三角帆蚌kinase X基因的克隆及功能初探  

Preliminary study on cloning and the functions of kinase X gene in Hyriopsis cumingii

作　　者：崔晓羽 董赛赛 段胜华 汪桂玲[1,2,3] 李家乐[1,2,3] CUI Xiaoyu;DONG Saisai;DUAN Shenghua;WANG Guiling;LI Jiale(Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries,Shanghai Ocean University,Shanghai 201306,China;National Demonstration Center for Experimental Fisheries Science Education y Shanghai Ocean University,Shanghai 201306,China;Shanghai Engineering Research Center of Aquaculture,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,水产科学国家实验教学示范中心,上海201306 [3]上海海洋大学,上海市水产养殖工程技术研究中心,上海201306

出　　处：《水产学报》2022年第4期537-545,共9页Journal of Fisheries of China

基　　金：国家重点研发计划(2018YFD0901406);国家自然科学基金(31772835);现代农业产业技术体系建设专项(CARS-49)。

摘　　要：三角帆蚌是我国特有的淡水育珠蚌,在养殖业中占有重要地位,在实际养殖过程中,雄性个体有着明显的产珠优势,因此对性别决定的相关研究至关重要。研究发现Sox9基因在许多物种中起到性别决定的作用,kinase X基因是蛋白激酶(PKA)合成中至关重要的基因,而Sox9基因很可能受到PKA激酶的调控。实验通过RACE法克隆kinase X基因的全长cDNA序列,使用荧光定量PCR的方法检测该基因在2龄雌雄三角帆蚌各组织中的相对表达量,利用RNA干扰技术研究干扰链对kinase X基因及下游基因Sox9基因表达的影响。结果显示,kinase X基因全长1652 bp,编码430个氨基酸;荧光定量PCR结果显示,kinase X基因在雄性性腺中表达量最高,雌雄间差异极显著;RNA干扰结果显示,合成的干扰链均对kinase X基因有着一定的干扰效率,其中干扰链1的干扰率最高,在雌性中的干扰率为83.1%,雄性中为81.9%,同时干扰链1干扰后Sox9基因的表达量在雌性中下降了90.3%,雄性中下降了56.6%,推测这两个基因可能参与性别决定过程。本实验为三角帆蚌性别决定和雄性单性化育种研究提供理论基础。Hyriopsis cumingii is a unique freshwater pearl mussel in our country.It occupies an important position in the aquaculture industry.In the actual breeding process,male individuals have a more obvious advantage in pearl production than female individuals.Therefore,research on the sex determination of H.cumingii is crucial.Previous studies have found that Sox9 gene plays a role in male sex determination in many species,kinase X gene is an important gene in the synthesis of protein kinase(PKA),and PKA plays a vital role in many physiological and biochemical processes,especially in the process of spermatogenesis.In this process,PKA has a very obvious effect on sperm capacitation.The kinase X gene and Sox9 gene are both located in cAMP-PKA pathway.And the relationship of the kinase X gene and Sox9 gene is upstream and downstream.It is speculated that the Sox9 gene may be regulated by PKA.The results showed that the kinase X gene was 1652 bp in length,the 3'UTR was 314bp,while the length of the 5'UTR was 45 bp,encoding 430 amino acids,and there was an STKYc structure domain which was located at 381-1179 bp,kinase X gene has a transmembrane domain and the Molecular weight(Mw)of the kinase X protein was 49.00 ku and the theoretical Isoelectric point(pI)was 8.22,among which Leu(L)was the most abundant amino acid,accounting for 9.8%;the homology analysis showed that H.cumingii had a high similarity with other shellfishes such as Mizuhopecten yessoensis,Chlamys farreri.The phylogenetic tree showed that H.cumingii had a close relationship with Crassocras gigas and Crassostrea virginica,but had a long genetic distance with mammals.The fluorescence quantitative PCR results showed that the kinase X gene was expressed in 6 tissues including gonads,gill,adductor muscle,foot,mantle,liver of the 2-year-old H.cumingii,and the kinase X gene was expressed the highest in the male gonads while expressed the lowest in the female liver.And in 1-3 years of H.cumingii,the expression of kinase X gene in male was all higher than that in femal

关 键 词：三角帆蚌 kinase X基因 SOX9基因 基因克隆 表达分析 RNAI 

分 类 号：Q785[生物学—分子生物学] S966.221[农业科学—水产养殖]