Cloning And Analysis Of Eukaryotic Translation Initiation Factor 5A And Cyclophilin A, Two Abundant Gene In The Tissue Of Silkgland In The Silkworm, Bombyx Mori

Based on the conservation of genome in evolution genes in different species with the same function often share homology with different degrees.The homology is instructive to clone silkworm novel genes and study their function. Besides, using homology among genes to clone new members of gene families becomes an efficient strategy. In addition, expression sequence tag data suggests a new way for finding tissue specific expression genes.The silk gland of B.mori is a long paired organ specialized in the synthesis and secretion of the silk protein fibroin.It represents one of the most active protein synthesizing systems among all organisms and is a model for research on physiological processes. The thesis includes three parts: in the first and second part of this study, it report the identification and characterization of two novel silkworm genes BmeIF5A and BmCyPA. In the third part, we describes the identification, classification, and functional analysis of silkworm cyclophilin gene family.In this study .using homologous strategy,we cloned a novel silkworm gene BmelF5A. We also cloned and identified the gene of silkworm using the protocol of 3 Rapid Amplification of cDNA Ends(3 RACE),termed it as BmeIF5A-2. It had the same ORF as the BmeIF5A,but a poly(A) tail.These results of the structure of the BmelF5A and BmeIF5A-2 indicated that BmelF5A gene is likely to contain two transcriptional types,and differences length of 3 untranslated region. An alignment of the coding sequence with the silkworm genome sequences reveal that there were 3 exons and 2 introns in BmelF5A. The deduced 160 amino acid sequence of the BmeIF5A coding a protein had a molecular mass of 17.5kDa and a pI of 5.16,which shares a conserved hypusine modified domain embedded in a 12 aminal acid sequence(STSKTGKHGHAK). 42 homologous sequences were aligned with the BmeIFSA sequence .It showed that the silkworm elF5A protein was conserved in almost eukaryotes comparing with other species. The phylogenetic analysis showed that BmeIF5A and three insects eIF5A gene are in the same clade. RT-PCR amplification in seven silkworm tissues revealed that BmeIFSA is expressed especially highly in silkgland,which indicated that the abundant may relate to the the synthesis of the silk protein fibroin of silkgland,and the level of this factor is likely to influence cell viability.We also cloned a novel silkworm gene BmCyPA. An alignment of the coding sequence with the silkworm genome sequences reveal that there were 2 exons and 1 intron in BmCyPA. The deduced 165 amino acid sequence of the BmCyPA coding a protein had a molecular mass of 19.4kDa and a pI of 8.79.Some of the homologous sequences were aligned with the CyPA sequence . This comparison shows that BmCyPA contained all of the residues participate in contact with the side chain of CsA and residues involved in peptidylprolyl cis-trans-isomerase activity. That means it may have peptidyl prolyl cis-trans-isomerase activity and can combine with CsA.RT-PCR amplification in seven silkworm tissues revealed that BmCyPA is expressed especially highly in silkgland, which suggesteded that it may play an important role in the fibroin folding,finally influence the rate of secretion of the silk protein fibroin.This paper describes the identification, classification, and transcriptional analysis of novel genes...