CN113057146A

CN113057146A - Application of bombyx mori BmUAP56 gene

Info

Publication number: CN113057146A
Application number: CN202110370111.1A
Authority: CN
Inventors: 蒋亮; 夏庆友; 谢恩玉; 郭慧珍
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2021-04-06
Filing date: 2021-04-06
Publication date: 2021-07-02
Anticipated expiration: 2041-04-06
Also published as: CN113057146B

Abstract

The invention discloses application of silkworm BmUAP56 gene, in particular to application of silkworm BmUAP56 gene in breeding silkworm varieties for resisting silkworm nuclear polyhedrosis virus and preparing medicaments for inhibiting silkworm nuclear polyhedrosis virus proliferation, wherein the nucleotide sequence of the BmUAP56 gene is shown as SEQ ID NO.3, the expression quantity of the BmUAP56 gene in silkworms is reduced through RNA interference or/and gene editing, or a reagent for inhibiting the combination of silkworm UAP56 protein and BmNPV virus protein 25K is used, and the combination force of silkworm UAP56 protein and BmNPV virus protein 25K is weakened through BmUAP56 gene mutation, so that the capacity of resisting silkworm nuclear polyhedrosis virus is obtained, and the application value is very good.

Description

Application of bombyx mori BmUAP56 gene

Technical Field

The invention relates to the technical field of biology, in particular to application of a bombyx mori BmUAP56 gene.

Background

The silkworm is a beneficial insect for producing silk and has important economic value. In many rural areas of our country, the silkworm industry is the main source of economic income for local economy, the prop industry and farmers, and the silk industry creates billions of yuan for silkworm farmers every year. However, silkworms face serious disease threats, and the nuclear polyhedrosis virus disease is the most common and serious silkworm disease in the silkworm industry production, and the pathogen causing the disease is the nuclear polyhedrosis virus (BmNPV), and the disease has extremely strong infectivity and is difficult to control. The BmNPV virus firstly infects the midgut cells of the silkworms by infecting the silkworms through a mouth test. In infected cells, gene expression and DNA replication of BmNPV follows an ordered time cascade model, including 4 stages: very early (0-4 h after infection, 0-4 hours post-infection, hpi), late early (5-7 hpi), late (8-18 hpi) and very late (> 18hpi), each subsequent stage being dependent on the previous stage, the gene expression product of the previous stage acting directly or indirectly in trans on the gene transcription of the subsequent stage.

As BmNPV has serious harm in silkworm industry production, researchers hope to find out key genes influencing the resistance of silkworms to viruses and then improve the resistance of silkworms to viruses by molecular biotechnology. The cloning and identification of the key gene full-length sequence influencing the resistance of the silkworm virus have important theoretical value and practical significance for clarifying the mechanism of resisting the silkworm virus and cultivating a resistant variety to be applied to silkworm production.

UAP56 belongs to ATP dependent RNA helicase DExD/H-box family member, and it interacts with proteins such as THO, Aly, etc. to form TREX complex, playing an important role in the process of mRNA nuclear export of eukaryote. UAP56 belongs to an essential gene of an organism, and its sequence is conserved among species. During the process of fighting against the host, the virus has evolved various strategies to enhance its infection capacity, including the utilization of key conserved genes of the host to help and promote the proliferation of the virus. However, it is not clear whether the BmUAP56 gene of silkworm participates in the infection of BmNPV at present.

Disclosure of Invention

In view of the above, the present invention aims to provide the application of bombyx mori BmUAP56 gene in breeding bombyx mori variety resisting bombyx mori nuclear polyhedrosis virus, which inhibits BmNPV by inhibiting BmUAP56 gene from binding with virus 25K protein.

In order to achieve the purpose, the invention provides the following technical scheme:

1. the application of silkworm BmUAP56 gene in breeding silkworm variety resisting silkworm nuclear polyhedrosis virus, and the nucleotide sequence of the silkworm BmUAP56 gene is shown in SEQ ID NO. 3.

Preferably, the bombyx mori nuclear polyhedrosis virus resistance is obtained by reducing the expression level of the BmUAP56 gene or mutation of the BmUAP56 gene in a bombyx mori silkworm body to reduce the binding force between the bombyx mori UAP56 protein and the BmNPV virus protein 25K.

Preferably, the means for reducing the expression level of the BmUAP56 gene is RNA interference.

Preferably, the nucleotide sequence of the RNA interference fragment for reducing the expression level of the BmUAP56 gene is shown as SEQ ID NO. 12.

2. The application of a reagent for inhibiting the combination of the UAP56 protein of silkworm and the protein 25K of BmNPV virus in the preparation of a medicament for inhibiting the proliferation of the nuclear polyhedrosis virus of silkworm, wherein the gene coding sequence of the UAP56 protein of silkworm is shown as SEQ ID NO. 3.

Preferably, the reagent for inhibiting the combination of the bombyx mori UAP56 protein and the BmNPV virus protein 25K is a small molecular compound, and the reagent reduces the component for reducing the expression of the bombyx mori UAP56 protein, or reduces the combination force of the bombyx mori UAP56 protein and the BmNPV virus protein 25K by mutating the BmUAP56 gene.

Preferably, the small molecule compound is an inhibitor CCT 018159.

Preferably, the component for reducing the expression of the UAP56 protein of the silkworm is a gene RNA interference fragment coding the UAP56 protein.

Preferably, the nucleotide sequence of the RNA interference fragment is shown as SEQ ID NO. 12.

The invention has the beneficial effects that: the full-length sequence of a key gene BmUAP56 which influences the resistance of the BmNPV of the silkworm is cloned, and the expression spectrum of the time tissue of the gene is detected by RT-PCR and quantitative PCR technologies, so that the gene is found to be expressed in each time and each tissue of the silkworm. In order to research the function of the gene, firstly, an inhibitor is used for detection, and CCT018159 can inhibit the proliferation of BmNPV virus; then, the direct combination of BmUAP56 and the 25K protein of BmNPV virus is proved by Co-IP, and CCT018159 can inhibit the combination between the 25K protein and BmUAP56 protein; further, a specific sequence of the BmUAP56 gene is used as a target to synthesize dsRNA to transfect the BmE cells of silkworms, so that the proliferation of BmNPV can be obviously inhibited. Therefore, the gene has important value in the research and application of silkworm antiviral molecule breeding, and the antiviral ability of silkworms can be improved by knocking out the gene through transgene interference or gene editing.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a time period expression diagram of RT-PCR detection of BmUAP56 gene (1-18 are egg two days, egg four days, egg six days, egg eight days, formin, first-instar silkworm, second-instar silkworm, third-instar silkworm, fourth-instar silkworm, fifth-instar silkworm, second-instar silkworm, fourth-instar silkworm, sixth-instar silkworm, eighth-instar silkworm and silkworm moth, respectively);

FIG. 2 is a tissue expression diagram of BmUAP56 gene detected by qPCR;

FIG. 3 is a cytotoxicity test chart of CCT018159 and 17-DMAG (CCT: means CCT 018159);

FIG. 4 is a diagram showing the inhibitory effect of different concentrations of CCT018159 and 17-DMAG on BmNPV (CCT: CCT 018159);

FIG. 5 shows the time points at which CCT018159 inhibits the proliferation of BmNPV virus (MOI 1: dose infected with BmNPV; DMSO: DMSO is a solvent for CCT018159, which is used herein as a negative control and represents 0. mu.M of CCT 018159; Nm: control not infected with BmNPV);

FIG. 6 is a Co-IP assay of BmUAP56 binding to BmNPV viral protein 25K;

FIG. 7 shows that CCT018159 inhibits the binding of BmUAP56 to 25K protein;

FIG. 8 shows that interference of BmUAP56 gene significantly inhibits BmNPV proliferation (A: BmUAP56 gene expression level detected by qPCR 48h after interference; B: BmNPV virus copy number detected by qPCR 48h after virus infection; C: BmNPV virus fluorescence observed 72h after virus infection).

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1: cloning of the coding region sequence of the BmUAP56 Gene

Designing a specific primer according to a silkworm genome sequence:

BmUAP56-CDS-F：5′-atggctgacaacgacgat-3′(SEQ ID NO.1)；

BmUAP56-CDS-R：5′-ctatcgtccttcgatgtagg-3′(SEQ ID NO.2)；

the amplification is carried out by taking the whole silkworm cDNA of 5-day-old silkworms as a template, and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min, followed by denaturation at 94 ℃ for 40 sec, annealing at 54 ℃ for 40 sec, extension at 72 ℃ for 1min10 sec for 30 cycles, and final extension at 72 ℃ for 10 min. The PCR product is identified and recovered by agarose gel electrophoresis, then is connected with a pMD19-T vector, the connection reaction is carried out under the action of T4 DNA ligase at 16 ℃ overnight, then DH5 ℃ competent cells are transformed, the obtained positive clone is sent to Shanghai Biotechnology Limited company for sequencing, and the sequencing result shows that the coding region sequence (SEQ ID NO.3) of the BmUAP56 gene is successfully cloned and consists of 1278bp nucleotide sequence, and the gene codes 425 amino acids and contains DEXDc and HELIC structural domains.

Example 2: detection of time-based tissue expression profiles of BmUAP56 gene

Taking cDNA of two days of eggs, four days of eggs, six days of eggs, eight days of eggs, newly-hatched silkworms, first-instar sleeping silkworms, second-instar starting silkworms, second-instar sleeping silkworms, third-instar starting silkworms, third-instar sleeping silkworms, fourth-instar starting silkworms, fourth-instar sleeping silkworms, fifth-instar starting silkworms, second-instar pupae, fourth-instar pupae, six-instar pupae, eight-instar pupae and silkworm moths as templates, and taking a specific primer of BmUAP56 gene:

BmUAP56-qRT-F：5′-cctcacgggaaacaggtg-3′(SEQ ID NO.4)：

BmUAP56-qRT-R：5′-tcttttagtttcacataatgttgctgc-3′(SEQ ID NO.5)：

performing RT-PCR detection, wherein the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 4 min, followed by denaturation at 94 ℃ for 40 sec, annealing at 56 ℃ for 40 sec, extension at 72 ℃ for 10 sec for 28 cycles, and final extension at 72 ℃ for 10 min. The silkworm housekeeping gene TIF-4A is used as an internal reference, and specific primers are used:

TIF-4A-qRT-F：5′-gaatggaccctgggacactt-3′(SEQ ID NO.6)；

TIF-4A-qRT-R：5′-ctgactgggcttgagcgata-3′(SEQ ID NO.7)；

performing RT-PCR detection, wherein the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 4 min, followed by denaturation at 94 ℃ for 40 sec, annealing at 56 ℃ for 40 sec, extension at 72 ℃ for 10 sec for 27 cycles, and final extension at 72 ℃ for 10 min.

The method is characterized in that cDNA of tissues such as blood, fat body, silk gland, trachea, Marek's tube, ovary and testis of 5-day-old 3-day-old silkworm larvae is used as a template, a specific primer BmUAP56-qRT-F/R (SEQ ID No.4, SEQ ID No.5) of BmUAP56 gene is used for qPCR detection, a specific primer TIF-4A-qRT-F/R (SEQ ID No.6, SEQ ID No.7) of silkworm housekeeping gene TIF-4A is used as an internal reference, and the operation is carried out according to the instructions of instruments and kits.

The results of RT-PCR and qPCR demonstrated that the BmUAP56 gene was expressed at all stages of silkworm development (FIG. 1) and in all tissues of silkworm (FIG. 2).

Example 3: effect of inhibitor CCT018159 on BmNPV proliferation

The small molecular compound CCT018159 is used as an inhibitor, and target genes of the small molecular compound CCT018159 comprise UAP56 and HSP 90; the small molecule compound 17-DMAG is a specific inhibitor of HSP90, and whether the small molecule compound is related to BmNPV proliferation needs further research.

0. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 80. mu.M, 100. mu.M of CCT018159 and 17-DMAG were added to BmE cells, and after 72 hours, the viability of the cells was measured by cell viability assay (MTS method), and the procedures of the instrument and the kit were followed. As a result, it was found that the cell survival rate gradually decreased with the increase in the concentration of CCT018159 drug, and that CCT018159 at a concentration of 10. mu.M or less did not produce significant toxicity, and 17-DMAG at a concentration of 80. mu.M or less did not produce significant toxicity (FIG. 3).

After the BmE cells are infected with the BmNPV-GFP virus with the green fluorescent label, CCT018159 and 17-DMAG at 0. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M and 80. mu.M are added respectively. DNA was extracted 48h after infection with the virus, and the DNA was purified using primers specific for the GP41 gene of BmNPV virus:

GP41-qRT-F：5′-cgtagtagtagtaatcgccgc-3′(SEQ ID NO.8)；

GP41-qRT-R：5′-agtcgagtcgcgtcgcttt-3′(SEQ ID NO.9)；

carrying out qPCR detection by taking a domestic silkworm housekeeping gene BmGAPDH as an internal reference and a specific detection primer thereof as follows;

BmGAPDH-qRT-F：5′-ccgcgtccctgttgctaat-3′(SEQ ID NO.10)；

BmGAPDH-qRT-R：5′-ctgcctccttgaccttttgc-3′(SEQ ID NO.11)；

the procedure was performed according to the instructions of the instrument and kit. The qPCR detection result shows that the virus content of BmNPV gradually decreases with the increase of the concentration of CCT018159, and the 17-DMAG treatment group has no similar trend (figure 4), which indicates that the CCT018159 can target UAP56 to inhibit the proliferation of BmNPV.

BmE cells are infected with BmNPV-GFP virus with a green fluorescent label, and 10 mu M of CCT018159 is added at 0, 4, 8, 12 and 24 hours after infection. DNA was extracted 48h after virus infection, RT-PCR and qPCR detection were performed with specific primer GP41-qRT-F/R (SEQ ID NO.8, SEQ ID NO.9) of BmNPV virus GP41 gene and specific primer BmGAPDH-qRT-F/R (SEQ ID NO.10, SEQ ID NO.11) of Bombyx mori housekeeping gene BmGAPDH, following the instructions of the instrument and kit. The result shows that the addition of CCT018159 within 12h after the challenge can obviously inhibit the proliferation of BmNPV (figure 5), and the CCT018159 can inhibit the virus at the later gene expression stage of the BmNPV.

Example 4: CCT018159 inhibits the combination of BmUAP56 and BmNPV virus protein

The BmUAP56 gene is connected to a PGEX-4T-1 vector through BamHI and NotI enzyme cutting sites to construct a BmUAP56 prokaryotic expression vector (PGEX-4T-1-UAP56), IPTG is used for induction expression and purification to obtain BmUAP56 recombinant protein, and the operation is carried out according to the instructions of instruments and kits. And then, carrying out a Pull down experiment by using total proteins and BmUAP56 proteins of the midgut sample of the bombyx mori infected with or not infected with BmNPV, carrying out mass spectrum identification on the difference bands, and screening 5 virus proteins comprising two late essential gene proteins E25 and 25K.

The interaction of BmUAP56 with 25K was identified by Co-immunoprecipitation (Co-IP). Respectively constructing Flag-tagged BmUAP56(UAP56-Flag) and His-tagged 25K (25K-His) incremental expression vectors, co-transfecting a BmE cell by UAP56-Flag +25K-His, transfecting by using GFP-Flag +25K-His as a control, collecting cell lysate 48h after transfection, enriching by using magnetic beads coupled with Flag antibodies, and then carrying out western blot detection on the enriched sample by using Flag and His antibodies, wherein the result shows that 25K is directly bound with BmUAP56 but not bound with GFP (figure 6), and the binding of E25 and BmUAP56 is not detected by using the same method.

Co-transfecting UAP56-Flag +25K-His to BmE cells, immediately adding CCT018159 with the concentration of 0, 5 and 10 mu M, lysing the cells by lysate containing drugs with corresponding concentration 48h after transfection, and performing Co-IP detection by magnetic beads coupled with Flag antibodies. The results show that the CCT018159 can inhibit the binding of BmUAP56 and 25K, and the inhibition effect is enhanced along with the increase of the concentration of the medicament (figure 7), and BmUAP56 is a target gene of the CCT 018159.

Example 5: interference with the effect of BmUAP56 gene on BmNPV proliferation

The specific sequence of BmUAP56 gene was selected as an interference target (SEQ ID NO.12), the corresponding dsRNA was synthesized (dsUAP56), and the dsRNA synthesized from Red fluorescent protein gene Red (dsRed, SEQ ID NO.13) was used as a control, and the procedures of the instrument and the kit were performed.

The dsUAP56 and dsRed are transfected into BmE cells respectively, RNA is extracted 48h after transfection and is reversely transcribed into cDNA, and qPCR detection is carried out by using a specific primer BmUAP56-qRT-F/R (SEQ ID NO.4, SEQ ID NO.5) of BmUAP56 gene and a specific primer TIF-4A-qRT-F/R (SEQ ID NO.6, SEQ ID NO.7) of an internal reference gene TIF-4A. The results showed that BmUAP56 was successfully interfered with, with significantly lower mRNA content than the control (fig. 8A).

BmNPV-GFP virus was infected 48h after transfection, DNA was extracted 48h after infection of the virus, and qCPR detection was performed with a specific primer GP41-qRT-F/R (SEQ ID NO.8, SEQ ID NO.9) for the GP41 gene of BmNPV virus and a specific primer BmGAPDH-qRT-F/R (SEQ ID NO.10, SEQ ID NO.11) for BmGAPDH, a housekeeping gene. The results showed a significant reduction in virus content in cells interfering with BmUAP56, only 43% of the control (fig. 8B).

Fluorescence was observed at 72h post-infection with virus and a significant reduction in viral fluorescence was found in cells interfering with BmUAP56 (fig. 8C), indicating that interfering with BmUAP56 significantly inhibited the proliferation of BmNPV.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> university of southwest

Application of <120> bombyx mori BmUAP56 gene

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> silkworm (Bombyx mori)

<400> 1

atggctgaca acgacgat 18

<210> 2

<211> 20

<212> DNA

<213> silkworm (Bombyx mori)

<400> 2

ctatcgtcct tcgatgtagg 20

<210> 3

<211> 1278

<212> DNA

<213> silkworm (Bombyx mori)

<400> 3

atggctgaca acgacgatct tctcgactac gaagatgaag aacaagcaga tcaacagact 60

gctgacgggt ctaccgaagt ggcgccaaag aaggaggtta aaggatccta cgtatccatc 120

cacagttctg gttttagaga cttcttactt aagccagaga tactccgcgc tattgttgat 180

tgcggttttg agcatccttc cgaagttcaa cacgaatgca ttccgcaagc cgtcctcggt 240

atggacatac tttgccaagc caagtccggt atgggtaaga cagccgtgtt tgttttggca 300

acattgcagc agctggaacc ctccgagagc cacgtttacg tgcttgtaat gtgccacacc 360

agagagttgg ctttccaaat aagtaaggag tacgagcgtt tctcgaaata tatgtcagga 420

gtcagagtat cagtattctt tggtggtatg ccaattcaga aagacgagga agtccttaag 480

acagcatgtc ctcacattgt agtgggaact cccggtagaa ttttggcatt agtaaacagt 540

aagaaactta atttgaaaca tttaaaacac ttcatccttg atgagtgtga taagatgctg 600

gaatctctag acatgaggcg agatgtgcag gagatattcc ggaacactcc tcacgggaaa 660

caggtgatga tgttctcagc cacattgagt aaagaaatca gacctgtgtg taagaaattt 720

atgcaagacc caatggaagt ttatgtagat gatgaagcca aactcaagct acatggactg 780

cagcaacatt atgtgaaact aaaagaaaat gagaagaata aaaaactctt cgaactactt 840

gatgtattgg agttcaatca ggtggttatt tttgtgaaat cagtacagcg ctgcatagct 900

ctggcacaac tgctcaccga ccaaaacttc cctgccattg gtatccacag aaatatgacc 960

caggatgagc gtctctcccg ctatcaacag ttcaaggact tccaaaaaag gatcctggtt 1020

gcaacaaatt tgtttggccg gggaatggac attgaaaggg taaatattgt cttcaactat 1080

gacatgcctg aagattcaga tacatatcta catcgtgtcg cccgcgcagg aaggtttggt 1140

accaaaggtc tggccattac tatgatatct gatgaaaatg atgccaagat cttaaaccaa 1200

gtacaagatc gtttcgatgt gaacattaca gagctgcctg aagaaattga gctctctacc 1260

tacatcgaag gacgatag 1278

<210> 4

<211> 18

<212> DNA

<213> silkworm (Bombyx mori)

<400> 4

cctcacggga aacaggtg 18

<210> 5

<211> 27

<212> DNA

<213> silkworm (Bombyx mori)

<400> 5

tcttttagtt tcacataatg ttgctgc 27

<210> 6

<211> 20

<212> DNA

<213> silkworm (Bombyx mori)

<400> 6

gaatggaccc tgggacactt 20

<210> 7

<211> 20

<212> DNA

<213> silkworm (Bombyx mori)

<400> 7

ctgactgggc ttgagcgata 20

<210> 8

<211> 21

<212> DNA

<213> Bombyx mori nuclear polyhedrosis virus

<400> 8

cgtagtagta gtaatcgccg c 21

<210> 9

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<212> DNA

<213> Bombyx mori nuclear polyhedrosis virus

<400> 9

agtcgagtcg cgtcgcttt 19

<210> 10

<211> 19

<212> DNA

<213> silkworm (Bombyx mori)

<400> 10

ccgcgtccct gttgctaat 19

<210> 11

<211> 20

<212> DNA

<213> silkworm (Bombyx mori)

<400> 11

ctgcctcctt gaccttttgc 20

<210> 12

<211> 436

<212> DNA

<213> silkworm (Bombyx mori)

<400> 12

cgtatccatc cacagttctg gttttagaga cttcttactt aagccagaga tactccgcgc 60

tattgttgat tgcggttttg agcatccttc cgaagttcaa cacgaatgca ttccgcaagc 120

cgtcctcggt atggacatac tttgccaagc caagtccggt atgggtaaga cagccgtgtt 180

tgttttggca acattgcagc agctggaacc ctccgagagc cacgtttacg tgcttgtaat 240

gtgccacacc agagagttgg ctttccaaat aagtaaggag tacgagcgtt tctcgaaata 300

tatgtcagga gtcagagtat cagtattctt tggtggtatg ccaattcaga aagacgagga 360

agtccttaag acagcatgtc ctcacattgt agtgggaact cccggtagaa ttttggcatt 420

agtaaacagt aagaaa 436

<210> 13

<211> 409

<212> DNA

<213> silkworm (Bombyx mori)

<400> 13

gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc 60

cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac 120

cgtgacccag gactcctccc tgcaggacgg ctgcttcatc tacaaggtga agttcatcgg 180

cgtgaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc 240

caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca aggccctgaa 300

gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg ccaagaagcc 360

cgtgcagctg cccggctact actacgtgga ctccaagctg gacatcacc 409

Claims

1. The application of silkworm BmUAP56 gene in breeding silkworm variety resisting silkworm nuclear polyhedrosis virus is characterized by that: the nucleotide sequence of the bombyx mori BmUAP56 gene is shown in SEQ ID NO. 3.

2. Use according to claim 1, characterized in that: the BmUAP56 gene expression quantity or BmUAP56 gene mutation is reduced in silkworm body, so that the binding force of the silkworm UAP56 protein and the BmNPV virus protein 25K is reduced, and the silkworm nuclear polyhedrosis virus resistance is obtained.

3. Use according to claim 2, characterized in that: the mode for reducing the expression amount of the BmUAP56 gene is RNA interference.

4. Use according to claim 3, characterized in that: the nucleotide sequence of the RNA interference fragment for reducing the expression quantity of the BmUAP56 gene is shown as SEQ ID NO. 12.

5. The application of the reagent for inhibiting the combination of the UAP56 protein of silkworm and the BmNPV virus protein 25K in the preparation of the medicament for inhibiting the proliferation of the nuclear polyhedrosis virus of silkworm is characterized in that: the gene coding sequence of the bombyx mori UAP56 protein is shown in SEQ ID NO. 3.

6. Use according to claim 5, characterized in that: the reagent for inhibiting the combination of the UAP56 protein of the silkworm and the BmNPV virus protein 25K is a small molecular compound, and a component for reducing the expression of the UAP56 protein of the silkworm or a component for reducing the combination force of the UAP56 protein of the silkworm and the BmNPV virus protein 25K by mutating the gene of the BmUAP 56.

7. Use according to claim 6, characterized in that: the small molecular compound is an inhibitor CCT 018159.

8. Use according to claim 6, characterized in that: the component for reducing the expression of the UAP56 protein of the silkworm is a gene RNA interference fragment for coding the UAP56 protein.

9. Use according to claim 6, characterized in that: the nucleotide sequence of the RNA interference fragment is shown as SEQ ID NO. 12.