Genomic Analysis About The SHsp, Cloning And Expression HSP20.8A And Fluorescent In Situ Hybridization In Silkworm, Bombyx Mori

The silkworm is the one of the most important economic insects in theworld. It has been domesticated for about 5000 years. The finished silkwormEST database in October 2002 and the finished whole genome sequencing inNovember 2003 provide abundant information which is of great importancenot only for sericulture but also for fundemental research of life science. Thesmall heat shock protein (sHSP) is a ubiquitous protein which can be foundnot only in prokaryote organism but also in eukaryote organism. Functionally,the sHSPs are involved in a variety of cellular processes, notably relate tocytoskeleton rearrangements and apoptosis. It endows cells with a transient,increased tolerance to heat and other stress. Prevent in vitro aggregation ofunfolding proteins. The sHSP bound to denatured protein and transferred thecomlex to adenosine triphosphate (ATP) dependent chaperones (such asHSP70) and refolded. In order to get an inventory about the sHSP of B.mori,the known sHSP sequences of human, mouse, D.melanogaster and B.moriwere used as query in B.mori database, the results was analyzed by comparinghethod. The known HSP20.8A's cDNA that accessed in NCBI (access numberAF315317) was used to construct into the baculovirus expression vector andhave it expressed in sf21 cell lines. The HSP20.8A gene was also constructedas the probe and hybridized it with the chromosome of B.mori. All the resultsare listed as below.1 The sHSP gene number and gene structure Use the 10 sHSPs of H.sapiens, 11 sHSPs from D.melanogaster, 8 sHSPfrom Mus and the 6 known sHSPs of B.mori as queries. A BLASTP researchwas performed in the silkworm genome database. The result is that there total - 85 -家蚕低分子量热激蛋白的基因组学分析与 HSP20.8A 的表达及 FISH 研究14 sHSPs in B.mori. Six genes are identical with the known sHSP sequences.The other 8 genes were the new found which have no identical sequence inNCBI. All the sHSP of B.mori have α-crystallin domain after scanned theconservative Domain of the sHSP genes, and the peptide length of conserveranges from 60 residues to 90 residues. Only three sHSP genes of B.mori haveintrons and the intron number for different sHSP gene ranges from 2 to 4. Theother 11 genes are intronless. A multi alignment was made regarding the 14sHSP. the result shows that the structure of the 14 sHSPs are mainly composedof three domains: the N-terminal domain which have poorly conservesequence and its length obviously variant; the alpha crystallin domain whichhave high similarity with the known conserve domain of most sHSPs, and theresidues length of this domain in B.mori ranges from about 60 residues to 90residues; The last domain is the short C-terminal domain.2 The tandem duplication phenomena about the sHSP It is interesting that 8 sHSP genes which have the character of tandemduplication locate at three scaffolds (Bmb002837, Bmb002838, Bmb002839and Bmb002840 locate at scaffold000419; Bmb007189 and Bmb007190locate at Scaffold001353; Bmb016798 and Bmb016799 locate atscaffold004261), and there no other functional genes in the same scaffold thatlocate between these sHSP genes. Another interesting thing is that there arefour genes of which the orientation is different from other four genes. Thereare no report about this special character.3 EST found of the sHSP gene Of the 14 genes, 13 have confirmed EST except for one gene, which geneID is Bmb036757. The EST number is different when it was transcripted bydifferent sHSP genes. Such as the EST found of Bmb002839 is 250, thenumber of which is much more than any other genes. The EST found ofBmb018979_Bmb018980 and Bmb002837 is 159 and 77 respectively. Indifferent tissues or organisms the transcription products of sHSP genes aredifferent such as of the total EST found 295 ESTs in unfertilized eggs, 71 in - 86 -山东农业大学硕士论文ovary, 58 in testis, 55 in silk gla...