CN113068661B

CN113068661B - Method for improving resistance to silkworm baculovirus infection

Info

Publication number: CN113068661B
Application number: CN202110370030.1A
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: 2022-07-15
Anticipated expiration: 2041-04-06
Also published as: CN113068661A

Abstract

The invention discloses a method for improving anti-bombyx mori baculovirus infection, and particularly relates to a method for treating bombyx mori by using a V-ATPase Vo complex inhibitor, wherein the bombyx mori is subjected to function loss by reducing expression of Bm Voa4 gene in the bombyx mori or mutating Bm Voa4 gene, research shows that the replication of viruses can be completely inhibited by using 4nM baveromycetin A1, and then research shows that the virus content in cells interfering with Bm Voa4 is remarkably reduced by selecting a target gene of baveromycetin A1, the virus content in the cells interfering with 24h and 48h after infection is respectively 74% and 68% of a control, but the virus content cannot be reduced by interfering with Bm Voa3 and interfering with Bm Voc, which shows that Bm Voa4 is a target gene of baveromycetin A1, and a reagent targeting with the Bm Voa4 gene can be used for research, development and production of medicines for preventing and development of bombyx nuclear polyhedrosis worthy of popularization.

Description

Method for improving resistance to silkworm baculovirus infection

Technical Field

The invention relates to the technical field of biology, in particular to a method for improving resistance to silkworm baculovirus infection.

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 a major source of economic income for local economy, both for the backbone industry and for 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 production of silkworms, and the pathogen causing the disease is nuclear polyhedrosis virus (BmNPV), which 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.

At present, no specific medicine is available for treating the silkworm nuclear polyhedrosis virus, the infection and the spread of diseases are mainly prevented by using a chemical disinfectant to strengthen strict disinfection in a silkworm raising period in production, and chlorine preparations, aldehyde preparations, surfactants, quicklime and the like are mainly used, so that a medicine suitable for treating the silkworm nuclear polyhedrosis virus is urgently needed.

Baveromycin a1(Bafilomycin a1, BafA), also known as NSC381866, is a macrolide antibiotic from Streptomyces griseus (Streptomyces griseus) of formula C35H58O 9. Baveromycin a1 is a potent and selective vacuolar ATP synthase (V-ATPase) inhibitor that blocks these proton pump activities in mammalian, plant or fungal cells with an IC50 in the 4-400nM range, and has not been found to have therapeutic effects against bombyx mori nuclear polyhedrosis virus. The V-ATPase consists of a conserved V1 complex and a non-conserved Vo complex, and the V1 and the Vo complexes consist of a plurality of subunits respectively. Bioinformatics analysis showed that 11 subunit members of the Vo complex were systematically identified from the bombyx mori genome database, including 4 members of the a subunit BmVoal, BmVoa2, BmVoa3, BmVoa 4. However, it is not clear whether the above genes are involved in resistance of silkworms to BmNPV.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for improving resistance to baculovirus infection of bombyx mori; the invention also aims to provide the application of the reagent targeting BmVoa4 gene expression in preparing the medicines for inhibiting the BmNPV proliferation.

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

1. a method for improving the resistance of silkworms to baculovirus infection, which comprises treating silkworms with a V-ATPase Vo complex inhibitor, by reducing the expression of BmVoa4 gene in the silkworms or by causing loss of function due to mutation of BmVoa4 gene.

Preferably, the method for reducing the expression of the BmVoa4 gene in silkworm is to reduce the expression of the BmVoa4 gene by using siRNA technology or shRNA technology.

Preferably, the V-ATPase Vo complex inhibitor is baverromycin A1.

Preferably, the BmVoa4 gene mutation method is deletion, frame shift or insertion mutation of the BmVoa4 gene by using CRISPR-Cas9 gene editing technology.

Preferably, the siRNA technique is the production of siRNA induced using dsRNA.

Preferably, the target sequence of the dsRNA is shown as SEQ ID NO. 20.

2. The application of the reagent targeting BmVoa4 gene expression or mutation in preparing the medicament for inhibiting BmNPV proliferation.

Preferably, the agent targeting BmVoa4 gene expression is an RNAi interference agent, and the agent targeting BmVoa4 gene mutation is a CRISPR-Cas9 gene editing agent.

Preferably, the RNAi-interfering agent is an siRNA fragment.

Preferably, the siRNA is induced by using dsRNA, and the target sequence of the dsRNA is shown as SEQ ID NO. 20.

The invention has the beneficial effects that: the invention finds that the baveromycetin A1 has the effect of inhibiting the proliferation of the bombyx mori nuclear polyhedrosis virus, when the treatment concentration of the baverromycin A1 is 1nM and 2nM, the virus DNA copy number is respectively reduced by 18.4 percent and 80.0 percent compared with a control in 24h after infection, and is respectively reduced by 30.8 percent and 88.4 percent compared with the control in 48h after infection, and the baverromycin A1 treatment of 4nM can completely inhibit the virus replication; then, a target gene of bavero mycin A1 is selected for research, the virus content in the cell interfering with BmVoa4 is obviously reduced, the virus content in the cell 24h and 48h after infection is respectively 74% and 68% of that in the control, but the virus content cannot be reduced by interfering with BmVoa3 and interfering with BmVoc, and the result shows that BmVoa4 is the target gene of bavero mycin A1, and the agent targeting the BmVoa4 gene can be used for research and development and production of medicines for preventing and treating the infection of the nuclear polyhedrosis virus of silkworms, and has popularization value.

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 graph showing the cytotoxicity of BafA and Tg;

FIG. 2 is a graph showing the effect of BafA and Tg on the proliferation of BmNPV virus;

FIG. 3 shows the effect of interfering BmVoc gene expression on BmNPV virus proliferation;

FIG. 4 is a graph of interference with the effect of BmVoa3 gene expression on BmNPV virus proliferation;

FIG. 5 is a graph of interference with the effect of BmVoa4 gene expression on BmNPV virus proliferation.

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: full-length sequences of the genes of clones BmVoa3 and BmVoa4

Firstly, designing a specific primer according to a silkworm genome sequence:

BmVoa3-CDS-F：5′-atgggggctatgttccg-3′(SEQ ID NO.1)；

BmVoa3-CDS-R：5′-ttaatcatctttattttcctcttg-3′(SEQ ID NO.2)；

BmVoa4-CDS-F：5′-atggggtctttgtttcgg-3′(SEQ ID NO.3)；

BmVoa4-CDS-R：5′-ttattcttctgcctgaccc-3′(SEQ ID NO.4)；

the amplification is carried out by taking the cDNA of a whole silkworm 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 55 ℃ for 40 sec, extension at 72 ℃ for 2min for 20 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 overnight at 16 ℃ under the action of T4 DNA ligase, then DH5 alpha competent cells are transformed, the obtained positive clone is sent to Shanghai Biotechnology Limited company for sequencing, and the sequencing result shows that the full-length sequences of BmVoa3 and BmVoa4 genes are successfully cloned, the full-length nucleotide sequence of the BmVoa3 gene is shown as SEQ ID NO.5, and the full-length nucleotide sequence of the BmVoa4 gene is shown as SEQ ID NO. 6.

Finally, we obtained 2517bp of the full-length BmVoa3 gene sequence and 2517bp of the full-length BmVoa4 gene sequence. The BmVoa3 gene encodes a 95.7kD protein, while the BmVoa4 gene encodes a 96.1kD protein.

Example 2: effect of inhibitor BafA on BmNPV proliferation

BmE cells were supplemented with 0nM, 10nM, 100nM, 1000nM Thapsigargin (Tg) (FIG. 1, A) and 0nM, 1nM, 2nM, 4nM, 8nM bafosfomycin A1(Bafilomycin A1, BafA) (FIG. 1, B), respectively, and cell viability was determined by cell viability assay (MTS method) after 72h, following the instructions of the apparatus and kit. As a result, it was found that neither drug at other concentrations, except 8nM of BafA, produced significant cytotoxicity on BmE cells.

BmE cells were pretreated with 0nM, 10nM, 100nM, 1000nM Tg and 0nM, 1nM, 2nM, 4nM BafA for 1h, respectively, and the drug was added to the pretreatment concentration immediately after infection of the cells with the green fluorescent-labeled BmNPV-GFP virus. DNA was extracted 24h and 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.7)；

GP41-qRT-R：5′-agtcgagtcgcgtcgcttt-3′(SEQ ID NO.8)；

qPCR detection is carried out, a domestic silkworm housekeeping gene BmGAPDH is used as an internal reference, and specific detection primers are as follows:

BmGAPDH-qRT-F：5′-ccgcgtccctgttgctaat-3′(SEQ ID NO.9)；

BmGAPDH-qRT-R：5′-ctgcctccttgaccttttgc-3′(SEQ ID NO.10)；

the procedure was performed according to the instructions of the instrument and kit. The qPCR assay results showed that Tg treatment did not reduce BmNPV virus content (fig. 2, a, fig. 2, C). BafA significantly reduced viral content, viral DNA copy number decreased by 18.4% and 80.0% respectively at 24h post-infection (fig. 2, B) and 30.8% and 88.4% respectively at 48h post-infection (fig. 2, D) after BafA treatment at 1nM and 2nM, and BafA treatment at 4nM completely inhibited viral replication.

Fluorescence was observed at 72h post-virus infection, with no significant difference in fluorescence between Tg-treated and control (fig. 2, E), whereas fluorescence in BafA-treated group was significantly less than control group, and the number of fluorescent cells gradually decreased with increasing drug concentration (fig. 2, F), indicating that BafA significantly inhibited the proliferation of BmNPV.

Target genes for BafA include the a, c subunits of the V-ATPase Vo complex and endoplasmic reticulum calcium ATPase (SERCA), Tg is a specific inhibitor of SERCA. The results show that BafA can obviously inhibit the proliferation of BmNPV, but Tg has no influence on the proliferation of BmNPV, which indicates that SERCA can not inhibit the proliferation of BmNPV, and BafA can target the a/c subunit of the silkworm V-ATPase Vo complex to inhibit the proliferation of BmNPV.

Example 3: detection of interference on the Effect of BmNPV Gene on BmNPV proliferation

Selecting a specific sequence of a BmVoc gene as an interference target (SEQ ID NO.11), synthesizing corresponding dsRNA (dsVoc), taking a specific sequence of a Red fluorescent protein gene Red as a target (SEQ ID NO.12), synthesizing dsRNA (dsRed) as a control, and operating according to the instructions of instruments and kits.

The dsVoc and dsRed were transfected into BmE cells, respectively, RNA was extracted 48h after transfection and reverse transcribed into cDNA, using primers specific for the BmVoc gene:

BmVoc-qRT-F：5′-cggcgtctgctatcatctt-3′(SEQ ID NO.13)；

BmVoc-qRT-R：5′-caggacagccacgacca-3′(SEQ ID NO.14)；

and specific primers of an internal reference gene TIF-4A:

TIF-4A-qRT-F：5′-gaatggaccctgggacactt-3′(SEQ ID NO.15)；

TIF-4A-qRT-R：5′-ctgactgggcttgagcgata-3′(SEQ ID NO.16)；

qPCR detection was performed, following the instrument and kit instructions. The results showed that BmVoc gene expression was successfully interfered with significantly lower mRNA content than the control (fig. 3, a).

BmNPV-GFP virus was infected 48h after transfection, DNA was extracted 24h (FIG. 3, B) and 48h (FIG. 3, C) after infection with the virus, and the BmNPV virus GP41 gene specific primer GP41-qRT-F/R (SEQ ID NO.7, SEQ ID NO.8) and the housekeeping gene BmGAPDH specific primer BmGAPDH-qRT-F/R (SEQ ID NO.9, SEQ ID NO.10) were tested by qPCR, showing that cells interfering with BmVoc could not significantly reduce BmNPV virus content.

Fluorescence was observed at 72h after infection with virus and no significant reduction in viral fluorescence was found in cells interfering with BmVoc gene expression (fig. 3, D), indicating that interfering with BmVoc gene expression does not inhibit the proliferation of BmNPV.

Example 4: detection of the influence of interference of BmVoa3 gene on BmNPV proliferation

The specific sequence of BmVoa3 gene (SEQ ID NO.5) was selected as the interference target (SEQ ID NO.17), and the corresponding dsRNA (dsVoa3) was synthesized and manipulated according to the instructions of the instrument and kit, with dsRed as the control.

dsVoa3 and dsRed were transfected into BmE cells, respectively, RNA was extracted 48h after transfection and reverse transcribed into cDNA, using primers specific for the BmVoa3 gene:

BmVoa3-qRT-F：5′-atcggtgaatgctgggtac-3′(SEQ ID NO.18，)；

BmVoa3-qRT-R：5′-tggtcctgttgaaggtggg-3′(SEQ ID NO.19)；

and a specific primer TIF-4A-qRT-F/R (SEQ ID NO.15, SEQ ID NO.16) of the internal reference gene TIF-4A for qPCR detection. The results showed that BmVoa3 gene expression was successfully interfered with significantly lower mRNA content than the control (fig. 4, a).

BmNPV-GFP virus was infected 48h after transfection, DNA was extracted 24h (FIG. 4, B) and 48h (FIG. 4, C) after infection with the virus, and the specific primers GP41-qRT-F/R (SEQ ID NO.7, SEQ ID NO.8) for the GP41 gene of the BmNPV virus and BmGAPDH-qRT-F/R (SEQ ID NO.9, SEQ ID NO.10) for the BmGAPDH gene were subjected to the QCPR assay, which showed that cells interfering with BmVoa3 could not significantly reduce the BmNPV virus content.

Fluorescence was observed at 72h post-infection with virus and no significant reduction in viral fluorescence was found in cells interfering with the expression of the BmVoa3 gene (fig. 4, D), indicating that interfering with BmVoa3 failed to inhibit the proliferation of BmNPV.

Example 5: detection of interference on the Effect of BmNPa 4 Gene on BmNPV proliferation

A specific sequence of BmVoa4 gene (SEQ ID NO.6) was selected as an interference target (SEQ ID NO.20), and a corresponding dsRNA (dsVoa4) was synthesized, with dsRed as a control.

dsVoa4 and dsRed were transfected into BmE cells, respectively, RNA was extracted 48h after transfection and reverse transcribed into cDNA, using primers specific for the BmVoa4 gene:

BmVoa4-qRT-F：5′-gccttcgggttctggat-3′(SEQ ID NO.21)；

BmVoa4-qRT-R：5′-gtggagccgtcgtagttgt-3′(SEQ ID NO.22)；

and a specific primer TIF-4A-qRT-F/R (SEQ ID NO.15, SEQ ID NO.16) of the internal reference gene TIF-4A for qPCR detection. The results showed that BmVoa4 gene expression was successfully interfered with significantly lower mRNA content than the control (fig. 5, a).

BmNPV-GFP virus was infected 48h after transfection, DNA was extracted 24h, 48h after infection of the virus, and qCPR detection was performed with a specific primer GP41-qRT-F/R (SEQ ID NO.7, SEQ ID NO.8) for the GP41 gene of BmNPV virus and a specific primer BmGAPDH-qRT-F/R (SEQ ID NO.9, SEQ ID NO.10) for the BmGAPDH gene. The results show a significant reduction in viral content in the cells interfering with BmVoa4, which was 74% and 68% of the control at 24h (fig. 5, B) and 48h (fig. 5, C) post infection, respectively.

Fluorescence was observed 72h after infection with virus and a significant decrease in viral fluorescence was found in cells interfering with the expression of BmVoa4 gene (fig. 5, D), indicating that interference with BmVoa4 significantly inhibited the proliferation of BmNPV.

The above results indicate that the BmVoa4 gene is a key gene affecting resistance of domestic silkworms, and is up-regulated after BmNPV infection, so as to promote virus infection rather than enhance host resistance to viruses. In the future molecular breeding work, the antiviral ability of the silkworms can be improved by knocking out the BmVoa4 gene through transgene interference or gene editing.

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 substitutions or changes made by the person skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the invention is subject to the claims.

Sequence listing

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<120> a method for improving resistance to infection by silkworm baculovirus

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tgggcggcca tctccgtctc catcctcgtg ctcatggagg ggctctcggc cttcctgcac 2400

acgctgcgtc tgcactgggt cgagttccag agcaagttct acggaggaga aggctacctc 2460

ttccagccct tctcgttcga gatcattcta gactcggcgg gtcaggcaga agaataa 2517

<210> 7

<211> 21

<212> DNA

<213> Bombyx mori nuclear polyhedrosis virus

<400> 7

cgtagtagta gtaatcgccg c 21

<210> 8

<211> 19

<212> DNA

<213> Bombyx mori nuclear polyhedrosis virus

<400> 8

agtcgagtcg cgtcgcttt 19

<210> 9

<211> 19

<212> DNA

<213> silkworm (Bombyx mori)

<400> 9

ccgcgtccct gttgctaat 19

<210> 10

<211> 20

<212> DNA

<213> Bombyx mori (Bombyx mori)

<400> 10

ctgcctcctt gaccttttgc 20

<210> 11

<211> 410

<212> DNA

<213> silkworm (Bombyx mori)

<400> 11

ctatcatctt cagcgccttg ggagctgcct atggaactgc caagtcagga actggtattg 60

ccgccatgtc ggtgatgagg cctgagctga tcatgaagtc gatcattcct gtcgtcatgg 120

cgggtattat tgccatctac ggtctggtcg tggctgtcct gattgctggt gccctccagg 180

agccagccaa ctaccccctt tacaaagggt tcatccactt gggtgctggt ttggctgtag 240

gattctctgg tctggctgcc ggtttcgcca taggcatcgt gggagatgca ggcgtgcgtg 300

gtactgctca gcagcctagg ttattcgtcg gaatgattct tattcttatt ttcgctgaag 360

tattgggtct ttacggactt atcgtcgcca tctacctgta cacaaaataa 410

<210> 12

<211> 409

<212> DNA

<213> silkworm (Bombyx mori)

<400> 12

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

<210> 13

<211> 19

<212> DNA

<213> Bombyx mori

<400> 13

cggcgtctgc tatcatctt 19

<210> 14

<211> 17

<212> DNA

<213> Bombyx mori

<400> 14

caggacagcc acgacca 17

<210> 15

<211> 20

<212> DNA

<213> Bombyx mori

<400> 15

gaatggaccc tgggacactt 20

<210> 16

<211> 20

<212> DNA

<213> Bombyx mori

<400> 16

ctgactgggc ttgagcgata 20

<210> 17

<211> 377

<212> DNA

<213> Bombyx mori

<400> 17

gaacggatgc tgtggcgtat ctcgagaggc aacgtcttcc tacgacgggc tgaactggac 60

aagccgctcg aagatcctgc tacgggtaac gagatctaca agaccgtgtt cgttgcgttc 120

ttccaaggcg aacagctaaa gtcccgcatc aagaaagtgt gcaccggttt ccacgcctcg 180

ctgtacccgt gcccgccttc gaacaccgaa cgacaggaca tggtcaaggg agtcaggacc 240

agactcgaag accttaacat ggtcttgaac caaacccgcg accacagaca acgtgtgctt 300

gcaagtgttg ccaaggagct gaccagctgg accataatgg tgaggaagat gaaggccatc 360

taccacaccc taaatct 377

<210> 18

<211> 19

<212> DNA

<213> Bombyx mori

<400> 18

atcggtgaat gctgggtac 19

<210> 19

<211> 19

<212> DNA

<213> Bombyx mori

<400> 19

tggtcctgtt gaaggtggg 19

<210> 20

<211> 451

<212> DNA

<213> Bombyx mori

<400> 20

gaggaacaag tcaccctact gggggaggag ggcctcatgg cgggagggca agcgctcaag 60

ctgggtttcg tcgcgggcgt gattctgagg gagagaatac cggcgttcga gcgcatgttg 120

tggcgggcct gcaggggaaa cgtgttcctg aggcaggctg agatcgacac gcccctggaa 180

gatccatcat catcggatca ggtttacaaa tcggtgttca tcatattctt ccaaggcgac 240

cagctgaaga cgcgcgtcaa gaaaatatgc gaaggtttca gggcgacgct gtatccgtgc 300

ccggagtcgc cggccgaccg ccgggagatg gccatgggcg tcatgaccag gatcgaggat 360

ctgaacaccg tcttaggtca gacccaggac catcgtcacc gcgtgctggt cgccgccgcc 420

aagaacatta agaactggtt cgtgaaagtg c 451

<210> 21

<211> 17

<212> DNA

<213> Bombyx mori

<400> 21

gccttcgggt tctggat 17

<210> 22

<211> 19

<212> DNA

<213> Bombyx mori

<400> 22

gtggagccgt cgtagttgt 19

Claims

1. A method for improving the resistance of silkworms to baculovirus infection is characterized in that: treating silkworms with a V-ATPase Vo complex inhibitor by reducing expression of the BmVoa4 gene in the silkworms or by loss of function by mutation of the BmVoa4 gene;

the method for reducing the expression of the BmVoa4 gene in silkworm is to reduce the mRNA level of the BmVoa4 gene by using siRNA technology; the siRNA technology is the generation of siRNA induced using dsRNA; the target sequence of the dsRNA is shown as SEQ ID NO. 20;

the V-ATPase Vo complex inhibitor is baveromycetin A1.

2. The method of claim 1, wherein: the method for mutating the BmVoa4 gene is to delete, shift or insertionally mutate the BmVoa4 gene by using CRISPR-Cas9 gene editing technology.

3. The application of the reagent targeting BmVoa4 gene expression or mutation in the preparation of the medicine for inhibiting BmNPV proliferation is characterized in that: the reagent targeting BmVoa4 gene expression is an RNAi interference reagent, the reagent targeting BmVoa4 gene mutation is a CRISPR-Cas9 gene editing reagent, the RNAi interference reagent is an siRNA fragment, siRNA is induced and generated by using dsRNA, and the target sequence of the dsRNA is shown as SEQ ID No. 20.