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The promoter of inducible expression gene bmpugt3 of Bombyx mori and its application

A technology of silkworm microparticles and induced expression, which is applied in the direction of microorganisms, animal cells, glycosyltransferases, etc., and has achieved good application prospects

Active Publication Date: 2022-07-22
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on microsomia-inducible promoters in silkworm

Method used

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  • The promoter of inducible expression gene bmpugt3 of Bombyx mori and its application
  • The promoter of inducible expression gene bmpugt3 of Bombyx mori and its application
  • The promoter of inducible expression gene bmpugt3 of Bombyx mori and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Obtaining the gene sequence of BmPUGT3 inducible expression gene of Bombyx mori

[0024] According to the silkworm genome database SilkDB ( https: / / silkdb.bioinfotoolkits.net / main / species-info / -1 ) and kaikobase ( https: / / kaikobase.dna.affrc.go.jp / ), the CDS sequence (SEQ ID NO: 1) of the BmPUGT3 (BGIBMGA010295) gene was obtained, the detection primers BmPUGT3-F and BmPUGT3-R of the gene were designed according to the gene sequence of BmPUGT3, the silkworm Actin 3 gene was an internal reference, and the primer was BmA3-F and BmA3-R.

[0025] BmPUGT3-F: 5'-atggagattggattaaaagtag-3' (SEQ ID NO: 2)

[0026] BmPUGT3-R: 5'-ttacttttgattggacaaaacc-3' (SEQ ID NO: 3)

[0027] BmA3-F: 5'-atggtgcgctcctccaagaacg-3' (SEQ ID NO: 4)

[0028] BmA3-R: 5'-ctacaggaacaggtggtggcgg-3' (SEQ ID NO: 5)

[0029] Normal silkworm Dazao varieties were raised with artificial feed in standard environment (temperature: 25°C, humidity: 80%), silkworms from 5 instars, part of which were fed w...

Embodiment 2

[0046] Cloning of BmPUGT3 promoter and construction of its expression vector

[0047] The position of the ATG translation initiation site of the BmPUGT3 gene was determined in Example 1, and then the genome sequence 1931bp before the ATG translation initiation site of the BmPUGT3 gene was obtained according to the silkworm genome database, using the website (https: / / www.fruitfly.org / seq_tools / promoter.html) analyzed this sequence, and found that there is a typical promoter structure region (SEQ ID NO: 16) in this sequence. According to this, the PPUGT3 promoter-specific primer PPUGT3-F-EcoR of BmPUGT3 gene was designed I. PPUGT3-R-BamH I amplified the sequence.

[0048] PPUGT3-F-EcoR I: 5'-ccg gaattc acctaaatcttctgtacgcc-3' (SEQ ID NO: 17)

[0049] PPUGT3-R-BamH I: 5'-cgc ggatcc cattgaagatgaccatatgaat-3' (SEQ ID NO: 18)

[0050] Utilize the kit (D3396, OMEGA) to extract the genomic DNA of the silkworm Dazao strain, and take the silkworm Dazao genomic DNA as a template,...

Embodiment 3

[0055] BmPUGT3 promoter functional verification

[0056] The pSL[PPUGT3-mCherry-SV40] vector constructed in Example 2 was transfected into BmN-SWU1 cell line by liposome, and the transfection ratio of liposome and plasmid was 2:1, ul:ug. Incubate at 28°C, add Bombyx mori larvae after 12 hours, and the number ratio of Bombyx mori larvae to cells is 10:1. PBS is added as a control, and red fluorescence is detected by fluorescence microscope after 72 h. The result is as Figure 5 As shown in A, B, C, D, in the cells transfected with pSL[PPUGT3-mCherry-SV40], the promoter PPUGT3 was activated and the expression of its downstream mCherry gene was induced when the silkworm microparticles were added, making the cells red However, the control group PBS had no red fluorescence, which indicated that the promoter PPUGT3 could be induced and activated by Bombyx mori to achieve the purpose of regulation.

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Abstract

The invention belongs to the technical field of silkworm transgenic, and in particular relates to a promoter of the Bombyx mori larvae inducible expression gene BmPUGT3 and its application. The nucleotide gene of the promoter is shown in SEQ ID NO: 16, and the promoter can drive external The source gene is expressed in Bombyx mori by induction of Bombyx mori larvae, which is not only suitable for molecular biology theoretical research such as gene function analysis, but also suitable for the use of genetic engineering to improve silkworm varieties, especially the breeding of silkworm varieties resistant to microparticle disease. application prospects.

Description

technical field [0001] The invention belongs to the technical field of silkworm transgenic, and in particular relates to a promoter of Bombyx mori larvae inducible expression gene BmPUGT3 and its application. Background technique [0002] The silk industry is a traditional advantageous industry in my country. It still dominates the international market and is an important source of income for millions of farmers. Silkworm microparticle disease (Pébrine) is the number one disease that endangers the safety of sericulture production. The fundamental reason is that the pathogen of the disease, Nosema bombycis, can be transmitted vertically through eggs, causing silkworm seeds to be poisoned. At present, the direct economic loss caused by microparticle disease in my country is over 100 million yuan each year, and the main silkworm seed farms in the country spend half of the total expenditure on silkworm seed production for the prevention and control of the disease. However, no s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66C12N15/65C12N5/10
CPCC12N9/1051C12N15/85C12N15/66C12N15/65C12N5/0601C12Y204/01017C12N2830/002C12N2800/105C12N2510/02
Inventor 李春峰于滨潘国庆周泽扬
Owner SOUTHWEST UNIV