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Bombyx mori bmpepck-2 gene and its application

A technology of silkworm and gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as strong infectivity and difficult to control

Active Publication Date: 2018-08-03
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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). highly contagious and difficult to control

Method used

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  • Bombyx mori bmpepck-2 gene and its application
  • Bombyx mori bmpepck-2 gene and its application
  • Bombyx mori bmpepck-2 gene and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1, clone the full-length sequence of BmPEPCK-1 and BmPEPCK-2 gene

[0018] First, specific primers were designed according to the silkworm genome sequence, BmPEPCK-1 full-length forward primer: 5'-atgctgcattttaaaaccaccccccgaaga-3' (SEQ ID NO.1), reverse primer 5'-gactcgtttaattttatatctttggt-3' (SEQ ID NO.2 ); BmPEPCK-2 full-length forward primer: 5'-atgctgcattttaaggccatcccc-3' (SEQ ID NO.3), reverse primer 5'-catgtgggataaataactaggaagag-3' (SEQ ID NO.4). Then, the cDNA of silkworm Dazao 5th instar and 3 days old was used as a template to amplify. The PCR reaction conditions were: 94°C pre-denaturation for 4 minutes, followed by denaturation at 94°C for 40 seconds, annealing at 57°C for 40 seconds, and extension at 72°C for 1 min and 40 seconds. 33 cycles, the final 72°C extension 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 act...

Embodiment 2

[0020] Example 2, Period tissue expression profile detection of BmPEPCK-1 and BmPEPCK-2 genes

[0021] The cDNA of ant silkworm, first instar sleeper silkworm, second instar sleeper silkworm, second instar sleeper silkworm, third instar sleeper silkworm, third instar sleeper silkworm, fourth instar sleeper silkworm, fourth instar sleeper silkworm, five-ling sleeper silkworm, mature silkworm and pupae were used as Template, with BmPEPCK-1 gene-specific primers PEPCK-1qRT (F: 5'-tcaaagaggaggtaggcga-3' (SEQ ID NO.7), R: 5'-gactcgtttaattttatatctttggt-3' (SEQ ID NO.8)) and BmPEPCK- 2 gene-specific primers PEPCK-2qRT (F: 5'-tcaaagaggaggtaggtaggcgaa-3' (SEQ ID NO.9), R: 5'-catgtgggataaataactaggaagag-3' (SEQ ID NO.10)) for RT-PCR detection, PCR amplification The growth conditions are: 94°C pre-denaturation for 4 minutes, followed by 94°C denaturation for 40 seconds, 57°C annealing for 40 seconds, 72°C extension for 10 seconds, a total of 38 cycles, and finally 72°C extension for 10 mi...

Embodiment 3

[0024] Example 3, Detection of the expression levels of BmPEPCK-1 and BmPEPCK-2 after BmNPV infection

[0025] The half-lethal dose of BmNPV virus was added orally to the 5th instar larvae of Dazai silkworm, and the midgut RNA of infected and non-infected silkworms was extracted at 6h, 12h and 24h after infection, and reversed into cDNA. The specific primer PEPCK-1qRT, the specific primer PEPCK-2qRT of the BmPEPCK-2 gene, and the specific primer TIF-4AqRT of the internal reference gene TIF-4A were used for qPCR detection, and the results were as follows: image 3 Shown in A. The results showed that BmNPV infection induced down-regulated expression of BmPEPCK-1 and BmPEPCK-2 in silkworm, and 24h after virus infection, the expression level of BmPEPCK-1 returned to normal while BmPEPCK-2 was still down-regulated.

[0026] BmE cells were infected with BmNPV virus, RNA was extracted at 0h, 3h, 6h, 12h and 24h after infection and reversed into cDNA. The specific primer TIF-4AqRT o...

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Abstract

The invention relates to a BmPEPCK-2 gene of domestic silkworms and application of the BmPEPCK-2 gene. The nucleotide sequence of the BmPEPCK-2 gene of the domestic silkworms is shown as SEQ ID NO.6; the gene can be induced by a BmNPV virus to be subjected to down-regulated expression; the BmPEPCK-2 gene is subjected to increment expression in a BmE cell and then is infected by a BmNPV-GFP virus; a result shows that the increment expression of the BmPEPCK-2 can remarkably inhibit the multiplication of the BmNPV virus, showing that the BmPEPCK-2 gene is a key gene influencing the resistance of the domestic silkworms; and theoretical foundation and target genes are provided for cultivation of transgenic resistance varieties of the domestic silkworms.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to the silkworm BmPEPCK-2 gene, and also relates to the application of the silkworm BmPEPCK-2 gene. Background technique [0002] The silkworm is an important economic insect, and silk is an important raw material for the silk industry. 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. [0003] Because BmNPV is a serious hazard in sericulture production, researchers have always hoped to find out the key genes that aff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12A01K67/04A61K31/7088A61P31/20
CPCA01K67/0333A01K2217/052A01K2227/70A61K31/7088C07K14/43586
Inventor 蒋亮徐国文郭慧珍夏庆友
Owner SOUTHWEST UNIV
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