Study On Gene Expression And Function Of Chitinase And Chitin Synthase In The Silkworm, Bombyx Mori

Chitin is the special and important biological polymer of insects and other arthropods nematodes and fungi, which supporting insect epidermis, trachea and the peritrophic membrane of intestinal epithelial cuticle. The metabolism of chitin in reliance on chitin synthesis and chitin degrading enzyme, as well as its strict coordination control chitin synthesis and degradation that compatible with the development of the organism. Insect growth and metamorphosis are strictly dependent on the structural changes of tissues and organs which contain the chitin. It is possible to control their growth and development, even their life when control the synthesis and degradation of chitin on insects and other invertebrates. Therefore, control the synthesis and degradation of chitin will become the research focus on specific new insecticides and fungicides.At least sixty percent of the agricultural pests belong to lepidoptera, so reseach on the safe and efficient prevention methods are important. Based on the silkworm genome database resource, we use RACE and molecular cloning, bioinformatics, RNAi technology to study the chitin synthase homologous gene BmChsA and BmChsB, and the chitinase gene BmChi-h and BmCht of the lepidoptera model insects of silkworm, the main results are as follows:1. We first cloned Bombyx mori chitinase homologous gene BmChsA and BmChsB cDNA, of which the lengths were 4943bp, 3055bp, respectively. Semi-quantative PCR indicates that BmChsA was specificity highly expressed in head and perisoma, and BmChsB was mainly expressed in midgut in high level; during ecdysis, the expression of BmChsA was up-regulated, and that of BmChsB was down-regulated.2. By RNAi technology, we investigates that 3rd instar molting silkworm was injected with 5μg per head BmChsA dsRNA or siRNA for 36h, 10% and 40% silkworms presented abnormal ecdysis, respectively. They turned out to be small size, half-ecdysis, and none-ecdysis. In these symptoms, the BmChsA transcription level of abnormal ecdysis larva was obviously lowed, and the effect of siRNA treated samples were significantly better than that of dsRNA. There was no special changes in phenotype by injecting 5μg dsRNA per head Bombyx mori chitinase gene BmChi-h and BmCht to 4th instar molted silkworms.3. After injecting 20-ecdysterone (20E) to 4th instar molted silkworm, the old epidermis started to dissolve and new epidermis regenerated at 12h; ecdysis precursor started at 36h, and parts of the individual have been molted, which was earlier than control group and juvenile hormone analogue Methoprene treated group for 36h, however, no significant differences in growth and development of 4th instar larva were appeared in Methoprene treated group and control group.4. Gene expression profiling of 4th instar molted silkworm shows that the expression levels of chitinase gene BmChi-h and BmCht in head and epidermis were obviously up-regulated by 20E, however, it suppressed the expression levels of chitin synthase gene BmChsA and BmChsB, but the suppression recoverd after 30h. The different affects of BmChsA and BmChsB treated by Methoprene indicated that differences maybe existed in metabolism regulate mechanism.5. Under the induction of different exogenous hormone, the Bombyx mori chitinase gene BmChi-h and BmCht in head and epidersim kept exactly similar tendency, which explained that those two genes played similar activity in silkworm. Chitin synthase gene BmChsA was specificity expressed in head and epidersim of larva, and BmChsB was only expressed in epidersim; In 4th instar larva epidersim, the expression level of BmChsA was higer than that of BmChsB, but at 4L-60h, the expression of BmChsB was restrained by Methoprene, while the expressions of BmChsA and BmChsB in epidersim were significantly enhanced by 20E at 4-60h. The conclusions above, which BmChsA and BmChsB were regulated by hormone, were similar to that of drosophila CHS-1 and CHS-2. We supposed their regulation mechanism were uniform.