中华鳖Dmrt1基因的克隆及表达分析  

Molecular cloning and expression analysis of Dmrt1 gene in Pelodiscus sinensis

作　　者：薛冰霜[1] 穆淑梅[1] 康现江[1] 张晗[1] 曹杰英[2] 徐二虎 

机构地区：[1]河北大学生命科学学院,河北保定071000 [2]河北省水产技术推广站,河北石家庄050000 [3]河北康态中华鳖良种有限公司,河北石家庄050000

出　　处：《河北农业大学学报》2014年第2期99-105,119,共8页Journal of Hebei Agricultural University

基　　金：河北大学研发基金项目(2003333112);石家庄市科技支撑计划项目(09150118A)

摘　　要：为研究Dmrtl基因对中华鳖（Pelodiscussinensis）性别分化的影响，采用RAcE方法获得该基因的eDNA全长序列，并利用荧光定量PCR技术分析了Dmrtt基因在中华鳖不同龄期及不同组织的表达情况。研究表明：中华鳖Dmrtl基因的cDNA序列全长为2335bp，包括164bp的5’非编码区（5’UTR）、1064bp的3’端非编码区（3’UTR），1107bp的开放阅读框（ORF），编码368个氨基酸，预测分子量为39．20kD，理论等电点（pI）为7．58。同源性分析发现，中华鳖Dmrtl基因的氨基酸序列与部分物种的相似性在66％～97％。荧光定量PCR分析显示，该基因在三龄成鳖精巢中的表达量最高，且显著高于稚鳖精巢的表达量（P〈O．05），但与1龄幼鳖、2龄成鳖精巢的差异不显著（P〉O．05）；在稚鳖和1龄幼鳖卵巢中有微量表达，且表达量差异不显著（P〉0．05），成鳖卵巢中无表达；肌肉和肝脏在各龄期均无表达；温度对稚鳖性腺的表达量影响不明显。以上结果表明，在发育过程中精巢Dmrtl基因的表达量呈上升趋势，卵巢也只在稚鳖与幼鳖中有微量表达，结果暗示该基因在性别分化和功能维持上可能具有重要作用。In order to study the influence of Dmrtl gene in Pelodiscus sinensis sexual differenti- ation, the full-length of Dmrtl cDNA sequence was obtained by the method of RACE, and the expression of the gene was analyzed by Real-time PCR in different ages and different tissues The total length of Drnrtl gene cloned in P. sinensis is 2 335 bp which contains 164 bp 5＇ un- translated regions （UTR） and 1 064 bp 3t-UTR, and 1 107 bp open reading frame （ORF） enco- ding 368 amino acids. The predicted molecular mass of Dmrtl protein was 39.20 kD, and thetheoretical isoelectric point was 7.58. The homology analysis showed that there were 66%- 97% similarities in amino acid sequence with some of previously reported other species. Real- time PCR revealed that the expression of the Dmrtl mRNA in testis of three-year-old turtles was significantly higher than newly hatched turtles （P〉0.05）, but there were not significantly different in relative expression level with one-year-old and two-year-old turtles （P〈0.05）. The mRNA of Dmrtl gene in ovary of new hatched and one-year-old turtles were weakly ex- pressed and the expression level was no significant difference （P〈0.05）. The mRNA of Drnrtl gene was not expressed in ovary of two- and three-year-old turtles, and there were not ex- pressed in muscle and liver of each ages. The effect of temperature on the gonad of newly hatched turtles was not distinct. The results suggest that the Dmrtl gene may play an impor- tant role in sexual differentiation and function maintain of P. sinensis.

关 键 词：中华鳖 DMRT1基因 荧光定量PCR 

分 类 号：Q786[生物学—分子生物学] Q781