CN107083396B

CN107083396B - Application of ZmPDIL gene in prevention and treatment of maize dwarf mosaic disease

Info

Publication number: CN107083396B
Application number: CN201710343961.6A
Authority: CN
Inventors: 周涛; 陈晖�; 谢吉鹏; 袁雯; 范在丰
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2017-05-16
Filing date: 2017-05-16
Publication date: 2020-06-23
Anticipated expiration: 2037-05-16
Also published as: CN107083396A

Abstract

The invention relates to application of ZmPDIL gene in prevention and treatment of maize dwarf mosaic disease, which utilizes a gene engineering method to improve the disease resistance of crops to maize dwarf mosaic disease by inhibiting the expression of ZmPDIL gene in maize. The invention discovers for the first time that silencing ZmPDIL gene can cause the SCMV replication and proliferation level to be obviously reduced, and shows that zein ZmPDIL and the coding gene thereof play an important role in SCMV infection and proliferation, are SCMV-resistant recessive antiviral genes coded by corns, and can be used for SCMV-resistant molecular breeding and other works of the corns. The invention provides basis for researching the function of plant protein in virus infection, corn antiviral breeding and other fields, improves the antiviral research level of corn in China, and promotes and ensures the production safety of corn in China.

Description

Application of ZmPDIL gene in prevention and treatment of maize dwarf mosaic disease

Technical Field

The invention relates to the technical field of biotechnology and crop disease control, in particular to application of ZmPDIL gene in maize dwarf mosaic disease control.

Background

Corn is a main food crop in China, but corn dwarf mosaic disease (MDMD) caused by SCMV (sugarcane mosaic virus) infection seriously threatens high and stable yield of corn in China. SCMV is a main pathogen causing maize dwarf mosaic disease in northern maize production areas in China, and usually causes more than 30 percent of yield loss of maize. Due to the emergence of SCMV-virulent strains, some of the original antiviral varieties lose resistance, resulting in greater harm in production. There is a great need to identify new disease-resistant genes or resistant materials so that SCMV and its hazards can be controlled continuously and more effectively, ensuring corn production safety.

Plant viruses, as an obligate parasite in cells, must rely on and utilize host factors to complete the life processes of virus uncoating, genome replication, protein expression, virus particle assembly and movement to neighboring cells during infection and propagation. Therefore, the identification of host factors involved in the infection and propagation processes of plant viruses can help us to deeply understand the infection and pathogenic mechanism of the viruses, thereby providing new materials and theoretical basis for the prevention and control of the virus diseases. In the post-genome era, various omics technologies are widely applied to interaction research between hosts and biotic stress or abiotic stress, and become one of the main means for searching virus pathogenic mechanisms and disease-resistant genes.

Disclosure of Invention

The invention aims to provide application of ZmPDIL gene in prevention and treatment of maize dwarf mosaic disease.

It is another object of the present invention to provide a method for inhibiting replication and proliferation of sugarcane mosaic virus in maize.

According to the invention, isotope labeling relative and absolute quantitative (iTRAQ) proteomics technology is utilized to screen proteins which are obviously and differentially expressed in a first upper leaf and a second upper leaf of a system infected leaf after SCMV infects corns, and a protein ZmPDIL which is obviously and differentially expressed in the two leaves is identified.

In order to realize the purpose, the invention provides the application of the ZmPDIL gene in the maize dwarf mosaic disease control, which utilizes a genetic engineering method to improve the disease resistance of crops to the maize dwarf mosaic disease by inhibiting the expression of the ZmPDIL gene in maize.

The maize dwarf mosaic disease refers to maize dwarf mosaic disease caused by SCMV infection.

In the present invention, the ZmPDIL gene is from maize, and the ZmPDIL gene is:

i) a nucleotide sequence shown as SEQ ID NO. 2; or

ii) the nucleotide sequence as shown in position 225-1766 of SEQ ID NO 2; or

iii) the nucleotide sequence shown in positions 225 and 1769 of SEQ ID NO 2; or

iv) a nucleotide sequence which hybridizes with any one of the sequences i) to iii) under stringent conditions of hybridizing at 65 ℃ in a 0.1 × SSPE containing 0.1% SDS or a 0.1 × SSC solution containing 0.1% SDS, and washing the membrane with the solution, and expressing the same functional protein, or

v) a nucleotide sequence which has more than 90% homology with any one of the sequences i) to iii) and expresses the same functional protein.

The protein encoded by the ZmPDIL gene is:

a) 1, SEQ ID NO; or

b) 1, and the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1.

The aforementioned use comprises introducing into maize a substance for inhibiting the expression of the ZmPDIL gene.

In the present invention, the substance for inhibiting the expression of ZmPDIL gene includes but is not limited to shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA.

Preferably, the substance for inhibiting the expression of the ZmPDIL gene is RNA encoded by the nucleotide sequence shown in 1898-2121 of SEQ ID NO. 2.

More preferably, the substance for inhibiting the expression of ZmPDIL gene is a recombinant brome mosaic virus carrying RNA encoded by the nucleotide sequence shown in SEQ ID NO:2, 1898-2121.

When the substance for inhibiting the ZmPDIL gene expression is a nucleic acid molecule, a vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA fragment encoding the nucleic acid molecule belongs to the protection scope of the invention.

Expression vectors carrying the desired fragment can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant viral vectors, direct DNA transformation, microinjection, electroporation and the like (Weissbach, 1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2)^ndEdition)。

The invention also provides a method for inhibiting the replication and proliferation of the sugarcane mosaic virus in corn, which is to introduce a substance for inhibiting the expression of ZmPDIL gene into the corn so as to inhibit the replication and proliferation of the sugarcane mosaic virus in the corn.

The invention also provides a medicament or a composition for preventing and treating maize dwarf mosaic disease, and the active component of the medicament or the composition is a substance for inhibiting ZmPDIL gene expression.

The invention further provides a medicament or a composition for inhibiting the replication and proliferation of the sugarcane mosaic virus in corn, wherein the active component of the medicament or the composition is a substance for inhibiting the expression of ZmPDIL gene.

In a preferred embodiment of the invention, the specific method for inhibiting the expression of the ZmPDIL gene in maize is as follows:

1. total RNA from maize inbred line Va35 was extracted and reverse transcribed to cDNA.

2. And (3) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and using a primer pair consisting of ZmPDIL-shaping-F and ZmPDIL-shaping-R to obtain a PCR amplification product.

ZmPDIL-silencing-F：5'-ATCCTAGGGGATCTGCGACGTGTT-3'

ZmPDIL-silencing-R：5'-TACCATGGTGCTGGTGGACACGTCAC-3'

3. The PCR amplification product of step 2 was digested with restriction enzymes Nco I and Avr II, and the digested product was recovered.

4. The vector pC13/F3-13m was digested with restriction enzymes Nco I and AvrII, and a vector backbone of about 5000bp was recovered.

5. And (4) connecting the enzyme digestion product in the step (3) with the vector skeleton in the step (4) to obtain a recombinant vector pC13/F3-13 m-PDIL.

6. The recombinant vector pC13/F3-13m-PDIL and the vector pC13/F1+2 were co-transfected into a leaf of nicotiana benthamiana, and cultured to obtain a leaf containing virions of recombinant Brome Mosaic Virus (BMV).

7. Purifying the recombinant BMV virus particles from the tobacco leaves of step 6.

8. In the two-leaf stage, a Va35 maize seedling which grows well and has 2 nd leaf (first true leaf) basically and completely unfolded is selected, a little carborundum is scattered on the 2 nd leaf, friction inoculation is carried out by purified recombinant BMV virus particles, and then ZmPDIL genes are silenced transiently.

The administration of SCMV after transient silencing of the ZmPDIL gene in maize leaves significantly inhibited SCMV accumulation after silencing of the ZmPDIL gene (as shown by significant reduction in SCMV genomic RNA levels and protein levels of CP), although the time of onset was not significantly different from that of SCMV administration and infection symptoms in maize leaves without silencing of the ZmPDIL gene. It is presumed that ZmPDIL may be an essential host factor in SCMV infection process, is recruited by virus in the infection process, participates in SCMV replication and proliferation process, and is a recessive anti-SCMV resistance gene coded by corn. The ZmPDIL gene may play an important role in controlling maize dwarf leaf disease caused by SCMV.

According to the invention, gene function tests and verification are firstly passed, and the silencing of ZmPDIL gene leads to the obvious reduction of SCMV replication and proliferation level, so that the zein ZmPDIL and the coding gene thereof play an important role in SCMV infection and proliferation, are SCMV-resistant recessive antiviral genes coded by corns, and can be used for SCMV-resistant molecular breeding and other works of the corns. The invention provides basis for researching the function of plant protein in virus infection, corn antiviral breeding and other fields, improves the antiviral research level of corn in China, and promotes and ensures the production safety of corn in China.

Drawings

FIG. 1 is a representation of the infection phenotype of SCMV in silencing ZmPDIL maize plants in example 3 of the present invention; wherein BMV-PDIL is ZmPDIL gene silencing plant, BMV-GFP is contrast.

FIG. 2 is a graph of the inhibition of SCMV replication and proliferation in maize plants by silencing the ZmPDIL gene in example 3 of the present invention; wherein (a) the relative accumulation of ZmPDIL in systemically infected leaves of the control group (BMV-GFP) and the treated group (BMV-PDIL) is 7 days and 14 days after SCMV vaccination; (b) relative accumulation of SCMV genomic RNA; (c) SCMV CP relative accumulation; (d) and (5) statistically analyzing the relative accumulation amount of the SCMV CP by using ImageJ software.

CBB in FIG. 2(c) represents Coomassie brilliant blue staining; the accumulation amount of the control group was set to 1, and 0.589 and 0.638 represent the accumulation amount of the protein in the treated group relative to the control group, respectively.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The biomaterials used in the present invention are as follows:

maize inbred line Va 35: see Ding XS, Schneider WL, chalivadi SR, ethyl. characteristics of a bromine molar virus strain and its as a vector for gene drawing in monoclonal antibodies hosts, mol Plant-Microbe interaction, 2006, 19: 1229-1239.

This cigarette produces: see Goodin MM, Zaitlin D, Naidu RA et al, Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions. mol plant-Microbe interaction, 2008,21(8): 1015. 1026).

Vector pC13/F1+ 2: see Zhu M, Chen Y, Ding XS, et al, size Elongin circuits with the viral genome-linked protein, VPg, of Surgane molar vision and defects viruses introduction, New Phytologist,2014,203: 1291-1304.

Vector pC13/F3-13 m-GFP: see Zhu M, Chen Y, Ding XS, et al, size Elongin circuits with the viral genome-linked protein, VPg, of Surgane molar vision and defects viruses introduction, New Phytologist,2014,203: 1291-1304.

Vector pC13/F3-13 m: see Zhu M, Chen Y, Ding XS, et al, size Elongin circuits with the viral genome-linked protein, VPg, of Surgane molar vision and defects viruses introduction, New Phytologist,2014,203: 1291-1304.

After the vector pC13/F1+2 and the vector pC13/F3-13m were co-transfected into tobacco, virus particles of Brome Mosaic Virus (BMV) were expressed. The virus particle of the brome mosaic virus consists of three RNA chains, a vector pC13/F1+2 contains RNA1 and RNA2, a vector pC13/F3-13m contains RNA3, and the two vectors are used for co-transfecting tobacco and then expanding and propagating the recombinant virus particle of BMV.

Sugarcane mosaic virus (SCMV): see Fan ZF, Chen HY, Liang XM, et al, comparative sequence of the genomic RNA of the predictive string of a type of virus infection in China, Arch Virol,2003,148: 773-.

Example 1 discovery of ZmPDIL protein and Gene encoding the same

The method comprises the steps of screening proteins which are significantly and differentially expressed in a first upper leaf and a second upper leaf of a system infected by SCMV (corn blast cell virus) after the SCMV infects corn by utilizing isotope labeling relative and absolute quantitative (iTRAQ) proteomics technology and method, and identifying a protein ZmPDIL which is significantly and differentially expressed in the two leaves, wherein the sequence is shown as SEQ ID NO: 1. The gene for coding ZmPDIL protein is named as ZmPDIL gene, and the sequence is shown as SEQ ID NO. 2.

Example 2 construction of recombinant plasmid pC13/F3-13m-PDIL

ZmPDIL-silencing-F：5'-ATCCTAGGGGATCTGCGACGTGTT-3'

ZmPDIL-silencing-R：5'-TACCATGGTGCTGGTGGACACGTCAC-3'

4. The vector pC13/F3-13m was digested with restriction enzymes Nco I and Avr II, and a vector backbone of about 5000bp was recovered.

5. And (4) connecting the enzyme digestion product in the step (3) with the vector skeleton in the step (4) to obtain a recombinant vector pC13/F3-13 m-PDIL. According to the sequencing result, the recombinant vector pC13/F3-13m-PDIL is structurally described as follows: double-stranded DNA molecules shown in 1898-2121 of SEQ ID NO 2 were inserted into the restriction sites of Nco I and Avr II of the vector pC13/F3-13 m.

The transient silencing vector of ZmPDIL gene consists of recombinant vector pC13/F3-13m-PDIL and vector pC13/F1+ 2.

Example 3 transient silencing experiment of ZmPDIL Gene

Viral particles for expanding BMV in Bunsen tobacco

1. Infiltrating the raw tobacco with agrobacterium carrying BMV vector

(1) Transforming the pC13/F3-13m-PDIL vector with correct sequencing into an agrobacterium tumefaciens C58C1 competent cell; meanwhile, the Agrobacterium strains carrying pC13/F1+2 and pC13/F3-13m-GFP, which were stored at-80 ℃ were streaked on LB plates (containing 50. mu.g/ml Kan, 100. mu.g/ml Rif), and cultured at 28 ℃ for about 48 hours. Picking a single colony on an LB plate, inoculating the single colony in 3-4 ml of LB liquid culture medium (containing 50 mu g/ml Kan and 100 mu g/ml Rif), and culturing at 28 ℃ and 180rpm for 14-18 h;

(2) 200-300 mul of bacterial liquid is taken according to the proportion of 1: 100 and transferred into 20-30 ml of LB liquid culture medium (containing 50 mu g/ml Kan and 100 mu g/ml Rif), and cultured for 10-12 h at 28 ℃ and 180 rpm;

(3) centrifuging at 4500rpm for 8min at room temperature, collecting thallus, suspending the precipitate with 20ml infiltration buffer (10mM MES, pH5.7, 10mM MgCl2, 200 μ M acetosyringone), measuring the concentration of the bacterial suspension with spectrophotometer, and adjusting the concentration of the bacterial suspension with infiltration buffer to OD₆₀₀＝2.0。

(4) Respectively mixing bacterial suspension carrying pC13/F1+2 with bacterial suspension carrying pC13/F3-13m-PDIL and pC13/F3-13m-GFP in equal volume, and standing at room temperature for 3 h. The lower epidermis of the lamina of the raw tobacco was infiltrated with a 1ml syringe without a needle.

(5) Culturing the infiltrated natural tobacco in a 22-24 ℃ artificial climate chamber for 3 days, then sampling, wrapping the sample with tinfoil paper, quickly freezing the wrapped sample in liquid nitrogen, and storing the wrapped sample at-80 ℃ for later use.

2. Detection of ZmPDIL inserts

Taking about 0.1g of agrobacterium-infiltrated natural tobacco leaves, extracting total RNA of the tobacco leaves by a TRIzol method, carrying out reverse transcription by an Oligo dT (5'-TTTTTTTTTTTTTTTTTT-3') primer, carrying out PCR amplification by using a reverse transcription product cDNA as a template and a primer XSD198/199(XSD 198: 5'-CTTGTGTTGCTGAGAAAC-3'; XSD 199: 5'-TCTTGTAAGAGGTCTGC-3'), carrying out electrophoretic analysis on a PCR product, and detecting whether an exogenous insert carried by a recombinant BMV virus in the agrobacterium-infiltrated natural tobacco leaves is lost or not by using pC13/F3-13m empty vector plasmid as a control. Grinding tobacco leaves carrying BMV recombinant virus with complete exogenous insert in liquid nitrogen, grinding into powder, and subpackaging into 2ml centrifuge tubes (about 0.4g per tube), and storing at-80 deg.C for use.

3. Crude purification of BMV virions

(1) Taking a proper amount of tobacco leaf sample powder (determined according to the number of maize plants to be inoculated) stored at-80 ℃, adding 800 mu l of BMV extraction buffer solution into each tube, uniformly mixing, and then incubating on ice for 20-30 min;

(2) centrifuging at 4 deg.C and 8000g for 10min, collecting supernatant, adding 40% PEG/NaCl solution with corresponding volume according to the ratio of adding 46 μ l 40% PEG/NaCl solution into each 200 μ l supernatant, mixing, placing on ice, and incubating at low speed on shaking table for 1 hr;

(3) centrifuging at 4 ℃ for 15min at 13000g, discarding the supernatant, and suspending and precipitating with 100 mul BMV storage buffer solution for each tube to obtain the roughly purified BMV virus particles for subsequent friction inoculation experiments.

4. Virion inoculation of Va35 maize and challenge inoculation of SCMV

Using NanoDrop^TMDetecting the concentration, OD, of BMV virions with a spectrophotometer₂₆₀The value of (d) is approximately equal to the concentration of virus particles (. mu.g/. mu.l). In the two-leaf stage, good-growing and 2 nd (first true) leaf substantially completely developed Va35 maize seedlings were selected, a little carborundum was scattered on the 2 nd leaf, and each leaf was frictionally inoculated with 20. mu.g of roughly purified BMV virus particles. BMV-PDIL and BMV-GFP (as a control) inoculated the same number of plants. Placing the inoculated corn plant in a transparent plastic cover, culturing in a lighting incubator at 18-20 ℃ for 6 days, removing the plastic cover, challenging and inoculating SCMV (Single leaf Virus) on a second leaf (first true leaf), placing the inoculated plant in the lighting incubator at 24 ℃/22 ℃ or a phytotron for continuous culture, and detecting the silencing efficiency of the target gene and the corresponding silencing efficiency of the target gene 7 days and 14 days after SCMV inoculationAccumulation of SCMV RNA and CP (coat protein).

II, transient silencing of ZmPDIL Gene reduces SCMV replication and accumulation levels

1. Virion inoculation of Va35 maize and challenge inoculation of SCMV

Using NanoDrop^TMDetecting the concentration, OD, of BMV virions with a spectrophotometer₂₆₀The value of (d) is approximately equal to the concentration of virus particles (. mu.g/. mu.l). In the two-leaf stage, good-growing and 2 nd (first true) leaf substantially completely developed Va35 maize seedlings were selected, a little carborundum was scattered on the 2 nd leaf, and each leaf was frictionally inoculated with 20. mu.g of roughly purified BMV virus particles. BMV-PDIL and BMV-GFP (as a control) inoculated the same number of plants. Placing the inoculated corn plant in a transparent plastic cover, culturing in a lighting incubator at 18-20 ℃ for 6 days, removing the plastic cover, challenging and inoculating SCMV (Single leaf virus) in a second leaf (first true leaf), placing the inoculated plant in the lighting incubator at 24 ℃/22 ℃ or a climatic chamber for continuous culture, and detecting the silencing efficiency of a target gene and the corresponding accumulation amount of SCMV RNA and CP 7 days and 14 days after SCMV inoculation respectively.

2. And on the 7 th day and the 14 th day after SCMV inoculation, a second system on the upper part is taken to infect the leaf, total RNA is extracted and is reversely transcribed into cDNA, the cDNA is taken as a template, a Kangji century fluorescence quantitative PCR reagent is adopted, a Ubiquitin gene is taken as an internal reference gene, RT-qPCR is used for identifying the relative expression quantity of the ZmPDIL gene and the content of SCMV genome RNA, and the Ubiquitin gene is taken as the internal reference gene.

Primer pairs for identifying the ZmPDIL gene were as follows:

ZmPDIL-qRT-F：5'-TGACGTTCCCAGTGAGTTTGA-3'

ZmPDIL-qRT-R：5'-GGTCTCCTTGCTCTTCTTGATG-3'

the primer pair for identifying the Ubiquitin gene is as follows:

Ubi-qRT-F：5'-GGAAAAACCATAACCCTGGA-3'

Ubi-qRT-R：5'-ATATGGAGAGAGGGCACCAG-3'

primer pairs for the identification of SCMV were as follows:

SCMV-CP-qRT-F：5'-GGCGAGACTCAGGAGAATACA-3'

SCMV-CP-qRT-R：5'-ACACGCTACACCAGAAGACACT-3'

data obtained 2^-△△CTThe method is used for analysis and comprises the following steps:

first, for the test group sample (test) and the control group sample (calibretor), C of the reference gene (ref) was used_TC of value-normalized target Gene (target)_TThe value:

△C_T(test)＝C_{T(target,test)}-C_T(ref,test)

△C_{T(calibrator)}＝C_{T(target,calibrator)}-C_{T(ref,calibrator)}

next, △ C of the control group sample was used_T△ C of value normalized experimental group sample_TThe value:

△△C_T＝△C_T(test)-△C_{T(calibrator)}

finally, expression level ratios were calculated:

2^-△△CTratio of expression amount

The relative content of SCMV genomic RNA is shown in FIG. 2(b), and the relative expression level of ZmPDIL gene is shown in FIG. 2 (a).

The results show that the accumulation of ZmPDIL in the experimental group is respectively reduced to about 15% and 40% and the accumulation of SCMV genomic RNA is about 80% and 70% compared with the control group 7 days and 14 days after SCMV inoculation.

5. Western blot analysis was performed on

day

7 and 14 after SCMV inoculation by infecting leaves with the second upper disc system and extracting total protein using SCMCPP antiserum as primary antibody and AP-labeled goat anti-rabbit as secondary antibody, as shown in FIG. 2(c) and FIG. 2 (d). The results show that the accumulation of SCMV CP is significantly reduced to about 0.8 times that of the control group on plants with ZmPDIL silencing efficiency of about 15% and 40%, respectively.

The results show that the SCMV is inoculated after the ZmPDIL gene is transiently silenced in the corn leaves, although the morbidity time of the SCMV is not obviously different from the morbidity time and infection symptoms of the SCMV inoculated on the corn leaves without the ZmPDIL gene (figure 1), the SCMV replication and proliferation level is remarkably reduced due to the silencing of the ZmPDIL gene (figure 2), and the corn protein ZmPDIL and the coding gene thereof play an important role in SCMV infection and proliferation, are SCMV-resistant recessive antiviral genes coded by the corn, and can be used for molecular breeding and other works of SCMV resistance of the corn.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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agagcgacga ggaagtcgtg attgctaaga tggacgcgac tgccaatgac gttcccagtg 1560

agtttgacgt ccaaggctac ccgaccttgt acttcgtgac cccgagcggg aaggtgacat 1620

cctacgacag cggcaggacg gcggacgaca tcgtcgactt catcaagaag agcaaggaga 1680

ccgcaggcgc cgccaccacc accaccaccc aggcgccacc ggcatccgag aaggcagccg 1740

ccgccgagcc cgtcaaggac gagctgtgag catccaaggt gtggacggct gtggctgcgt 1800

aagcatggga ggaaaacctg gcgctaacgt gcctccaacg cctagttttt gcgggaccag 1860

agggagagag agagagggac ggagttagag agagagggga tctgcgacgt gttttgctct 1920

gcggcggcat gtctgtgtat cccattgagg ttatgtacac aattacaccg cggtcggtgg 1980

gtgtttttta gttaattatc acaagtgaca gtagaaagga acagattgtg atgaggttga 2040

atggatgaat aaatcgctag tccgccccgc ctcgttggat gcgtcaatga aacaggcgct 2100

tttgtgacgt gtccaccagc actaacgctg gaagcaaact cgaacggaga 2150

Claims

The application of the ZmPDIL gene in the prevention and treatment of maize dwarf mosaic disease is characterized in that the disease resistance of crops to the maize dwarf mosaic disease is improved by inhibiting the expression of the ZmPDIL gene in maize by utilizing a genetic engineering method;

wherein, the nucleotide sequence of the ZmPDIL gene is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein a substance for inhibiting the expression of the ZmPDIL gene is introduced into maize.
3. The use according to claim 2, wherein the substance for inhibiting the expression of the ZmPDIL gene comprises shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA.
4. The use as claimed in claim 2 or 3, wherein the substance for inhibiting the expression of ZmPDIL gene is RNA encoded by the nucleotide sequence shown in SEQ ID NO 2, 1898-2121.
5. A method for inhibiting replication and proliferation of sugarcane mosaic virus in corn, characterized in that a substance for inhibiting ZmPDIL gene expression is introduced into corn, thereby inhibiting replication and proliferation of sugarcane mosaic virus in corn; wherein ZmPDIL gene is defined as described in claim 1.
6. The method of claim 5, wherein the substance for inhibiting the expression of the ZmPDIL gene comprises shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA.
7. The method as claimed in claim 5 or 6, wherein the substance for inhibiting the expression of ZmPDIL gene is RNA encoded by the nucleotide sequence shown in 1898-2121 of SEQ ID NO 2.