Promoter of nosema bombycis inducible expression gene BmPUGT2 and application of promoter

A technology of silkworm microparticles and inducible expression, applied in microorganisms, animal cells, glycosyltransferases, etc., to achieve the effect of good application prospects

Active Publication Date: 2021-03-19
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few reports on mi

Method used

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  • Promoter of nosema bombycis inducible expression gene BmPUGT2 and application of promoter
  • Promoter of nosema bombycis inducible expression gene BmPUGT2 and application of promoter
  • Promoter of nosema bombycis inducible expression gene BmPUGT2 and application of promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Silk silkworm micropelic induced Bmpugt2 gene expression

[0022] Depending on the silkworm genomic database Silkdb (https: / / silkdb.bioinfotoolkits.net / main / species-info / -1) and NCBI (https: / / www.ncbi.nlm.nih.gov), get BMPUGT2 (AK378453.1) Gene's CDS sequence (SEQ ID NO: 1), such as figure 1 As shown, BMPUGT2 gene sequence diagram, the hemorrhage bold ATG represents the translation start site, the TGA indicates the termination codon, the gray frame represents the non-translation zone, and the detection primer of the gene is designed according to the gene sequence of Bmpugt2. Bmpugt2-f and bmpugt2 -R, the silkworm ACTIN 3 gene is innergin, the primer is BMA3-F and BMA3-R.

[0023] Bmpugt2-f: 5'-AtcattcacagTacgtat-3 '(SEQ ID NO: 2)

[0024] Bmpugt2-R: 5'-Gaaacattattattgt-3 (SEQ ID NO: 3)

[0025] BMA3-F: 5'-AtggggCgctcctccaagaacg-3 '(SEQ ID NO: 4)

[0026] BMA3-R: 5'-ctacaggaacaggtggggTGGCGGG-3 '(SEQ ID NO: 5)

[0027] Made of normal silkworm breeding in standard environment...

Embodiment 2

[0029] BMPUGT2 promoter carrier construction

[0030] Depending on the silkworm genomic database (https: / / silkdb.bioinfotoolkits.net / main / species-info / -1), the genomic sequence of the first 1247 bp of the BMPugt2 gene (SEQ ID NO: 6), such as image 3 As shown, the lowercase the 5'utr area of ​​BMPugt2, the upper-write slope bold ATG represents the translation start site, using the website to analyze this genome sequence (https: / / www.fruitfly.org / seq_tools / promoter .html) found that in this sequence contains a typical promoter structure area, the region-specific primer PPUGT2-F-ECORI and PPUGT2-R-BamHi amplification of the sequence, the sequence is as follows:

[0031]

[0032]

[0033] The genomic extract kit (D3396, Omega) extracts the genomic DNA of the silkworm lottery, and uses primer PPUGT2-F-EcoRi, PPUGT2-R-BAMH and Q5 high-fidelity enzyme (M0493, NEB) to carry out PCR Amplification, its amplification conditions were: 98 ° C pre-degeneration 30s; 98 ° C denaturation 10s,...

Embodiment 3

[0036] BMPUGT2 promoter function verification

[0037] The PSL [PPUGT2-MCHERRY-SV40] expression vector constructed in Example 2 was transfected into the BMN-SWU1 cell line according to the plasmid: liposome = 1: 2, Ug: ul, and added silkworm microparticles, and silkworm. The ratio ratio of the fine particles and cells was 10: 1 to add PBS for control, and the red fluorescence was detected by fluorescence microscopy after 72 h. Such as Figure 4 As shown, in the cells transfected with PSL [PPUGT2-MCHERRY-SV40], when the silkworm micropel is added, the promoter PPUGT2 is activated and induces the expression of the downstream MCHERRY gene, and the cells are red-fluorescent, and the control group PBS is No red fluorescence, thereby explaining that the promoter PPUGT2 is the purpose of activating activation, achieving regulation, which can be activated by silkworm fine particles.

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Abstract

The invention belongs to the technical field of bombyx mori transgenosis, and particularly relates to a promoter of a nosema bombycis inducible expression gene BmPUGT2 and application of the promoter.A nucleotide gene of the promoter is shown as SEQ ID NO:6, and the promoter can drive an exogenous gene to be expressed in bombyx mori under induction of nosema bombycis, the promoter is suitable formolecular biology theoretical research such as gene function analysis and the like, meanwhile is suitable for bombyx mori variety improvement by utilizing genetic engineering, particularly suitable for bombyx mori nosema-disease-resistant variety cultivation, and has a good application prospect.

Description

Technical field [0001] The present invention belongs to the field of silkworm transgenic technology, and in particular, the promoter of a silkworm microparticle induced expression gene Bmpugt2 is used. Background technique [0002] Microsporosis is caused by intracellular specialty parasitic microsporium, endangered human health, economic animal industry development and ecological environment stability. As the traditional advantageous industry in my country, the silk industry has also been affected by the disease of microsporne disease. At present, my country's direct economic losses brought about hundreds of dollars per year, the main silkworm field for preventing control of the disease is half a total expenditure of the total expenditure. However, silkworm materials that have not been found to be resistant to this disease have not yet been found. [0003] With the breakthrough and perfect technology of genetic technology, genetically modified technology has become a powerful to...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/65C12N15/64C12N5/10
CPCC12N9/1051C12N15/85C12N15/65C12N15/64C12N5/0601C12Y204/01017C12N2830/002C12N2800/105C12N2510/02
Inventor 李春峰于滨潘国庆周泽扬
Owner SOUTHWEST UNIVERSITY
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