采用RT-PCR方法从黄牛睾丸组织中克隆睾丸特异乳酸脱氢酶.C(LDH-C)基因的cDNA序列,结果发现2种Ldhc剪接体,根据Ldhc基因在进化上的保守性,参照普通牛Ldhc基因结构分析了黄牛Ldhc基因的选择性剪接体的结构,结果显示,剪接体存在外显子缺失:其中剪接体1缺失第7个外显子,剪接体2缺失第4和第7两个外显子.因为LDH-CA酶的NAD+结合结构域由Ldhc的外显子2-4编码,酶的活性中心由外显子4编码.所以剪接体1包含完整酶的NAD+结合结构域和酶的活性中心,剪接体2失去了大部分NAD+结合结构域和酶的活性中心.对黄牛睾丸组织和精子LDH同工酶的电泳分析未检测到潜在的Ldhc剪接体的表达,推测睾丸组织Ldhc选择性剪接的功能是调控该基因在睾丸中表达的方式之一.
Total RNA was extracted from cattle testes and reverse transcribed and amplified by RT2PCR. The two splice variants of LDH2Cwere obtained. Compared with the full length ORFof bovine LDH2C,as a result,The two cloned splice variants were missing exons,In detail,variants 1(missing the coding sequence of exon 7);Variant 2(missing the coding sequence of exons 4 and 7)lacks a large part of the NAD binding domain and the entire active site. Native poly2 acrylamide gel electrophoresis was performed to determine if the splice variants can be translated into proteins. However, native PAGE detected no specific bands from cattle testes and spermatozoa. This study suggests the alternative splicing of LDH2C might be involved in regulation of LDH2C expression.
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