTTTCTGTATCTTGATCGCAATAATCTTAGTGGTCTCATTCCTGCAAGTGTTGGTGGGTTGATAGAGCTCGTAGATCTGAGGATGTCATATAATAACTTGTCTGGTACCATTCCAGAGTTGCTTGGGAACTGTAGTAAGCTGGAATATCTGGCTTTGAACAACAACAAGTTAAATGGTTCTTTGCCAGCAAGTCTCTATCTACTCGAGAATCTTGGTGAGCTATTTGTCAGTAACAACAGCCTTGGAGGGAGGCTTCATTTTGGTTCTAGCAACTGCAAGAAATTGGTTTCTTTAGATCTCTCGTTCAATGATTTCCAAGGCGGTGTTCCACCTGAGATAGGCAACTGCAGTAGCCTTCACTCTTTAGTCATGGTGAAATGCAACTTGACAGGTACAATCCCATCATCAATGGGTATGTTGAGAAAGGTTTCGGTTATTGACCTTTCCGATAATCGTCTCTCGGGGAATATCCCTCAAGAGCTTGGGAACTGCAGCAGCTTGGAAACCTTGAAGCTGAACGACAACCAGCTCCAAGGCGAGATACCACCTGCATTGAGTAAGCTAAAGAAGCTACAAAGCCTGGAGCTTTTTTTTAATAAGCTGTCCGGTGAGATTCCTATTGGCATATGGAAGATTCAGAGTCTGACACAGATGCTCGTTTATAACAACACTCTCACCGGGGAACTACCAGTTGAAGTAACTCAGCTGAAGCACCTTAAGAAGCTTACACTGTTTAACAACGGCTTTTATGGAGATATACCAATGAGTTTAGGCCTGAATCGAAGCTTAGAGGAGGTGGACCTTCTTGGTAACCGTTTTACAGGGGAGATACCACCCCATCTCTGCCATGGACAGAAGTTGAGATTGTTCATCTTGGGTTCTAATCAGCTTCATGGTAAGATACCAGCGTCTATTCGTCAGTGTAAGACCCTTGAGCGAGTCAGACTTGAAGATAACAAACTTTCAGGTGTTCTTCCGGAATTCCCTGAGAGTCTTAGTCTTTCCTATGTGAACCTCGGAAGCAATAGCTTTGAAGGATCCATCCCGCGCAGCTTGGGAAGCTGTAAAAATCTCTTGACTATTGACCTTTCTCAAAACAAACTCACGGGTCTGATACCTCCAGAACTGGGAAATCTGCAAAGCCTTGGACTGTTGAACCTTTCACATAATTATCTGGAAGGTCCTCTGCCATCCCAGCTATCAGGCTGTGCGAGACTTCTGTATTTTGATGTTGGATCCAACTCATTGAACGGTTCTATTCCATCAAGCTTCAGAAGCTGGAAAAGCTTGTCCACTTTAGTTCTCAGTGACAATAATTTTCTAGGAGCTATTCCACAGTTCTTGGCAGAGCTTGACCGACTCTCAGATCTGCGGATAGCTCGAAATGCTTTTGGAGGTAAGATTCCTTCCTCGGTTGGCTTGTTGAAGAGTCTACGCTATGGCTTAGACCTCAGTGCGAACGTATTTACGGGTGAGATTCCAACCACACTGGGGGCTCTTATCAATCTTGAACGTCTCAACATATCCAACAACAAGTTGACAGGGCCTTTATCGGTTCTTCAAAGTCTTAAGTCATTGAATCAAGTTGACGTCTCGTATAATCAGTTCACGGGTCCAATACCCGTAAATCTGTTATCAAATTCTTCAAAGTTTTCTGGAAATCCAGACCTCTGCATTCAAGCTTCTTACTCAGTGAGTGCCATAATCCGCAAAGAGTTTAAATCTTGCAAAGGTCAAGTCAAACTTAGCACGTGGAAGATCGCCCTTATAGCAGCTGGGTCCTCACTATCCGTATTGGCTTTGCTTTTTGCTCTCTTTTTGGTTTTATGCCGGTGCAAAAGAGGAACCAAGACAGAAGATGCTAATATCCTCGCAGAGGAAGGTCTGTCCTTGTTGCTGAACAAAGTTCTAGCAGCCACTGACAATCTAGATGACAAGTACATCATTGGAAGAGGAGCTCATGGAGTTGTTTACAGAGCTTCTTTAGGATCAGGCGAAGAATACGCCGTGAAGAAACTCATCTTTGCGGAACACATTCGCGCAAACCAAAATATGAAGCGGGAGATCGAAACAATCGGGCTAGTCAGGCACAGAAATCTCATTCGGTTAGAAAGATTTTGGATGAGGAAAGAAGATGGCTTAATGCTGTATCAGTACATGCCCAATGGAAGCCTACACGACGTTTTGCACAGAGGTAATCAAGGAGAAGCAGTTCTTGACTGGTCTGCACGGTTCAACATAGCCCTTGGGATTTCACATGGACTGGCGTATTTACATCATGATTGTCATCCACCAATAATTCACCGCGACATCAAACCAGAGAACATACTCATGGACTCGGATATGGAGCCTCACATTGGAGATTTCGGATTGGCTCGGATTCTAGATGACTCAACAGTTTCAACGGCCACTGTTACTGGCACAACTGGGTACATTGCACCAGAAAATGCGTACAAGACGGTGAGGAGCAAGGAATCAGATGTTTACAGTTATGGAGTTGTTTTGCTCGAGCTGGTAACAGGAAAGAGAGCACTGGACAGATCTTTCCCGGAAGATATCAACATTGTGAGCTGGGTCAGATCTGTATTAAGCAGCTACGAGGATGAAGACGATACTGCTGGTCCAATCGTTGATCCAAAACTTGTGGATGAGCTTCTGGATACGAAGCTCAGGGAACAAGCAATCCAAGTCACAGACTTGGCTCTTAGATGTACAGACAAGAGGCCGGAGAACAGACCATCGATGAGAGATGTGGTGAAAGATTTGACTGATTTGGAAAGTTTTGTAAGAAGCACTTCGGGTTCAGTTCACTAG;

the PEPR2 protein is derived from Arabidopsis thaliana, and the protein sequence of the PEPR2 protein at the coding region 1088aa is specifically MRNLGLLEITLLCSLFVYFRIDSVSSLNSDGLALLSLLKHFDKVPLEVASTWKENTSETTPCNNNWFGVICDLSGNVVETLNLSASGLSGQLGSEIGELKSLVTLDLSLNSFSGLLPSTLGNCTSLEYLDLSNNDFSGEVPDIFGSLQNLTFLYLDRNNLSGLIPASVGGLIELVDLRMSYNNLSGTIPELLGNCSKLEYLALNNNKLNGSLPASLYLLENLGELFVSNNSLGGRLHFGSSNCKKLVSLDLSFNDFQGGVPPEIGNCSSLHSLVMVKCNLTGTIPSSMGMLRKVSVIDLSDNRLSGNIPQELGNCSSLETLKLNDNQLQGEIPPALSKLKKLQSLELFFNKLSGEIPIGIWKIQSLTQMLVYNNTLTGELPVEVTQLKHLKKLTLFNNGFYGDIPMSLGLNRSLEEVDLLGNRFTGEIPPHLCHGQKLRLFILGSNQLHGKIPASIRQCKTLERVRLEDNKLSGVLPEFPESLSLSYVNLGSNSFEGSIPRSLGSCKNLLTIDLSQNKLTGLIPPELGNLQSLGLLNLSHNYLEGPLPSQLSGCARLLYFDVGSNSLNGSIPSSFRSWKSLSTLVLSDNNFLGAIPQFLAELDRLSDLRIARNAFGGKIPSSVGLLKSLRYGLDLSANVFTGEIPTTLGALINLERLNISNNKLTGPLSVLQSLKSLNQVDVSYNQFTGPIPVNLLSNSSKFSGNPDLCIQASYSVSAIIRKEFKSCKGQVKLSTWKIALIAAGSSLSVLALLFALFLVLCRCKRGTKTEDANILAEEGLSLLLNKVLAATDNLDDKYIIGRGAHGVVYRASLGSGEEYAVKKLIFAEHIRANQNMKREIETIGLVRHRNLIRLERFWMRKEDGLMLYQYMPNGSLHDVLHRGNQGEAVLDWSARFNIALGISHGLAYLHHDCHPPIIHRDIKPENILMDSDMEPHIGDFGLARILDDSTVSTATVTGTTGYIAPENAYKTVRSKESDVYSYGVVLLELVTGKRALDRSFPEDINIVSWVRSVLSSYEDEDDTAGPIVDPKLVDELLDTKLREQAIQVTDLALRCTDKRPENRPSMRDVVKDLTDLESFVRSTSGSVH;

the AtPep1 is derived from an elicitor small peptide of Arabidopsis, and the protein sequence of the AtPep1 protein at the coding region 23aa is specifically ATKVKAKQRGKEKVSSGRPGQHN;

the geminivirus in the step one can be particularly beet severe kojic virus (BSCTV), and the pathogenic factor coded by the beet severe kojic virus (BSCTV) is C4, namely BSCTV C4;

the gene sequence of the BSCTV C4 gene at a 264bp coding region is specifically ATGAAAATGGGGAACCACATCTGCATGCCCTTATTCAATTCGAAGGAAAAGTCCAGATCCGTAATGCCCGTTACTTCGATCTGCAACATCGAAGTACCAGCAAACAATTCCACTGCAATATTCAGGGAGCTAAATCCAGTTCCGACGTCAAGTCCTACGTCTCAAAGGACGGAGATCACATCGACTGGGGTGAATTTCAGGTCGATGGAAGATCTGCACGCGGAGGTCAACAGACGGCTAATGATGCTGCAGCAGAGGCATTA;

the protein sequence of the BSCTV C4 protein in the coding region 87aa is specifically MKMGNHICMPLFNSKEKSRSVMPVTSICNIEVPANNSTAIFRELNPVPTSSPTSQRTEITSTGVNFRSMEDLHAEVNRRLMMLQQRH.

Experiments prove that under the treatment condition of AtPep1 small peptide, the over-expression PEPR2 can promote the endocytosis of pathogenic factor C4 protein of BSCTV, and the method for synergistically improving the resistance of plants to geminivirus by utilizing PEPR2 and AtPep1 has important significance for agricultural production by inhibiting the infection of virus by influencing the function of C4.

Example 2:

application of PEPR2 and AtPep1 in synergistic effect in resisting virus infection

1) PEPR2 and AtPep1 synergistically influence the localization of BSCTV-encoded C4 protein in plant cells

At the early stage, the fact that the PEPR2 protein encoded by Arabidopsis thaliana can interact with the C4 protein of BSCTV (which is not published at present) is found through yeast two-hybrid screening, so that the influence of the PEPR2 and the corresponding small peptide AtPep1 on the positioning of the C4 protein in plant cells is analyzed, and the specific operation steps are as follows:

s101, using Arabidopsis cDNA as a template, and carrying out PCR (the information of the used primers is shown as the following: PEPR2-F primer: 5'-ACTACTAGTTTTCAGTCTCTTGAGCTCTAATCT-3', because the 5 'end of a PEPR2 coding region has a high-order structure, the primers are designed in a 5' non-coding region adjacent to an initiation codon; PEPR2-R primer: 5'-AGTGTCGACAGTGAACTGAACCCGAAGTGCTTC-3'), so that the gene sequence of the amplified PEPR2 is the gene sequence at the 3267bp of the coding region;

s102, cloning the PEPR2 gene to pCang-MYC (MYC label is added on the basis of pCambia1300 vector, the vector is kanamycin-resistant in plants; similar construction can be carried out by using other commercial plant overexpression vectors), and fusing the vector with MYC protein label to construct a recombinant expression vector pCang-PEPR 2-MYC;

s103, at the same time, BSCTV (ATCC PVMC-6; previously called BCTV-CFH strain, the strain stores the linearized BSCTV double-stranded DNA on a plasmid pCFH) is used as a template, a PCR method (the used primer information is as follows: C4-GFP-F primer: 5'-AGTCCATGGTGATGAAAATGGGGAACCAC-3'; C4-GFP-R primer: 5'-AGTAAGCTTGATGCCTCTGCTGCAGCATCATTAG-3') is used for amplifying the C4 gene of the BSCTV, and the BSCTV is connected to pCambia1300-GFP (GFP, CAMBIA, Canberra, Australia is are added on the basis of pCambia1300 vector; other commercial vectors with fluorescent tags can be adopted for similar construction), so as to obtain a recombinant expression vector pCambia 1300-C4-GFP;

s104, respectively transforming the two recombinant plasmids into agrobacterium EHA105 to obtain recombinant bacteria;

s105, co-over-expressing C4-GFP and PEPR2-MYC in Nicotiana benthamiana leaves of the Nicotiana benthamiana by using the cultured recombinant bacteria through an injection method; C4-GFP and pCang-MYC empty vector Agrobacterium were co-injected as control samples;

s106, collecting tobacco leaves 48 hours after the agrobacterium is injected, and respectively injecting AtPep1 small peptide (the amino acid sequence is ATKVKAKQRGKEKVSSGRPGQHN) with the final concentration of 100 nM;

s107, detecting a GFP fluorescent signal by using a laser confocal microscope, and analyzing the positioning of the C4-GFP in the tobacco cells;

the localization of C4-GFP in the empty vector control sample on the tobacco plasma membrane was not altered in the case of S108, AtPep1 small peptide treatment for 0 hours and 30 minutes (see figure 1A); in the sample of the over-expression PEPR2-MYC, the localization of C4-GFP was slightly changed without treating AtPep 1; when treated with AtPep1 for 30 min, C4-GFP produced significant endocytosis (see FIG. 1B).

In FIGS. 1A and 1B, the upper left corner of each of the four graphs is the GFP fluorescence signal, the upper right corner is the chlorophyll autofluorescence control, the lower left corner is the bright field cell profile, and the lower right corner is the signal overlay.

The above results indicate that the synergistic effect of PEPR2 and AtPep1 promotes the change in the subcellular localization of C4 protein, which may affect the function of C4 in geminivirus infection, since previous studies indicate that the localization of C4 on the plasma membrane is critical to its pathogenicity.

2) PEPR2 and AtPep1 affect the ability of C4 protein in plant callus induction

Since previous studies showed that the constitutive expression of the C4 gene causes arabidopsis thaliana to form callus and transgenic plants cannot be obtained, earlier studies have obtained a pet 8-C4 arabidopsis thaliana plant by constructing C4 in an estrogen-inducible expression vector pet 8 and by genetic transformation (the seed is from the laboratory of the seqi researchers of the institute of genetics and developmental biology, academy of china academy of sciences, and can be used for measuring the inhibitory effect of the technology used in the present invention on the BSCTV disease determinant C4);

under normal culture conditions, the plant can grow normally; when estrogen is applied to induce the expression of the C4 gene, the plant forms callus; thus, C4 protein activity can be analyzed on the basis of the ability to induce callus;

to investigate the effect of PEPR2 and AtPep1 on C4 function, specific study steps were as follows:

s101, transforming the plasmids of the pCang-PEPR2-MYC and pCang-MYC empty vectors (control) into agrobacterium EHA105 to obtain recombinant bacteria;

s102, transforming the cultured recombinant bacteria into homozygote plants of pER8-C4 by a flower drop dyeing method, respectively obtaining homozygote offspring of the plants of pCang-PEPR2-MYC/pER8-C4 and pCang-MYC/pER8-C4 through selfing, identifying the expression quantity of PEPR2, and selecting 2 independent PEPR2 overexpression Arabidopsis strains for subsequent analysis;

s103, spreading the seeds of the plants on an MS culture medium (without or with the AtPep1 small peptide with the final concentration of 100 nM) containing 2 mu M estrogen for germination, and analyzing the plant phenotype 1 week after germination;

s104, under the condition of not containing the small AtPep1 peptide, compared with the empty vector of pCang-MYC, the influence of over-expression of PEPR2-MYC on the function of C4 is little; the capacity of C4-produced calli was completely inhibited by the addition of 100nM of the above AtPep1 small peptide (see FIG. 2A), FIG. 2A is a photograph of the plant taken 1 week after germination;

s105, when the plants grow for 3 weeks on the corresponding culture media, comparing under a microscope: in the absence of AtPep1, pCang-PEPR2-MYC slightly inhibited the formation of C4 calli (calli were smaller in volume); the function of C4 in inducing callus formation was completely abolished by adding 100nM of the above AtPep1 small peptide (see FIG. 2B), FIG. 2B is a photograph of the plant taken 3 weeks after germination.

The above results indicate that the application of small peptides of AtPep1 inhibited the ability of C4 protein to induce callus (disease decision) in the case of over-expression of PEPR 2.

3) PEPR2 and AtPep1 synergistically affect infection of geminivirus BSCTV to plants

Since the above results indicate that the simultaneous over-expression of PEPR2 and the application of AtPep1 can inhibit the function of C4, and C4 is a pathogenic factor in the BSCTV infection process, the influence of PEPR2 and AtPep1 on BSCTV infection is analyzed, and the specific operation steps are as follows:

s101, transforming infectious plasmids (pCambia 1300-BSCTV1.8copy; from the institute of genetics and developmental biology, Xian investigator laboratory) of BSCTV into an agrobacterium EHA105 strain to obtain recombinant agrobacterium EHA 105-BSCTV;

s102, simultaneously, the plasmids pCang-PEPR2-MYC and pCang-MYC (control) are also transformed into agrobacterium EHA105 respectively;

s103, respectively suspending the agrobacterium tumefaciens in 10mM MgCl2 solution, and respectively mixing the bacterial solutions of pCang-MYC and pCang-PEPR2-MYC with EHA105-BSCTV bacterial solution to obtain two bacterial solutions, namely bacterial solution 1: pCanG-PEPR2-MYC (concentration OD600 ═ 1)/BSCTV (concentration OD600 ═ 0.01) and bacterial solution 2: pCanG-MYC (concentration OD600 ═ 1)/BSCTV (concentration OD600 ═ 0.01);

s104, after standing for 3 hours, injecting the bacterial liquid into the Nicotiana benthamiana leaves by an agrobacterium injection method;

s105, in the treatment of adding the AtPep1, the AtPep1 is diluted in a10 mM MgCl2 solution and injected into the tobacco lamina by the same method;

s106, observing the leaf rolling and twisting symptoms of the stem tip after the tobacco continues to grow for two weeks;

s107, the control plant (pCang-MYC, without AtPep1) has serious curved top symptom; overexpression of PEPR2 alone or application of AtPep1 alone was not sufficient to inhibit BSCTV infection; meanwhile, the tobacco excessively expressing PEPR2 in the leaf blades and applied with AtPep1 has no obvious symptoms (see figures 3A and 3B);

s108, collecting stem apex tissues of the treated plants, extracting DNA by a CTAB method, amplifying a C4 gene (C4 gene is used as a representative fragment for detecting a BSCTV genome) by PCR by using the C4 primer to detect the accumulation condition of the BSCTV at the top of the plants, and an ACTIN gene of tobacco is used as an internal reference, so that the accumulation of the DNA of the BSCTV is obviously reduced in a sample which is subjected to over-expression of PEPR2 and synergistic treatment of AtPep1 compared with other samples (see the attached figure 3C).

The experimental results show that the method enhances the resistance of the plant to the geminivirus.

The technical scheme proves that the combined use of the over-expression of PEPR2 and AtPep1 can influence the subcellular localization, pathogenic function and geminivirus infection of pathogenic factor C4 protein of geminivirus BSCTV from various aspects;

due to the fact that single PEPR2 is overexpressed or AtPep1 is applied, virus infection cannot be effectively inhibited, the AtPep1 treatment can be carried out on plants with the PEPR2 overexpressed in specific time and space, the antiviral capacity of the plants is improved through the application of exogenous small peptides while the disease-resistant ways of the plants are not continuously activated, and the defects of other constitutive continuously activated disease-resistant way technologies are overcome;

meanwhile, the technical scheme specifically recognizes through AtPep1-PEPR2 to inhibit the activity of the C4 protein, so that the mechanism is clearer, and the influence of other side effects is avoided;

because the C4 protein sequence has conservation among various geminiviruses, the technical scheme can also have effect on controlling diseases of other types of geminiviruses.

In the technical scheme, different types of plant expression vectors are utilized to carry out over-expression on the PEPR2 gene of the arabidopsis thaliana, so that the effect is possible, meanwhile, transgenic plants of the arabidopsis thaliana of pCang-PEPR2-MYC or other host plants can be utilized, and the resistance of various plants to BSCTV and other geminiviruses is improved by applying AtPep1 small peptide externally;

in addition, the above method can be used for preventing plants which are not diseased.

And finally: the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.

Claims

1. A method for inhibiting geminivirus infection by the synergistic effect of Arabidopsis PEPR2 protein and AtPep1 small peptide is characterized in that: the method specifically comprises the following steps:

step one, selecting a plant with geminivirus;

2. The method for inhibiting geminivirus infection through synergistic effect of arabidopsis PEPR2 protein and Atpep1 small peptide according to claim 1, wherein the method comprises the following steps: the PEPR2 gene and the PEPR2 protein are both derived from arabidopsis thaliana.

3. The method for inhibiting geminivirus infection through synergistic effect of arabidopsis PEPR2 protein and Atpep1 small peptide according to claim 1, wherein the method comprises the following steps: the AtPep1 is derived from elicitor small peptide of Arabidopsis thaliana.

4. The method for inhibiting geminivirus infection through synergistic effect of arabidopsis PEPR2 protein and Atpep1 small peptide according to claim 1, wherein the method comprises the following steps: the geminivirus in the first step can be beet severe kojic virus (BSCTV), and the pathogenic factor coded by the beet severe kojic virus (BSCTV) is C4, namely BSCTV C4.