Construction Of Tissue-specific-expression CDNA Library Of Eyestalk Optic Ganglion In Eriocheir Sinensis

Eriocheir sinensis is a kind of health aquatic products which is delicious and nutritious. In recent years, breeding E. sinensis rises around the country, the problem of neoteny shows up in breeding cultivate process, and causes much damage in production. These problems relate to E. sinensis secreting neuropeptide hormone abnormally. Hormone which is secreted by crustacean eyestalk neurosecretory cells is important to regulation and control its growth and development.This experimentation devoted to construct the cDNA library of E. sinensis eyestalk speciality expression with SSH, for clone tissue-specific-expression cDNA, research the function, probe into molecular mechanism of developmental and lay the foundation for explan the molecular mechanism of sexual precocity in E. sinensis.In this experimentation, we distilled total RNA of high quality in eyestalk and muscle in E. sinensis. The total RNA was reversed transcription, then we got the cDNA, and the Rsa I efficiency for eyestalk and muscle cDNA is high; the adaptor ligation efficiency of tester cDNA satisfied the need of following experimentation. Beta-actin is substracted after twice hybridization and suppress PCR; substractive efficiency enhanced 215 times; that enriches eyestalk speciality expression gene. The cDNA library of E. sinensis eyestalk speciality expression was constructed. Connected pured RCR product with pMD19-T, tranceformed E. coli. JM109, filtrated the library with resistance filtration, and then we got 324 positive cloning. Choose PCR clone product randomly, the resulet prove that at least 75% clone could amplify, PCR amplification segment mostly center in 200bp-500bp, and cDNA library is high quality.Select 3 groups positive PCR product to sequence. Contrast BLAST:there is no similarity to 227bp, and we need to check up if it is a new sequence; the 309bp sequence is MIH segment; the 438bp is possible to be CHH variant precursor. It proved that this method is feasible to construct the cDNA library of E. sinensis eyestalk speciality expression. If we go on filtrating the possive chone, finally we can get genes of E. sinensis eyestalk speciality expression. In this experimentation we constructed the cDNA library of E. sinensis eyestalk speciality expression with SSH successfully, and provide material to new gene.