草鱼LAT2cDNA基因克隆、序列分析及组织表达检测

Molecular Cloning,Sequence Analysis and Expression Detection of LAT2 from Grass Carp

目的:克隆草鱼LAT2cDNA基因,分析基因生物信息及在不同组织的表达情况。方法:采用RT-PCR方法从草鱼前肠组织克隆LAT2cDNA基因,用生物软件对基因序列进行基因生物信息分析;采用RT-PCR方法检测LAT2基因在组织中的表达情况。结果:成功克隆草鱼LAT2cDNA基因,基因长1 216bp,编码404个氨基酸;与斑马鱼的同源性高达92.5%,而与哺乳类动物的同源性在75.2%～85.2%之间;对其构建的基因系统进化树与传统形态分类相吻合;预测的12跨膜结构中第1到第6个跨膜区与其它动物类似,其中完成转运功能的主要跨膜部位与其它动物同源性高达92%;并在草鱼前肠、中肠、后肠、肝、肾、心、脑、肌肉和鳃组织均检测到基因的表达。结论:为进一步探讨鱼类氨基酸吸收转运代谢及氨基酸转运载体基因表达机理奠定基础。

Objective：To clone and analyse the LAT2cDNA gene of grass grap,detect the gene expression in several tissue.Method：By using RT-PCR method,LAT2 gene of grass carp was cloned from foregut＇s totoal RNA.Gene sequences was analysed with biological software,and detected in several organizations expression with RT-PCR.Result：Sequencing results showed that the gene included 1216 bp,encoded a preprotein of 404 aa residues;Nucleotide sequence analysis showed that the sequence homology was 92.5% identity with the sequence of zebrafish,and between 75.2% and 85.2% with the sequence of animals;The evolution tree built by kimura way from the LAT2 homology of grass carp and other animals was consistent with that of traditional taxonomic results;Predicted protein from nucleotide sequence of LAT2 had 12 transmembrane regions that included 6 transmenbrane regions from the first to the sixth are similar with others animals,and the homology of main function area was 92%;The LAT2 mRNA was detected in foregut,mindgut,hindgut,live,kidney,heart,brain,muscle and gill.Conclusion：The research laid a foundation for further study on the metabolism of fish amino acid absorbed and transshipment,and the metabolism of LAT2 gene expression.