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Enhanced Expression of Antiviral Factor Bmlipase-1 by PiggyBac-based Transgenic Technology in Silkworm, Bombyx Mori

Author: ChenJie
Tutor: ZhaoPing；XiaQingYou
School: Southwestern University
Course: Special Economic Animal Feeding
Keywords: Transgenic system Pancreatic lipase Non-transgenic Silkworm Gene transfer vector Promoter Challenge Viral resistance GM technology Antiviral protein
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 24
Quote: 2
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Abstract

Lipase (Lipase), the international biochemical Federation named CODEN EC.3.1.1.3, the name of the Chinese system triacylglycerol acyl hydrolase. The lipase is widely exist in animals, plants and micro-organisms belonging to the α / β hydrolase family. The main role of the lipase in the organism is the hydrolysis of triacylglycerols, diacylglycerols, monoacylglycerols, glycerol, as well as free amino acid ester bond. Silkworm fat enzyme 1 (BmLipase-1) has been confirmed with a strong resistance to the role of endogenous protein of Bombyx mori nuclear polyhedrosis virus (BmNPV), and its expression and stress due to virus attacks and raised. The thesis of this study was to investigate the incremental expression of endogenous viral resistance protein Bmlipase-1 viral resistance, in order to provide new ideas for silkworm breeding for disease resistance. Based on a detailed map of the silkworm genome and whole genome sequence identified silkworm lipase gene and analysis of the the relevant gene structure and expression characteristics. Further, by the piggyBac transposon-based transgenic technology, constructed actin A4 promoter and viral gene IE1 promoter start BmLipase-1 expression vectors, microinjection, to obtain transgenic individuals, and transgenic lines for virus resistance detection. The main findings of this paper are as follows: 1. Silkworm lipase bioinformatics analysis based on sequence similarity and functional domains conserved in the silkworm genome database retrieval possible silkworm lipase gene. Successfully identified in the silkworm, 30 members of the lipase gene family, chromosomal location analysis show that the entire family members dispersed chromosome the positioning analysis showed Bmlipase-1 is located on chromosome 5. Analysis Bmliase2 gene may duplicate genes and Bmlipase1 mutually combining evolutionary tree. Chip data show silkworm lipase is mainly divided into three types, an expression in the fat body and gonads a expression in the intestine and the blood, an expression in the silk gland. Viral resistance the Bmlipase1 with Drosophila melanogaster lipase and the lipase protein homologies were 56% and 21%, respectively, tissue microarray data analysis showed that its expression in the midgut of fifth instar 3 days and blood to the midgut The highest expression level the developmental stages chip data after its Shangcu expression BmNPV induced chip data showed that its expression is not affected by the virus induced changes. 2. Incremental expression of Bmlipase-1 transgenic expression vector construct to fifth instar 3 days in the midgut cDNA was cloned from the Bmlipase-1 KCDS-simple by pMD19T, L4440, and pSLfa1180fa and piggyBac [3 × p3 EGFP afm] and other basic carrier successfully constructed the incremental expression Bmlipase-1 transgenic expression vector pBac IE1Pqipase-sv40-3 × P3-EGFPafm] and PBac [A4P-lipase-sv40-3, x P3-EGFPafm]. Gene transfer vector detected by restriction enzyme digestion and confirmed by sequencing correctly. 3. Genetically gene-positive individuals to obtain and molecular detection of the use of constructed transgenic vectors, select stagnation fertility silkworm varieties large manufacturing as a receptor material transgenic injection filter containing the marker gene EGFP-positive individuals, and multi-generational culture, successfully obtained to the IE1 promoter start Bmlipase-1 expression the the transgenic lines IEI-Bmlipase-1 and for the the promoter start Bmlipasel expression transgenic lines the A4-Bmlipase-1 to A4. By southern hybridization, RT-PCR and quantitative PCR technologies such as genetically modified strains of transgenic positive copy number and mRNA expression levels were detected. The results show that there are three copies of A4-Bmlipasel, IE1-Bmlipasel containing a copy. At the transcriptional level, turn creating a big variety the gene system Bmlipase-1 mRNA expression was significantly higher than that of non-transgenic and the A4-Bmlipasel turn gene system Bmlipase-1 mRNA expression is higher than the IEI-Bmlipase1 transgenic system. The research results show that, IEI-Bmlipase1 transgenic lines 3 instars before Bmlipase-1 mRNA expression significantly increased expression after 4 instar little gap between the non-transgenic system. The transgenic positive strains resistant virus detection in order to verify that the incremental expression of BmNPV silkworm virus resistance, we are of the the IEI-Bmlipase1 obtained transgenic lines challenge testing. Established after ID50 test the the two virus attack concentration of 2.66 × 10 ~ 4 and 3.46 × 10 ~~ 4. Exchange that toxicity results were analyzed statistically, that Bmlipase-1 in the first 8 days after challenge, the concentration of 2.66 × 10 ~ 4 transgenic group 1 compared to the same concentration of the antiviral effect of the control group 1 was significantly reduced mortality 20 %, while the concentration of 3.46 × 10 ~ 4 transgenic group 2 also have some antiviral effects of mortality by about 15%. Graph 5-8 days after the challenge transgenic group compared with the control group of the same concentration and delayed the time of death. Through the material within 24 hours after the exchange that poison quantitative PCR analysis the transgenic group the lipase-1mRNA of levels higher than the control group, combined with the challenge data presumably the obvious resistance lipase incremental expression, which the overall level of such delay the death rate of the transgenic lines. And because of a variety of reasons such as secondary infection, leading to late silkworm that mortality becomes very close to a large number of deaths only. In summary, this paper based on the silkworm genome-wide fine mapping and genome-wide expression microarray data, system identification and analysis of the silkworm lipase gene. Successfully constructed increment expression virus the resistance protein Bmlipase-1 piggBac gene transfer vector microinjection success ie1 promoter to start the Bmlipase-1 expression ie1 lipase-1 transgenic lines A4 promoter starts Bmlipase-1 expression The A4 lipase-1 transgenic lines, the amount of gene expression analysis showed that the virus resistant transgenic system protein Bmlipase-1 expression was significantly higher than the non-transgenic system. Virus attack transgenic system test results show that, compared to non-transgenic, transgenic system of BmNPV showed varying degrees of resistance to improve. The research results show that, based on transgenic technology, incremental expression of endogenous viral resistance protein is also a means to enhance host resistance, disease resistance breeding some reference.

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