文章摘要
三角帆蚌kinase X基因的克隆及功能初探
Preliminary study on the functions of kinase X gene in Hyriopsis cumingii
投稿时间:2020-10-22  修订日期:2021-03-03
DOI:
中文关键词: 三角帆蚌  kinase X基因  Sox9基因  基因克隆  表达分析  RNAi
英文关键词: H.cumingii  kinase X gene  Sox9 gene  gene cloning  expression analysis  RNAi
基金项目:国家重点研发计划“蓝色粮仓科技创新”(2018YFD0901400);国家自然科学基金(31772835);现代农业产业技术体系建设专项资金(CARS-49)
作者单位邮编
崔晓羽 上海海洋大学 201306
董赛赛 上海海洋大学 
段胜华 上海海洋大学 
汪桂玲 上海海洋大学 201306
李家乐 上海海洋大学 
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中文摘要:
      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。本研究通过RACE法克隆kinase X基因的全长cDNA序列,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,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1652bp,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。
英文摘要:
      Hyriopsis cumingii is a unique freshwater breeding 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 important. 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 play 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 are upstream and downstream. It is speculated that the Sox9 gene may be regulated by PKA. The results showed that the kinase X gene was 1652bp in length, the 3’UTR is 314bp, while the length of the 5’UTR is 45bp, encoding 430 amino acids, and there was an STKYc structure domain which is located at 381 ~ 1179 bp, kinase X gene has a transmembrane domain and the Molecular weight (Mw) of the kinase X protein was 49.00ku and the theoretical Isoelectric point (pI) was 8.22, among that 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 shellfish such as Mizuhopecten yessoensis, Chlamys (Azumapecten) 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 H.cumingii , the kinase X gene was expressed the highest in the male gonads while expressed the lowest in the female liver. And in 1~3 year of H.cumingii, the expression of kinase X gene in male is all higher than that in female, and the difference between male and female is extremely significant. Among them, the expression of kinase X gene in 2 year H.cumingii is highest among 1 ~3 year. Due to the high expression of the kinase X gene of the 2 year H.cumingii near the sexual maturity stage, the sperm occurrence rate is high; after 7 days of interference, the expression of kinase X gene decreased. RNA interference results show that the synthesized 3 interference chains have a certain interference efficiency on the kinase X gene, and the interference rate of the interference chain 1 is the highest. The interference rate is 83.1% in females and 81.9% in males, while interference chain 2 has an interference rate of 76.0% in females and 25.7% in males; interference chain 3 has an interference rate of 24.0% in females and 67.7% in males. At the same time, the Sox9 gene expression decreased by 90.3% in females and 56.6% in males after the interference of interfere chain 1. That improved the synthesis of kinase X gene was positively correlated with the synthesis of Sox9 gene. It is speculated that the two may work together to participate in the sex determination process through the cAMP-PKA-mediated signaling pathway. In this experiment, through preliminary functional exploration of the kinase X gene, it was found that the upstream and downstream relationship between kinase X gene and Sox9 gene and the two were positively correlated, and it effectively interfered with the kinase X gene, which is important in cAPM-PKA of H.cumingii. This study provides a theoretical basis for the study of sex determination and male parthenogenesis of H.cumingii.
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