| Expressed Sequence Tag (EST) is important structural information of gene in the genome of organisms. Large-scale EST sequencing of cDNA libraries from specific Tissues, organ or cell can be directly used to analyze the expressed gene structure and charateristics, discover new gene,and construct the gene expressed profile of many tissue by ESTs.Silkworm is an important economic insect and a model organism of Lepidoptera insect In this study, silkland, middle gut, fat body, blood, egg(embryonic period) and cultured cell were selected to construct cDNA libraries. It is expected to discover a lot of genes associated with sex regulation, metamorphosis, embryonic development and metabolism by Large-scale EST sequencing and analyzing of these cDNA libraries.In addition, the function of fat body is protein storage, providing energy, Intermediary metabolism, it also was model Tissue during silkworm metamorphosis period. The function genes associated with it will be acquired and molecular mechanism of metamorphosis will be analyzed.1. Construction of silkworm cDNA libraryTotal RNA and mRNA of high quality were isolateted ,Double-stranded cDNA with EcoRI And XhoI overhang extension were synthesized and recovered. They were recombinanted with Plasmid. The cDNA library were obtained by transforming the recombinant into the host cell HB101.The recombination rate of these library was an average 93%.Primary sink size obtained 1.23X105 and its insert size ranged from 0.4-1.5kb.2. Large-scale EST sequencing and data analysiscDNA clone from the silkworm cDNA library were partial sequenced (EST sequencing),81625 ESTs sequence were obtained after removing the vector sequence,repeat sequence and germ DNA.The length of EST sequences ranged front 0.25-0.55kb.24678 unigene contained 11627 contig and 13951 singletons were gained by assembling, contig length was 0.2-1.2kb.We predicted that most of coding genes of silkworm were less than 1.2kb.The parameter of bioinformatics analysis of EST were: blastx, E30%;The parameter of bioinformatics analysis of EST were: blastx, E30%; blastn,E25%. There were 7944 unigene(32605 ESTs) matched with known genes, 4045 unigene (16087 ESTs) matched known sequences and 16734 UniGene(32933 ESTs) matched no sequences.The ESTs of known gene of silkworm have significant similarities with gene of silkworm and Drosophila melanogaster, Manduca Sexta, Anopheles gambiae, Homo sapiens and Mus Musculus in GeneBank. A lots of Functional gene associated with sex regulation, metamorphosis, embryonic develop and metabolism etc. were discovered.3. Study on gene expressed profile of fat body3.1 Characterization of gene expressed profile of fat body6337 unigene contained 4262 contig and 2705 singletons were gained. There were 1226 unigene (9264 ESTs) matched known genes,1341 unigene(2871EST) matched with known sequences and 3770 UniGene (6895 EST) matched no any sequences.Among the 502 known genes, there were 117 high abundant genes,234 middle abundant genes and 151 low abundant gene.All known genes of fat body grouped into cytoskeletal, extracellular matrix, Energy metabolism , hormones and hormonal regulation, signal transduction and cell regulation, transcription and translation, membrane-associated and substantial metabolism.3.2. Gene Expressed difference of fat bodyThe categories of gene most highly expressed in fat body during larval period were those participating in substance metabolism, and the next were those related with transcription and translation, signal transduction and cell regulation, energy metabolism; During pupal state, genes highly expressed were related with substance metabolism, cytoskeletal, transcription and translation, membrance-associated.Gene expression in fat body showed difference during metamorphosis development states. Among the eight kind of genes, expression of gene linked to cytoskeletal, extracellular matrix, energy metabolism ,signal transduction and cell regulation, transcription and tra... |
PDF Full Text Request