凡纳滨对虾卵巢类围食膜蛋白基因克隆及其在早期胚胎发育中的表达

Cloning and expression analysis of shrimp ovarian peritrophin-like gene during early embryonic development in Litopenaeus vannamei

围食膜蛋白最早是从昆虫围食膜中解离得到的,在保护昆虫免受外源微生物侵染过程中发挥了重要作用。为了解围食膜蛋白在对虾免疫过程中的作用,实验前期进行了凡纳滨对虾转录组分析,并克隆获得了凡纳滨对虾2条类围食膜蛋白基因LvPT1和LvPT2,其编码的2条氨基酸序列相似性极高,同已报道的其他虾类围食膜蛋白具有较高同源性。利用Real-time PCR方法进行组织表达分析发现,LvPT1和LvPT2与对虾卵巢围食膜蛋白(SOPs)类似,在卵巢中高度表达,利用半定量PCR方法,分析LvPT2在对虾早期发育中的表达情况。结果显示,LvPT2在受精卵时期表达量最高,随着发育进行,表达量逐渐降低,至无节幼体孵出后无明显表达。研究表明,LvPT1和LvPT2与虾SOPs应为同一类围食膜蛋白,可能参与了受精卵细胞的形成,并在保护对虾早期胚胎发育时免受外界病原微生物的侵染中发挥着重要作用。

Peritrophin was one type of proteins which was the important structural component of peritrophic membrane （PM） lining the midgut in invertebrate. It was first separated from insect, and played an important role in protecting insects from invasion by microorganisms. Recently, many peritrophin-like genes were identified from tissues not only intestine in crustacean, which implied that the role ofperitrophin-like proteins might be diverse. In this study, two cDNA sequences encoding peritrophin-like protein named as LvPT1 and LvPT2 were found and cloned from Litopenaeus vannamei. Their deduced amino acid sequences exhibited high similarity to each other and most other shrimp peritrophins which have been reported. Tissue expression analysis by Real-time PCR revealed that LvPTI and LvPT2 were mainly expressed in shrimp ovary, similar with shrimp ovarian peritrophins （SOPs）. Semi-quantitative RT-PCR was used for analyzing the expression of LvPT2 during the early development of L. vannamei. The results showed that LvPT2 expression was the highest in fertilized eggs, and decreased significantly in developing process. There was no obvious expression of LvPT2 in nauplius hatched out from the hatching membrane. Therefore, we presumed that LvPT1 and LvPT2 might be the same kind of peritrophin to play the similar role with other shrimp SOPs, in participating in the formation and protection of fertilized egg, and protecting shrimp embryo against pathogen infection.