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Application of bmuap56 gene in silkworm

A silkworm and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve problems such as unclear, achieve the effect of inhibiting proliferation and improving the antiviral ability of silkworm

Active Publication Date: 2022-03-18
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether the BmUAP56 gene of silkworm is involved in the infection of BmNPV is still unclear.

Method used

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  • Application of bmuap56 gene in silkworm
  • Application of bmuap56 gene in silkworm
  • Application of bmuap56 gene in silkworm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Cloning the coding region sequence of BmUAP56 gene

[0028] Design specific primers based on silkworm genome sequence:

[0029] BmUAP56-CDS-F: 5'-atggctgacaacgacgat-3' (SEQ ID NO.1);

[0030] BmUAP56-CDS-R: 5'-ctatcgtccttcgatgtagg-3' (SEQ ID NO. 2);

[0031] Amplify the cDNA of the silkworm at 5 instar and 3 days old as a template. The PCR reaction conditions are: 94°C pre-denaturation for 4 minutes, then 94°C denaturation for 40 seconds, 54°C annealing for 40 seconds, and 72°C extension for 1min10 seconds, a total of 30 Cycle, 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 ligase, ligated overnight at 16°C, and then transformed into DH5°C competent cells, and the positive clones were obtained and sent to Shanghai Sequencing by Sangon Biotechnology Co., Ltd., the sequencing results...

Embodiment 2

[0032] Example 2: Period tissue expression profile detection of BmUAP56 gene

[0033] Eggs for two days, eggs for four days, eggs for six days, eggs for eight days, ant silkworms, first-instar dormant silkworms, second-instar dormant silkworms, second-instar dormant silkworms, third-instar dormant silkworms, third-instar dormant silkworms, fourth-instar dormant silkworms, four-instar dormant silkworms The cDNAs of the instar sleeping silkworm, the fifth instar silkworm, the second day of pupae, the fourth day of pupae, the sixth day of pupae, the eighth day of pupae and silkworm moth were used as templates, and the specific primers of BmUAP56 gene were used:

[0034] BmUAP56-qRT-F: 5'-cctcacgggaaacaggtg-3' (SEQ ID NO.4):

[0035] BmUAP56-qRT-R: 5'-tcttttagtttcacataatgttgctgc-3' (SEQ ID NO.5):

[0036] For RT-PCR detection, PCR amplification conditions were: 94°C pre-denaturation for 4 minutes, followed by 94°C denaturation for 40 seconds, 56°C annealing for 40 seconds, 72°C e...

Embodiment 3

[0042] Example 3: Effect of Inhibitor CCT 018159 on BmNPV Proliferation

[0043] As an inhibitor, the small molecule compound CCT018159 has been reported in the literature to target genes including UAP56 and HSP90; the small molecule compound 17-DMAG is a specific inhibitor of HSP90, and whether it is related to the proliferation of BmNPV needs further study.

[0044] Add 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 100 μM CCT018159 and 17-DMAG to BmE cells, and use cell viability assay (MTS method) to detect cell viability after 72 hours, according to the specifications of the instrument and kit manual to operate. The results found that the cell viability gradually decreased with the increase of CCT018159 drug concentration, CCT018159 at a concentration of 10 μM and below did not produce obvious toxicity, and 17-DMAG at a concentration of 80 μM and below did not produce obvious toxicity ( image 3 ).

[0045] After BmE cells were infected with BmNPV-GFP virus labeled...

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Abstract

The invention discloses the application of the silkworm BmUAP56 gene, and specifically discloses the application of the silkworm BmUAP56 gene in cultivating silkworm varieties resistant to silkworm nuclear polyhedrosis virus and preparing medicines for inhibiting the proliferation of silkworm nuclear polyhedrosis virus. The nucleotide sequence is shown in SEQ ID NO.3, and the expression level of the BmUAP56 gene in the silkworm body can be reduced by RNA interference or / and gene editing, or a reagent that inhibits the binding of the silkworm UAP56 protein to the BmNPV viral protein 25K can also be passed through the BmUAP56 gene The mutation weakens the binding force between silkworm UAP56 protein and BmNPV virus protein 25K, thereby obtaining the ability to resist silkworm nuclear polyhedrosis virus, which has good application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of the silkworm BmUAP56 gene. 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. In infected cells, the gene expression and DNA replication of BmNPV follow an orderly time cascad...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12
CPCA01K67/0339C12N9/78C12Y306/04013A61K31/4155A61K31/713A61P31/12A01K2227/706A01K2267/02
Inventor 蒋亮夏庆友谢恩玉郭慧珍
Owner SOUTHWEST UNIV
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