文蛤CDK7基因克隆及其在不同品系生长发育中的表达分析

【目的】掌握文蛤（Meretrix meretrix）细胞周期蛋白依赖性激酶7（CDK7）基因（MmCDK7）的时空表达及在不同品系生长发育中的表达规律，从分子水平探究红壳色文蛤新品系的生长优势，为筛选文蛤生长相关基因及揭示其生长发育机制提供理论依据。【方法】利用RACE克隆MmCDK7基因cDNA序列，通过BLAST、ScanProsite、NetPhos3.0 server及ExPASy等在线软件进行生物信息学分析，使用实时荧光定量PCR检测MmCDK7基因在文蛤不同组织和不同发育时期的表达情况，并比较同一养殖条件下红壳色文蛤（简称红文蛤）和黄壳色文蛤（简称黄文蛤）的壳长、壳长相对增长率及MmCDK7基因表达差异。【结果】MmCDK7基因cDNA序列全长1296bp，其中，5'端非编码区（5'-UTR）为83bp，3'端非编码区（3'-UTR）为196bp，开放阅读框（ORF）为1017bp，共编码338个氨基酸残基。MmCDK7蛋白分子量约38.32 D，理论等电点（pI）为8.78，包含丝氨酸/苏氨酸蛋白激酶催化结构域（S_TKc）、酪氨酸激酶催化结构域（TyrKc）及与细胞周期蛋白结合有关的激酶结构域NRTALRE；而S_TKc结构域中有包含蛋白激酶ATP结合位点区域、丝氨酸/苏氨酸蛋白激酶活化位点区域及T-loop环。MmCDK7氨基酸序列与虾夷扇贝CDK7氨基酸序列高度同源，其相似性为75.00%；基于CDK7氨基酸序列相似性构建的系统发育进化树显示，文蛤与中国真蛸、长牡蛎、厚壳贻贝及虾夷扇贝等软体动物先聚为一支。MmCDK7基因在性腺、水管、外套膜和肝胰腺等组织中均有表达，以性腺中的相对表达量显著高于其他组织（P<0.05，下同）；MmCDK7基因在2种文蛤的8个发育时期均有表达，均以多细胞时期的相对表达量最高。在同一养殖条件下，除9月18日外，其他采样时间点均表现为红文蛤的壳长显著大于黄文蛤，红文蛤相对于黄文蛤的壳长增长率在2.79%~32.37%；除11月15日和11月30日外，其他采样时间点均表现为红文蛤的MmCDK7基因相对表达量显著高于黄文蛤的相对表达量。【结论】MmCDK7基因属于CDK家族成员，在细胞分裂旺盛的性腺及多细胞时期的相对表达量最高，且在生长速度较快红文蛤中的相对表达量多数情况下显著高于黄文蛤，故推测MmCDK7基因参与调控文蛤的早期生长发育过程。

Cloning of CDK7 gene in Meretrix meretrix and its expression analysis on growth and development of different strains

【Objective】To master the spatiotemporal expression of cyclin dependent kinase 7（CDK7）gene（MmCDK7） in Meretrix meretrix and the expression law in the growth and development process of different strains ，so as to explore the growth advantages of new strains of red clam at the molecular level. It provided a theoretical basis for screening growth related genes of M. meretrix and revealing its growth and development mechanism.【Method】The cDNA sequence of MmCDK7 gene was cloned by RACE. Bioinformatics analysis was carried out by BLAST，ScanProsite，NetPhos 3.0 server and ExPASy. The expression of MmCDK7 gene in different tissues and different developmental stages of M. meretrix was detected by real-time fluorescence quantitative PCR. The differences of shell length，relative growth rate of shell length and MmCDK7 gene expression between red clam and yellow clam were compared under the same culture conditions.【Result】The total length of the cDNA sequence of MmCDK7 gene was 1296 bp，of which the 5 ' untranslated region（5'-UTR）was 83 bp，the 3 ' untranslated region（3'-UTR）was 196 bp，and the open reading frame（ORF）was 1017 bp，encoding 338 amino acid. The molecular weight of MmCDK7 protein was about 38.32 kD and the theoretical isoelectric point（pI）was 8.78. It included serine / threonine protein kinase catalytic domain（S_TKc），tyrosine kinase catalytic domain（TyrKc）and kinase domain NRTALRE related to cyclin binding. And S_ TKc domain contained protein kinase ATP binding site，serine / threonine protein kinase activation site and T-loop loop. The amino acid sequence of MmCDK7 was highly homologous with that of Mizuhopecten yessosensis，and its similarity was 75.00%. The phylogenetic tree constructed based on the amino acid sequence similarity of CDK7showed that M. meretrix and molluscs such as Octopus sinensis，Crassostrea gigas，Mytilus coruscus and M. yessosensis first gathered into one branch. MmCDK7 gene was expressed in gonad，siphon，mantle and hepatopancreas，and the relative expression in gonad was significantly higher than that in other tissues（P<0.05，the same below）. MmCDK7 gene was expressed in 8 developmental stages of two species，and the relative expression in multicellular stage was significantly higher than that in other stages. Under the same culture conditions，except September 18，the shell length of red clam was significantly longer than that of yellow clam，and the growth rate of red clam relative to yellow clam was 2.79%-32.37%. Except November 15 and November 30，the relative expression of MmCDK7 gene in red clam was significantly higher than that in yellow clam at other sampling time points.【Conclusion】MmCDK7 gene belongs to CDK family. The relative expression of CDK7 gene in gonad and multicellular stage with vigorous cell division is the highest，and the relative expression of CDK7 gene in red clam with fast growth rate is significantly higher than that in yellow clam in most cases. Therefore，it is speculated that MmCDK7 gene is involved in regulating the early growth and development of M. meretrix.