Cloning And Expression Analysis Of Epithelial Chitin Synthase Gene In Bombyx.mori

Insect cuticle plays a vital role during its growth and development, and the structural and functional integrity of the cuticle strongly depend on the chitin in the cuticle. Although many insect chitin synthase (CHS)genes have been reported, there is no information for silkworm chitin synthase (BmCHS).Researches on the expression pattern of chitin synthase, in vitro gene silencing, and phenotypic analysis of genetic mutant domestrated that chitin synthase is responsible for chitin synthesis in the insect cuticle. Abnormalties in the chitin synthesis will directly affect the cuticle structure and lead to the abnormal distribution of cuticular proteins and their functions. Therefore, abnormalties in the chitin synthesis will disturb the growth of insect at every metamorphesis developmental stage.The silkworm is an important ecnomic insect and the model organism of lepidotera. Chitin synthase is an important target gene for insect control because chitin does not exist in the vertebrates. Studies on the silkworm chitin synthase will increase our knowledge of the mechanism of silkworm chitin synthesis and lay a foundation for its application in the insect control.This study analyzed the sequence and its structure of BmCHS1 based on the information from the silkworm genome and microarray database. BmCHS1 cDNA was cloned and its spatial and temporal expression patterns were analyzed using RT-PCR.The results are as follows:1.cDNA cloning of BmCHSl geneOne genomic fragment responsible for the chitin synthesis was identified from the silkworm genome database through bioinformatic analysis.The cDNA sequence of the gene, named as BmCHS1,was cloned using the molecular cloning technologies according to its EST sequence. The whole length of BmCHS1 cDNA sequence is 5293bp inclucing 4188bp of ORF,726bp of 5'UTR and 379bp of 3'UTR.2. The gene structure of silkworm BmCHS1Silkworm BmCHSl was located on the chromosome 4 containing 21 exons and 20 introns. The analysis showed that BmCHSl was a 13-transmembrane-helix membrane protein with a theoretic pI 6.32 and a molecular weight of 159.5KD.SignalP 3.0 Server predicted that the first 17 peptids of this protein was a signal peptide. There were 7 transcriptional regulation sites upstream the gene including hunchback and Br-C.3. Evolutionary analysis of silkworm BmCHS1Multiple sequence alignment of different insect CHS 1 proteins indicated that CHS 1 showed higher homology to those chitin synthases expressed in insect cuticles. Silkworm BmCHSl has the highest similarity with SeCHS 1 compared to other insect chitin synthases. It was further found that insect chitin synthases are distinct from those present in fungi.4. The spatial and temporal expression patterns of silkworm BmCHS1RT-PCR analysis of BmCHS1 gene expression in the tissues from the 5th larvae day 3 was carried out. The results showed that BmCHS1 was mainly expressed in those tissues from the ectoderm, such as head, trachea, and epidermis. No expressional signal was detected in other tissues. The microarray data showed that BmCHS1 was expressed through the whole life of silkworm and was upregulated during each molting and metamorphosis stage, indicating that the expression of BmCHS1 may be regulated by the titer of ecdysteroid hormone.