Histopathology Study And CDNA Library Construction And Identification Of Liver From Chinemys Reevesii Marinated In Malachite Green

Turtles have important uses and economic value in medicine, view and admire, edibility and scientific research. The culture of turtle can add natural resources, keep ecological equilibrium and meet the edibility as well as medicine demands of humanity. In aquiculture of China, Chinemys reevesii is the commonest animal. In artificial propagation, the emergence of illnesses increasingly by the increase in cultural density and the pollution of cultural environment. The illnesses of turtles have affected the development of turtle breeding. The prevention and cure of turtle's illness is important increasingly.Malachite green is commonly used in disease prevention of aquatic animals. As we all know, to human, malachite green has teratogenic, carcinogenic etc side effects, long-term use will cause pollution and other negative consequences, according to the agricultural products pollution-free quality standards, malachite green is banned drug in aquaculture. In the turtle's diseases, saprolegniasis is the most common one. it effects most to baby turtles, and it's very difficult to treat, except malachite green, the treatment effect of other medicines are not good. In this case, some farmers still use malachite green to control saprolegniasis. Therefore, the discussion about the harm of malachite green to turtles is very significant.Through the application of paraffin section technology, we study the histopathological changes after turtle was affected by malachite green, the results showed that the turtle liver was affected most by malachite green, liver cell appeared steatosis and tissue appeared big empty spots, cytoplasm appeared large vacuoles, partial liver cell dissolve necrosis. Pathological changes on other tissue and organs are not obvious. In view of this situation, we use SMART technology to construct the cDNA library for turtle liver which was affected by malachite green.By using RNAiso Reagen to extract total RNA from the malachite green affected turtle liver, reverse it to first-strand of cDNA, and then reverse it to second-strand cDNA by LD-PCR, proteinase K digestion, Sfi I enzyme digestion, through CHROMASRIN-400 column, recycling cDNA which more than 500bp, cDNA connect with Trip1Ex2 carrier, and connect the carrier invitropacking to construct cDNA library. Titer of the library is 7.5×109 pfu/ml, insert segments average length is longer than 1.0kb, and recombinant frequency is 94.38%.Constructing the cDNA library can lay a foundation for further study such as genome sequencing, related gene cloning and screening etc, through comparative analysis with the normal gene sequence of turtle, then clarify the teratogenic and carcinogenic mechianisms of malachite green.