Cloning Of LHβ And LHR Gene And Their Tissues Expressing Analysis In Andrias Davidianus

Chinese giant salamander(Andrias davidianus) belongs to a national second class protected animal which has been listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in Appendix I, it belongs to the Amphibia, Caudata, Cryptobranchidae. As special status, it not only has a high scientific value, but also has important economic value in terms of medicine, edible and ornamental. Study on Chinese giant salamander, however, mainly concentrated in the population distribution and ecological protection, embryonic development and reproductive system structure and process of fertilization, physiological, biochemical and genetic basis research, but rarely for the study of its molecular aspects. In this study, take the RT-PCR and RACE to cloning LHβ and LHR gene3’end cDNA sequence of the Chinese giant salamander, and select a number of LHP and LHR genes sequences of other representative species to establish phylogenetic relationship tree, in order to further explore the provide the basis for the origin of the giant salamander and the evolution in the Amphibia. Also using semi-quantitative method to analyze the expression of these two genes in different tissues, to provide a theoretical basis for research of the LHβ and LHR gene function and molecular evolution in the amphibian, and to provide a basis for LHβ and LHR gene expression in vitro and further application in improving the giant salamander breeding. The main findings are as follows:1. According conserved sequence of LHP genes and LHR gene in other species which have been reported in the NCBI to primer, the Chinese giant salamander pituitary as material, and to extract total RNA, then reverse transcription into cDNA, and take PCR amplification, respectively, obtained a193bp and182bp fragment. After sequencing, the nucleotide and amino acid blast to conservative fragment of the Chinese giant salamander LHβ gene and LHR gene proved to be successful cloning. 2. In this study, using RT-PCR and RACE, a conserved sequence were designed for two specific primers Chinese giant salamander LHβ gene and LHR cDNA from the3’end sequences of unknown, and three specific primers the5’end sequence of the unknown. Sequencing and splicing, respectively obtained490bp and1069bp. Sequence analysis showed that the Chinese giant salamander LHβ gene3’end cDNA sequences coding for76amino acids of LHR gene3’end of the cDNA sequence encoding374amino acids.3.By RT-PCR and RACE techniques, designing three specific primers on the basis of the obtained3’terminal sequence for amplified the Chinese giant salamander LHP gene and LHR cDNA of5’end of the unknown sequence. After sequencing, only the LHP gene got about400bp. Splicing LHβ gene sequences, which got its full-length cDNA893bp. Analysis showed that the Chinese giant salamander LHβ gene cDNA sequence encoding130amino acids.4. According to the Chinese giant salamander LHβ full-length cDNA sequences and LHR gene3’cDNA sequence to design specific primers, which amplified fragment size of349bp and204bp respectively; Chinese giant salamander β-actin gene as reference gene, according to the known sequence to design specific primers, amplifiying fragment size of123bp. By semi-quantitative method,building an optimal system to study the LHβ and LHR gene expression in different tissues, the analysis showed that, LHP, of LHR gene expression in all the tissues, which the highest expression in the testis was61%、75%, respectively. Expression of LH in amphibians is similar to some other species, not only luteinizing and promote the development of germ cell function, may also have other mechanisms or functions. It may provide new evidence for the hypothesis which the expression of the gonadotropin extrapituitary may be a common physiological phenomenon.