马氏珠母贝(Pinctada fucata martensii)PmHEX基因的分子克隆及表达分析

Molecular Cloning and Expression Analysis of PmHEX Gene from Pinctada fucata martensii

β-N-乙酰-己糖胺酶（beta-N-acetylhexosaminidase/beta-Hexosaminidase,HEX）是一种糖苷水解酶,在几丁质降解、N-糖基化修饰、糖复合物代谢等过程发挥重要作用。为了探究PmHEX在马氏珠母贝贝壳形成中的作用,本研究利用RACE技术克隆获得马氏珠母贝PmHEX（Pinctada fucata martensii HEX,PmHEX）cDNA全长序列并通过qRT-PCR检测其在不同组织的表达模式。结果表明,PmHEX序列全长3 812 bp,其中5＇UTR为22 bp,3＇UTR为364 bp,开放阅读框（ORF）为3 426 bp,编码1 141个氨基酸;预测其相对分子量为125.92 kD,理论等电点为9.06;SMART软件分析PmHEX蛋白质序列,发现它具有典型的GH20和GH20b结构域和一个碳水化合物结合结构域;多序列比对显示PmHEX与其它物种的HEX具有较高的保守性,与珠母贝HEX序列相似度高达54%;q RT-PCR表达分析显示PmHEX在外套膜各区表达量较高,且在边缘区的表达量显著高于其他组织。综上所述,PmHEX可能参与马氏珠母贝贝壳的形成过程。

β-N-acetylhexosaminidas e（HEX） is a glycoside hydrolase, which plays an important role in chitin degradation, N-glycosylation modification and glycoconjugate metabolism. In order to investigate the role of PmHEX in the formation of Pinctada fucata martensii shells, the full length sequence of PmHEX cDNA in Pinctada fucata martensii was obtained by RACE technology and its expression patterns in different tissues were detected by qRT-PCR. The results showed that the full length of PmHEX sequence was 3 812 bp, including a 5＇UTR of 22 bp and a 3＇ UTR of 364 bp. The open reading frame（ORF） was 3 426 bp, encoding 1 141 amino acids.The predicted relative molecular weight was 125.92 kD and the theoretical isoelectric point was 9.06. SMART program analysis indicated that PmHEX protein sequence contained typical Glyco_hydro_20（GH20） and Glyco_hydro_20 b（GH20 b） domains and a CHB-HEX domain. Multiple sequence alignment showed that PmHEX and HEX of other species were highly conservative, the similarity of PmHEX and HEX sequences in Pinctada margaritifera was as high as 54%. The q RT-PCR analysis presented that the expression of PmHEX in the mantle was higher, especially the expression in marginal zone was significantly higher than that in other tissues（p〈0.05）. In conclusion, PmHEX might participate in the formation of Pinctada fucata martensii shells.