THE MOLECULAR CLONING OF OBESE cDNA OF ERYTHROCULTER MONGOLICUS AND ITS ORGANIZATION EXPRESSION SPECIFICITY

In this study,Erythroculter mongolicus were taken from the Nanhu Lake, the length and weight of male and female were about 16 centimeters, 112 grams and 18 centimeters, 120 grams, respectively. Tissues such as adipose, liver, heart, spleen, muscle, brain, ovary, kidney and spermary had been obtained from the fish and reserved.After washed with distilled water, total RNAs of each tissue samples were extracted by one step method and the extracted RNA was identified by Eppendorf Bio-photometer.All the total RNA that ratios of OD280/OD260 were diluted to 0.5μg/μL .The primers designed according to the consensus region of human and mouseob genes were employed to perform RT-PCR(reverse transcriptase-polymerase chain reaction). The product of RT-PCR from total RNA ofE.mongolicus fat tissue and pBluescript SK(+)vector were both digested by EcoR Ⅰand Hind Ⅲ, then linked and transformed intoE.coli competent cell.The positive recombinant plasmid was sequenced and got a 438bp coding region DNA sequence.Compared to the nucleotide sequences ofob genes in human, pig and mouse, theob genes of E .mongolicus had a homology as 82.9%, 84.0% and 99.1%, respectively.The nucleotide sequences ofob genes were very conservative among the four animal species.In comparison in the leptin amino acid sequence with human, pig and mouse, the homologies were 80.8%, 78.8%and 95.9%respectively.The result ofob gene tissue express specificity by RT-PCRmethod indicated thatob gene was expressed mainly in adipose and liver, less in heart, spleen, muscle, brain, ovary, slightly in kidney and none in spermary and intestines inE.mongolicus