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A kind of method to improve resistance to silkworm baculovirus infection

A technology of baculovirus and silkworm, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of unclear BmNPV resistance and achieve the effect of inhibiting replication

Active Publication Date: 2022-07-15
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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  • A kind of method to improve resistance to silkworm baculovirus infection
  • A kind of method to improve resistance to silkworm baculovirus infection
  • A kind of method to improve resistance to silkworm baculovirus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning of full-length gene sequences 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 whole silkworm cDNA from the 5th instar 3 days of silkworm was used as a template for amplification. The PCR reaction conditions were: 94°C pre-denaturation for 4 minutes, then 94°C denaturation for 40 seconds, 55°C annealing for 40 seconds, and 72°C extension for 2 min for 20 seconds, a total of 30 cycle, with a final extension at 72°C for 10 minutes. The PCR products were identified and recovered by agarose gel electrophoresis, and then ligated with the pMD19-T vector. The ligation reaction was carried out under the action of T4 DNA liga...

Embodiment 2

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

[0034] Add 0nM, 10nM, 100nM, 1000nM of thapsigargin (Thapsigargin, Tg) to BmE cells respectively ( figure 1 , A) and 0nM, 1nM, 2nM, 4nM, 8nM of Bafilomycin A1 (Bafilomycin A1, BafA) ( figure 1 , B), after 72h, 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 kit. It was found that with the exception of BafA at 8 nM, 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 of Tg and 0nM, 1nM, 2nM, and 4nM of BafA for 1 h, respectively. After the cells were infected with BmNPV-GFP virus labeled with green fluorescence, the corresponding concentration of drugs for pretreatment was added immediately. . DNA was extracted at 24h and 48h after infection with the virus, and specific primers for the GP41 g...

Embodiment 3

[0044] Example 3: Detecting the effect of interference with BmLoc gene on BmNPV proliferation

[0045] Select the specific sequence of 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), synthesize dsRNA (dsRed) as the target For control, follow the instructions of the instrument and kit.

[0046] dsVoc and dsRed were transfected into BmE cells respectively, RNA was extracted 48 hours after transfection and reverse transcribed into cDNA, using specific primers for 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 the 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] For qPCR detection, fo...

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Abstract

The invention discloses a method for improving resistance to Bombyx mori baculovirus infection, specifically treating Bombyx mori with a V-ATPase Vo complex inhibitor, reducing the expression of Bm Voa4 gene in Bombyx mori or making Bm Voa4 gene mutation to cause loss of function, Studies have shown that bafilomycin A1 treatment with 4nM can completely inhibit virus replication, and then the target gene of bafilomycin A1 was selected for research. The virus content at and 48h were 74% and 68% of the control, respectively, but the interference of Bm Voa3 and Bm Voc could not reduce the virus content, indicating that Bm Voa4 is the target gene of bafilomycin A1, and by targeting the Bm Voa4 gene The reagent can be used for the research, development and production of drugs for preventing and treating Bombyx mori nuclear polyhedrosis virus infection, and has promotion value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving resistance to Bombyx mori baculovirus infection. Background technique [0002] Bombyx mori is a beneficial insect that produces silk and has important economic value. In many rural areas in my 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 for silkworm farmers every year. However, silkworms are facing serious disease threats. Nuclear polyhedrosis virus disease is the most common and most serious type of silkworm disease in sericulture production. The pathogen causing the disease is nuclear polyhedrosis virus (BmNPV). are extremely contagious and difficult to control. BmNPV virus mainly infects silkworm through oral test, firstly infecting silkworm midgut cells. [0003] At present, there is no specific drug for the treatment of silkworm nuclear p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/12A01K67/033
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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