细角滨对虾酚氧化酶原cDNA克隆及序列结构分析

cDNA Clone of Prophenoloxidase for Litopenaeus Stylirostris and Sequence Structure Analysis

采用RT-PCR原理和长片段扩增技术克隆细角滨对虾酚氧化酶原基因。结果表明,细角滨对虾血淋巴细胞内存在2个pro PO基因。pro PO gene 1的c DNA序列包含有372氨基酸,前190个氨基酸为一个M家族血蓝蛋白,是一个铜结合位点区域,191-372为一个C家族的血蓝蛋白,是一个免疫球蛋白样的区域。pro PO gene 2的2个功能位点之间的序列有重叠,pro PO gene 2 c DNA序列的6-935bp包含了第一个功能位点,928-1464bp则包含了第二个功能位点。系统进化树比对分析发现2个基因之间的序列差异非常大。细角滨对虾和凡纳滨对虾的pro PO gene 2同处于一个密切相关的群,pro PO gene 1则和其他几种对虾的pro PO gene处于一个群。pro PO gene 2与pro PO gene 1在对虾免疫活动中是否存在不同的功能还有待于进一步的研究。

Prophenoloxidase （proPO） is one of the important factors on humoral immunity of shrimp, so far there are no reports for Litopenaeus stylirostris. Depend on techniques of RT-PCR and long fragment amplification cloning, prophenoloxidase gene of L. stylirostris was cloned. The results show that, there are two proPO genes in the lymphocytes of L. stylirostris. ProPO gene 1 cDNA sequence contains 372 amino acids, the first 190 amino acids are a family of M hemocyanin, a copper binding site region, 191-372 is one of the C family of hemocyanin, is an immunoglobulin like region. There are sequence overlap between the 2 functional sites of proPO gene 2, which means that 6-935bp contains the first functional sites, while 928-1464bp contains second functional sites. The phylogenetic tree alignment analysis showed that sequence structures of two genes is very different. ProPO gene 2 of L. stylirostris and L. vannamei was in a closely related group ; but proPO gene 1 of L. stylirostris and L. vannamei was in another group with other several shrimp. The function of ProPO gene 2 and proPO gene 1 in shrimp immune activity still needs further study.