Method for improving bombyx mori baculovirus infection resistance

A technology of baculovirus and silkworm, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve problems such as unclear BmNPV resistance

Active Publication Date: 2021-07-06
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether the above genes are involved in the resistance of silkworms to BmNPV is still unclear.

Method used

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  • Method for improving bombyx mori baculovirus infection resistance
  • Method for improving bombyx mori baculovirus infection resistance
  • Method for improving bombyx mori baculovirus infection resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Cloning the gene full-length sequence of BmVoa3 and BmVoa4

[0026] First, specific primers were designed according to the silkworm genome sequence:

[0027] BmVoa3-CDS-F: 5'-atgggggctatgttccg-3' (SEQ ID NO.1);

[0028] BmVoa3-CDS-R: 5'-ttaatcatctttattttcctcttg-3' (SEQ ID NO.2);

[0029] BmVoa4-CDS-F: 5'-atggggtctttgtttcgg-3' (SEQ ID NO.3);

[0030] BmVoa4-CDS-R: 5'-ttattcttctgcctgaccc-3' (SEQ ID NO.4);

[0031] The cDNA of the silkworm at 5 instar and 3 days old was amplified as a template. The PCR reaction conditions were: pre-denaturation at 94°C for 4 minutes, followed by denaturation at 94°C for 40 seconds, annealing at 55°C for 40 seconds, and extension at 72°C for 2 minutes and 20 seconds, a total of 30 seconds. cycle, with a final extension at 72°C for 10 minutes. The PCR product was identified and recovered by agarose gel electrophoresis, and then ligated with the pMD19-T vector. The ligation reaction was performed under the action of T4 DNA l...

Embodiment 2

[0033] Example 2: Effect of Inhibitor BafA on BmNPV Proliferation

[0034] Add thapsigargin (Thapsigargin, Tg) of 0nM, 10nM, 100nM, 1000nM respectively in BmE cells ( figure 1 , A) and Bafilomycin A1 (Bafilomycin A1, BafA) of OnM, 1nM, 2nM, 4nM, 8nM ( figure 1 , B), 72h later, the cell viability was detected by the cell viability assay (MTS method), and the operation was performed according to the instructions of the instrument and the kit. It was found that except for 8nM BafA, the other concentrations of the two drugs did not produce significant cytotoxicity to BmE cells.

[0035] BmE cells were pretreated with 0nM, 10nM, 100nM, 1000nM Tg and 0nM, 1nM, 2nM, 4nM BafA for 1h, and after the cells were infected with the BmNPV-GFP virus labeled with green fluorescence, immediately add the corresponding concentration of pretreatment drugs . Extract DNA at 24h and 48h after infection with the virus, and use specific primers for the GP41 gene of BmNPV virus:

[0036] GP41-qRT-F:...

Embodiment 3

[0044] Embodiment 3: Detection interferes with the impact of BmVoc gene on BmNPV proliferation

[0045] Select the specific sequence of the BmVoc gene as the interference target (SEQ ID NO.11), synthesize the corresponding dsRNA (dsVoc), use the specific sequence of the red fluorescent protein gene Red as the target (SEQ ID NO.12), and synthesize the dsRNA (dsRed) as For control, operate according to the instructions of the instrument and kit.

[0046] Transfect dsVoc and dsRed into BmE cells respectively, extract RNA 48 hours after transfection and reverse transcribe into cDNA, using specific primers of BmVoc gene:

[0047] BmVoc-qRT-F: 5'-cggcgtctgctatcatctt-3' (SEQ ID NO.13);

[0048] BmVoc-qRT-R: 5'-caggacagccacgacca-3' (SEQ ID NO.14);

[0049] And specific primers for internal reference gene TIF-4A:

[0050] TIF-4A-qRT-F: 5'-gaatggaccctgggacactt-3' (SEQ ID NO.15);

[0051] TIF-4A-qRT-R: 5'-ctgactgggcttgagcgata-3' (SEQ ID NO. 16);

[0052] Perform qPCR detection and o...

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Abstract

The invention discloses a method for improving bombyx mori baculovirus infection resistance. The method comprises the steps that bombyx mori is treated by specifically using a V-ATPase Vo complex inhibitor, and by reducing the expression of a Bm Voa4 gene in the bombyx mori or mutating the Bm Voa4 gene to cause function loss, the research shows that virus replication can be completely inhibited by using 4nM bafilomycin A1 for treatment; and then, a target gene of the bafilomycin A1 is selected for research to find that the virus content in cells interfering with the Bm Voa4 is remarkably reduced, the virus contents 24 hours and 48 hours after infection are 74% and 68% of those of a control group correspondingly, but the virus contents cannot be reduced by interfering with the Bm Voa3 and the Bm Voc, so that the Bm Voa4 is the target gene of the bafilomycin A1. A reagent targeting the Bm Voa4 gene can be used for research, development and production of drugs for preventing and treating bombyx mori nuclear polyhedrosis virus infection, and has popularization value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving resistance to silkworm baculovirus infection. Background technique [0002] The silkworm is a beneficial insect that produces silk and has important economic value. In many rural areas of our country, sericulture is the pillar industry of the local economy and the main source of income for farmers. The silk industry generates tens of billions of yuan in income for silkworm farmers every year. However, silkworms are facing serious disease threats. Nuclear polyhedrosis virus disease is the most common and most harmful type of silkworm disease in sericulture production. The pathogen that causes the disease is nuclear polyhedrosis virus (BmNPV). is highly contagious and difficult to control. BmNPV virus mainly infects silkworm through oral test, and first infects midgut cells of silkworm. [0003] At present, there is no specific drug for the treatment of silkwo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/033C12N15/85C12N15/12
CPCA01K67/0339C07K14/47C12N15/8509C12N2310/20A01K2267/02
Inventor 蒋亮夏庆友谢恩玉郭慧珍
Owner SOUTHWEST UNIV
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