Functional Analysis Of Gene Promoter And Preliminary Research On Innate Immunity Of Malpighian Tubule From Silkworm, Bombyx Mori

The thesis is mainly consisted of two research aspects.1. Functional analysis of tissue- or spatio- specific gene promoter from silkworm,Bombyx moriThe domesticated silkworm, Bombyx mori, is not only one of the most importanteconomic insects, but also a model of the lepidopteran insects. Silkworm is one member ofthe holometabolous class of insects, which possesses four distinguished developmentalstages, i.e., egg, larva, pupa, and adult. The whole developmental course of silkworm resultsfrom the tissue- or spatio- specific expression of a large number of genes, which iscontrolled by promoter.In this paper, the regulation promoter regions of ovary non-coding RNA gene (ONCR),nonsusceptibility to DNV-2 gene (Nsd-2) and Bombyx mori hatching enzyme-like gene(BmHEL) are cloned from genomic DNA from silkworm. A series of luciferase and EGFPreporter plasmids, driven by ONCR, Nsd-2 and BmHEL promoters, are constructed,respectively. Via the transient expression systerm in BmN cells or silkworm transfected by ahelium burst biolistic device, the functional and characteristic analysis of the promoter areinvestigated. Under normal condition, transcriptional activities of ONCR, Nsd-2 andBmHEL promoter are very low in BmN cells, indicating that the BmN cells maybe lack oftissue-specific transcriptional factors. We also have a try to analyze the transcriptionalactivities on tissue level, but can not get significant results, which need further investigation.The current results will be helpful to apply of the cloned promoters in stable transformationcell system or transgenic silkworm.2. Preliminary research on innate immunity of Malpighian tubules (MT) fromsilkwormTo date, fat body is considered to be the major immune-responsive organ in silkworm.To the best of our knowledge, there has been no report on innate immunity of MT fromsilkworm.In this paper, the innate immunity of MT from silkworm was investigated, accordingto the reports on innate immunity of MT from Drosophila melanogaster. E.coli killingassay results shows that tubules have antibacterial activity, suggesting that MT of silkwormis also capable of sensing an immune insult. Recombinant virus BmNPV-EGFP was constructed and infected the silkworm through injection method. The investigation ofinfective stages indicates that the virus infects MT at the last stage. Two-dimensionalpolyacrylamide gel electrophoresis (2-DE) is used to separate the proteins of the MT, andthe results show that the BmNPV infection led to the change of proteomics of MT.Furthermore, spots that increase dramatically in the infected one are identified by means ofmatrix assisted laser adsorption ionization time of flight mass spectrometry(MALDI-TOFMS), and 1 protein spot has a good match with H+ transporting ATP synthasebeta subunit (β-H+ ATPase) of Bombyx mori. The quantitative real-time PCR resultsindicated that the expression ofβ-H~+ ATPase gene is different between the infected and thecontrol, suggesting thatβ-H+ ATPase maybe plays an important biological role in immunefunction of MT of the silkworm. The present results give the basic information for furtherresearch on the innate immunity of silkworm MT.