Screening And Cloning Tail-regeneration Related Genes Of The Lizerd (Eremias Multiocellata) By Suppression Subtractive Hybridization

The phenomenon of Eremias multiocellata 's tail autotomy is a natural protective adaption when it confronts to the enemy or danger. After the tail autotomized, the disconnected position had strong regeneration capacity. It is the core of life science, which refers to the reconstruction of tissue and organ, cell proliferation, cell differentiation and dedifferentiation, and the regulation of physiology-biochemistry. Expression of abundant genes is altered in both quantity and quality during regeneration. To finding out their differences and their internal association is the premise of elucidating the exact molecular mechanism of tail-regeneration. It is very important to clarify the differential expressed genes in musculature separated from original tail and regernerated tail of Eremias multiocellata, so we achieved the purpose by suppressive subtractive hybridization technique (SSH), a new method for screening differential expressed genes.We investigated the Eremias multiocellata, which is a species of terrestrial ectothermic vertebrates and distributing in the desert and hemieremion area in the northwest of China. The tails autotomized by external stimulus including pressing, squeezing and drawing. Regneration was completed under different environmental conditions in 4-10 weeks. mRNA was extracted from the original tail's musculature (dirver) and regernerated tail's musculature (tester), then reverse transcripted into cDNA. The cDNA was cut with Rsa I enzyme and conjuncted to the adaptor. Subtractive library was constructed by suppressive subtractive hybridization technique, then the product was conjuncted with T/A carrier and transformed into Escherichia coli (E coli).Fianlly, sequence analysis and homology blasr was performed in the positive clone picked up randomly.The cDNA subtractive library of differential expressed genes was constructed successfully. Ten positive clones which contained inserted fragment were picked-up randomly to make sequence analysis and altogether 8 kinds of up-regulated expressed genes obtained. Through homology blast, we found that 6 sequences were different but high homologous to reported genes and 2 sequences were unknown-function genes.Among the genes, some directly related to tail-regeneration of Eremias multiocellata. The main function of them are coding protein involved in the regulation of cell growth, material metabolism and cell apoptosis. Cloning these genes established the base of making further study the function of related genes in tail-regeneration.