池蝶蚌HsPif基因的克隆及表达分析

CLONING AND EXPRESSION ANALYSIS OF HSPIF FROM HYRIOPSIS SCHLEGELII

为了探究池蝶蚌(Hyriopsis schlegelii)酸性基质蛋白Pif基因的基本功能,研究通过RACE-PCR技术首次在池蝶蚌中获得了Pif基因的cDNA全长序列,命名为HsPif,其全长为3457 bp, 5′端非翻译区(5′UTR)为485 bp,3′端非翻译区(3′UTR)为363 bp,开放阅读框(ORF)3072 bp,共编码1023个氨基酸,生物信息学分析结果显示HsPif蛋白含有一个von-Willebrand因子A型结构域和三个几丁质结合结构域;氨基酸组成成分结果表明天冬氨酸含量最高,组氨酸含量最低;HsPif蛋白亲水指数为–0.566,为亲水蛋白。构建系统进化树分析显示Pif基因保守性较高,与三角帆蚌(Hyriopsis cumingii)Pif相似度最高,且与其他贝类在同一个大支上。组织荧光定量PCR结果表明:HsPif基因主要在外套膜中表达;分子原位杂交显示杂交反应主要在外套膜上皮细胞发生。进行植核手术后进行qPCR检测HsPif基因的表达量变化,实验结果表明, HsPif基因可能与珍珠层的分泌有关,有助于进一步了解珍珠形成的机制,为珍珠养殖提供参考。

At present, there are few researches on the pearl breeding mechanism of the high-quality freshwater mussel Hyriopsis schlegelii, and the Pif gene is closely related to the formation of shellfish pearls. In order to explore the preliminary function of Pif gene of Hyriopsis schlegelii, the full-length cDNA sequence of Pif gene was obtained for the first time in Hyriopsis schlegelii by RACE-PCR technique, which is named HsPif. The full-length untranslated region(5’UTR) of 5’-terminal untranslated region of Pif gene was 485 bp, 3’ untranslated region(3’UTR) and 363 bp, open reading frame(ORF) 3072 bp, which encodes a total of 1023 amino acids. It was speculated that the bioinformatics analysis of two proteins encoding HsPif97 and HsPif80 showed that HsPif protein contained one von-Willebrand factor A(VWA) domain and three chitin binding domains(ChtBD2). The results of amino acid composition showed that the content of aspartic acid was the highest, accounting for 12.5%, and the content of histidine was the lowest, accounting for 1.32%. The α helix content in the protein secondary structure accounted for 19.63%, the irregular crimp accounted for 55.09%, and the extension chain accounted for 16.86%. The hydrophilic index of HsPif protein was –0.566. Phylogenetic tree analysis showed that HsPif and Hyriopsis cumingii Pif were the most conservative in one branch and on the same large branch as other shellfish. The results of tissue fluorescence quantitative PCR showed that HsPif gene was mainly expressed in the mantle, and in situ hybridization showed that the hybridization reaction mainly occurred in the mantle epithelial cells. The changes of HsPif gene expression were detected by qPCR after grafting. The results showed that HsPif gene expression changed with the development of pearl sac. The expression level of HsPif gene decreased significantly on the 7th and 90th day after nuclear insertion, and significantly increased on the 15th, 60th and 120th day.There was no significant change between the 30th day and the control group. The above experimental results suggest that Hs Pif gene may be related to the secretion and formation of pearls, which is helpful to further understand the mechanism of pearl formation and provide reference for pearl culture.