Analyses On ESTs And Gene Expression Profile Of Several Tissues Of Bombyx Mori

The "non-bias" cDNA libraries were constructed by directional cloning from 5'-end of 12 distinct tissues at different developmental stages in our laboratory; and then random sequencing was carried out with a large number of cDNA clones from each library. This investigation is based on the analyses of large scale EST data, with the aims of finding important functional genes associated with sex differentiation, metamorphosis, embryonic development and substance metabolism and obtaining the information of gene expression related to tissue-specific and tissue differentiation, providing the bases for exploiting benefit gene resources and effective control of lepidoptera pest1 The clustering of gene expression profile based on EST dataThe clustering method of gene expression profile based on large scale EST data was adopted for the first time to extract important information from a large amount of sequence data obtained in our research .The similarity in expression profile between contigs (EST>5) or between cDNA libraries was estimated by Pearson's coefficient. A gene or library pairwise Euclidean distances matrix was computed from the correlation coefficient matrix and then used to build their associate dendrograms according to the UPGMA algorithm implemented in the neighbor program. The dendrogram reveals the similarities of contigs or libraries. Similar contigs or libraries are clustered together. Clusters of contigs sharing related expression patterns were obtained by use of this method for further research and the tissues sharing similar profile were recognized with this analysis as well.A rigorous statistical test established by Audic and Claverie was used to assess the reliability of the identification of differentially expressed genes from EST counts sampled from pairwise comparison, so as to discover the differentially expressed genes. A lot of genes with differential expression patterns between two tissues or stages were obtained by this analysis.These methods were applied in this research successfully. It is expected to build a foundation for further data analysis in the future.2 The characteristics of large-scale EST data2.1 cDNA Library Construction and Initial AnalysisThe experimental material was a domestic strain "Dazao". The twelve "non-bias" cDNA libraries prepared from important tissues of 5th instar larvae, including silk gland, midgut, fat-body, hemocytes, ovary, testis and embryo, cultured cell. The large- scale EST sequencing was made by directional cloningmethod from 5'end and 81,625 validity ESTs were obtained. By assembling with Pharp program, 24,678 unigenes containing 11627 contigs and 13051 singletons were gainedThe homologous analysis of ESTs shows that 7,944 unigenes have significant homology with data in GenBank and other databases. Among these unigenes, 3,899 unigenes (32,605 EST) match known genes with function annotation, 4,045 unigenes (16.087EST) match known sequences without function annotation, and 16,734 unigenes (32,933ESTs) are new sequences. A total of 60% ESTs are functionally unknown sequences.2.2 The Clustering Results of Gene Expression ProfilesThe Clustering of gene expression profile for 3,064 contigs (EST>5) and 12 libraries was made separately and their dendrograms built respectively. The results showed that the- contigs sharing similar expression patterns were grouped into a cluster in each library, from which the library-specific genes were obtained and applied to further research. The dendrogram of libraries showed that the expression profiles of cultured cell, midgut and unfertilized embryo were significantly different from those of other tissues. Tissues between female and male fatbody, female and male hemocytes, silk gland and ovary exhibited obvious similarity in expression profiles. This is an interesting phenomenon that is worth of further study.2.3 Genes Expressed in AH TissuesAnalysis on contigs expressed in all tissues showed that this fraction contained 12 known genes and 7 functionally unknown sequences. The kno...