三角帆蚌KLHL10基因的特征和表达分析  

Characterization and expression analysis of KLHL10 gene in freshwater mussel Hyriopsis cumingii

作　　者：董赛赛 崔晓羽 段胜华 夏思宇 刘斐斐 葛静远 汪桂玲[1,2,3] 李家乐[1,2,3] DONG Saisai;CUI Xiaoyu;DUAN Shenghua;XIA Siyu;LIU Feifei;GE Jingyuan;WANG Guiling;LI Jiale(Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai 201306,China;Shanghai Engineering Research Center of Aquaculture,Shanghai Ocean University,Shanghai 201306,China;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学农业农村部淡水水产种质资源重点实验室,上海201306 [2]上海海洋大学上海市水产养殖工程技术研究中心,上海201306 [3]上海海洋大学水产科学国家级实验教学示范中心,上海201306

出　　处：《上海海洋大学学报》2021年第3期389-398,共10页Journal of Shanghai Ocean University

基　　金：国家自然科学基金(31772835);国家重点研发计划(2018YFD0901406)。

摘　　要：为了探究KLHL10基因在三角帆蚌性别分化中的作用,利用RACE(Rapid-amplification of cDNA ends)克隆了其cDNA全长,使用实时荧光定量分析比较其在6个不同组织(性腺、鳃、肝胰腺、斧足、闭壳肌、外套膜)、早期发育阶段(1~8月龄)性腺及12、24、36月龄雌雄性腺中表达水平的差异,运用RNA干扰(RNAi)对其功能进行初步探究。结果显示KLHL10基因cDNA全长为2361 bp,其中5′非编码区长93 bp,3′非编码区长447 bp,开放阅读框(ORF区)长1821 bp,编码606个氨基酸;qRT-PCR结果显示KLHL10基因在精巢中高表达;早期发育阶段在6月龄时表达量最高;12、24、36月龄的表达结果显示,KLHL10基因在精巢中的表达量均高于同时期在卵巢中的表达量(P<0.05)。同时,设计KLHL10基因的3条dsRNA干扰链,结果显示RNA干扰能有效减少KLHL10基因在性腺组织的表达量。根据以上结果推测KLHL10基因在三角帆蚌中是雄性相关基因,其可能参与三角帆蚌的性别分化与精巢发育。In order to investigate the role of the KLHL10 gene in the sex development of Hyriopsis cumingii,the full length of its cDNA was cloned using RACE,and KLHL10 expression levels in six tissues(gonad,gill,adductor muscle,foot,mantle,and hepatopancreas)were determined.Further,the quantitative real-time PCR was performed to compare expressions levels between 1-8 months of age and 12-,24-,and 36-month-old H.cumingii,and its function was explored using RNA interference.The results showed that the full length of the KLHL10 gene was 2361 bp,and the 5′non-coding region was 93 bp,the 3′non-coding region was 447 bp,and the open reading frame was 1821 bp which encoded 606 amino acids.qRT-PCR results showed that the gene was highly expressed in the testis,and was expressed at the highest level at 6 months of age in early stage.Moreover,in 12-,24-,and 36-month-old individuals,KLHL10 gene expression levels in the testis were higher than those in the ovaries(P<0.05).At the same time,the double-stranded RNA(dsRNA)interference experiments were also performed using three dsRNA strands.The results showed that the expression of KLHL10 in gonad was effectively reduced.According to the above results,it is speculated that the KLHL10 gene is a male-related gene in H.cumingii,which may participate in sex differentiation and testis development of H.cumingii.

关 键 词：三角帆蚌 KLHL10基因 性别分化 QRT-PCR RNA干扰 

分 类 号：S917[农业科学—水产科学]