凡纳滨对虾核自身抗原精子蛋白基因克隆及其表达  

Molecular cloning and expression analysis of the gene encoding nuclear autoantigenic sperm protein from white prawn (Litopenaeus vannamei)

作　　者：颜婕[1] 刘红[1] 方昱[1] 蔡生力[1] 

机构地区：[1]上海海洋大学水产与生命学院上海,201306

出　　处：《中国水产科学》2016年第5期1073-1079,共7页Journal of Fishery Sciences of China

基　　金：上海市教育委员会重点科研创新项目(12ZZ160)

摘　　要：利用cDNA末端快速扩增技术克隆出凡纳滨对虾(Litopenaeus vannamei)核自身抗原精子蛋白(NASP)基因,NASP基因c DNA全长2258 bp,其中包括92 bp 5′端非编码区,147 bp 3′端非编码区及2019 bp开放阅读区,编码673个氨基酸,预测分子量为74.18 k D。该序列已提交至Gen Bank,序列号为KT274811。在NCBI上进行序列比对后发现,其与斑节对虾(Penaeus monodon)NASP序列相似性最高,为90%。采用荧光定量PCR方法测定了不同发育时期卵巢与肝胰腺中NASP m RNA相对表达水平,结果表明,NASP在卵巢中的表达量高于肝胰腺中的表达量,且在Ⅱ期表达量最高,Ⅲ期表达量最低。此外,通过构建系统进化树比较了凡纳滨对虾NASP与其他物种间的遗传距离。实验结果为进一步研究该基因在凡纳滨对虾卵巢发育中的作用提供了依据。Many genes that play roles in shrimp vitellogenesis, such as that encoding nuclear autoantigenic sperm protein （NASP）, have been identified by suppression subtractive hybridization. In shrimp, NASP plays an important role in ovarian maturation. In this study, the NASP gene was cloned from Litopenaeus vannamei using the rapid amplification of eDNA ends technique. The full-length eDNA of NASP consisted of 2258 bp with a 92-bp 5＇-untranslated region （UTR）, a 174-bp 3＇-UTR, and a 2019-bp open reading frame, which encoded a protein of 673 amino acids with a predicted isoelectric point of 4.46 and a predicted molecular weight of 74.18 kDa. The sequence has been submitted to GenBank under the accession number KT274811. The putative protein had two conserved regions： SHNi-TPR and TPR_2. A sequence alignment analysis revealed that the L. vannamei NASP showed the highest similarity to the NASP of Penaeus monodon （GenBank accession number： FJ040859.1）. We constructed a phylogenetic tree based on NASP sequences to evaluate the evolutionary relationships of NASPs between L. vannamei and other species. In the phylogenetic tree, the NASP ofL. vannamei was in the same branch as that of P. monodon. The transcript levels of NASP were investigated in the ovary and hepatopancreas of L. vannamei at difi%rent overy developmental stages by real-time fluorescent quantitative PCR. The results showed that NASP transcripts were present at all stages, with the highest levels at the second stage and the lowest levels at the third stage. The NASP transcript levels were higher in the ovary than in the hepatopancreas, in which the transcript level was negligible. These results provide the basis for further research on the role of NASP in ovarian development in L. vannamei.

关 键 词：凡纳滨对虾 NASP 基因克隆 序列分析 荧光定量PCR 

分 类 号：S917[农业科学—水产科学]