内蒙古白绒山羊LDH-A基因cDNA的克隆与特性分析

Cloning and Characterization Analysis of LDH-A Gene cDNA in Inner Mongolia Cashmere Goat

【目的】克隆内蒙古白绒山羊乳酸脱氢酶A(LDH-A)基因并分析其基本表达模式。【方法】采用RT-PCR和3′RACE技术克隆基因,将得到的基因cDNA序列及其编码的氨基酸序列进行生物信息学分析。利用RT-PCR方法进行组织表达检测。【结果】获得了内蒙古白绒山羊LDH-A基因全长cDNA序列。该基因cDNA全长1 649 bp,包含一个996 bp的ORF和642 bp的3′UTR。SMART分析表明ORF编码的蛋白质具有Ldh_1_N domain和Ldh_1_C domain,Psite分析表明有L-乳酸脱氢酶活性位点,PSORT程序分析将其定位于细胞质中。LDH-A基因在脾、肾、睾丸和肌肉组织中均有表达。【结论】内蒙古白绒山羊LDH-A基因编码的蛋白具有典型的乳酸脱氢酶结构,cDNA核苷酸序列与牛的LDH-A基因(NM_174099.2)具有较高的同源性。在脾、肾、睾丸和肌肉组织中均有表达。

[Objective] The full length of LDH-A gene cDNA in Inner Mongolia Cashmere Goat was cloned and expression pattern analyzed. [Method] RT-PCR and 3＇ RACE （Rapid Amplification of cDNA Ends） methods were used to clone the gene. The nucleotide sequence was analyzed by BLAST and ORF Finder while the amino acid sequences was analyzed by SMART and Psite on line. Tissue expressions were analyzed by RT-PCR. [Result] The full length of LDH-A gene cDNA in Inner Mongolia Cashmere Goat was cloned for the first time. The LDH-A gene was 1 649 bp in length, including an ORF of 996 bp and a 3＇-UTR of 642 bp. SMART analysis suggested that the ORF encoding protein contained a Ldh 1 N and a Ldh_l_C domain. Psite analysis showed that there was lactate dehydrogenate active site in the protein, which localized in cytoplasm according to PSORT program analysis. LDH-A gene was expressed in the tissues of spleen, kidney, testicle and muscle. [Conclusion] The LDH-A protein was encoded by LDH-A gene had typical structure of lactate dehydrogenase in Inner Mongolia Cashmere Goat. The goat LDH-A gene sequence shared high identity with bovine LDH-A gene （NM_174099.2） and was expressed in the tissues of spleen, kidney, testicle and muscle.