马氏珠母贝生长激素诱导跨膜蛋白基因GHITM克隆与表达分析

Molecular Cloning and Expression Analysis of Growth-hormone Inducible Transmembrane Protein Gene (GHITM) from Pinctada martensii

生长激素诱导跨膜蛋白(growth hormone-induced transmembrane protein,GHITM)是一类在进化上高度保守的跨膜蛋白,参与机体生长发育、老化及先天免疫等过程。为探究GHITM在马氏珠母贝生长发育过程中的作用,本实验利用c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE),克隆得到马氏珠母贝GHITM(Pinctada martensii GHITM,Pm-GHITM)基因c DNA全长序列,并对其序列特征以及在组织中的表达进行分析。结果显示:Pm-GHITM基因c DNA全长为1 468 bp,其中,开放阅读框(open reading frame,ORF)为1 017 bp,编码338个氨基酸,5'UTR长62 bp,3'UTR长389 bp,分子量约为35.48 ku,等电点(p I)为9.87;多序列及系统进化树分析表明Pm-GHITM与其他物种的GHITM有较高的同源性,与太平洋牡蛎(Crassostrea gigas)的GHITM相似度最高,为67%;结构域分析结果表明Pm-GHITM序列中包含一个高度保守的BI-1(Bax inhibitor-1)结构域;荧光定量数据指出Pm-GHITM在马氏珠母贝的血淋巴、外套膜、足、鳃、性腺、闭壳肌以及肝胰腺中均有表达,在性腺的表达量最高,肝胰腺和闭壳肌次之,血淋巴中的表达量最低(p<0.05)。本研究可以为进一步探究Pm-GHITM在马氏珠母贝的生长发育过程中的作用做铺垫。

Growth hormone-induced transmembrane protein （GHITM） was a class of evolutionarily conserved tr- ansmembrane protein and involved in various biological process, such as growth, development and innate immunity. To explore the function of GHITM in the growth ofP. martensii, in this study, using rapid amplification of cDNA ends （RACE） technology, we had obtained the full length sequence of Pinctada martensii GHITM （Pm- GHITM） cDNA and analyzed its sequence characteristic and function. Our results showed that the cDNA full length of Prn-GHITM was 1 468 bp, including 62 bp of 5＇ UTR, 389 bp of 3＇ UTR, and 1 017 bp of open reading frame （ORF） encoding 338 amino acids, with an estimated molecular mass of 35.48 ku and theoretical isoelectric point of 9.87. Multisequencing alignment and phylogenetic analysis results showed that Pm-GHITM was highly homologous with that from other species and had 67% sequence identity with that from Crassostrea gigas. Amino acid sequence analysis showed Pm-GHITM had an conservative domains. Meanwhile, real-time PCR analysis showed that Pm-GHITM was ubiquitously expressed in all tissues detected, including adductor muscle, foot, mantle, hemolymph, hepatopancreas, gonads and gills, with the highest expression level in gonads followed by hepatopancreas and adductor muscle, and lowest in hemolymph. Therefore, our data provided the basis for further research in the mechanism underlying the growth in pearl oyster.