长牡蛎中I84蛋白酶抑制因子家族的扩张和功能分化

蛋白酶抑制因子是极其多样的蛋白质或多肽，能抑制蛋白酶的水解活性。研究发现蛋白酶抑制因子能通过抑制病原蛋白酶活性，从而抑制病原的入侵。I84蛋白酶抑制因子家族是MEROPS数据库中新增的一个家族，其部分成员在免疫防御过程中的作用得到了一定的揭示。为探究I84蛋白酶抑制因子家族在长牡蛎中的分布和功能状况，实验鉴定了长牡蛎中23个潜在的I84家族基因，根据系统进化树分析，挑选了5个同源基因进行时空表达和功能分析。首先，利用克隆技术验证了长牡蛎中5个I84家族同源基因CgSi3、CgSi5、CgSi6、CgSi16和CgSi19可表达性。结果显示，5个基因在外套膜、闭壳肌、性腺、血淋巴细胞、肝胰腺和鳃等6个组织中均表达，但肝胰腺中表达量显著高于其他组织。同时，5个基因在长牡蛎幼体不同发育阶段表达模式不同，其中各基因在受精卵时期均不表达，CgSi3表达量则在眼点幼虫期显著上升后下降，而CgSi6在壳顶幼虫期开始表达后，表达量随长牡蛎发育而持续增加。另外，对牡蛎进行病原相关模式分子(PAMPs)注射后，5个基因也表现出不同的表达模式，其中LPS和PGN注射后CgSi6表达量变化明显，而poly (...

I84 protease inhibitor family show significant expansion and functional differentiation in Crassostrea gigas

Protease inhibitors are extremely diverse proteins or small peptides that inhibit the proteolytic activities of proteases. Protease inhibitors could inhibit the invasion of pathogens by inactivating pathogen proteases. The I84 protease inhibitor family is a new family in MEROPS database, and the role of its members in host defense has been revealed to some extent. In this study, 23 potential I84 family genes were identified in the Pacific oyster Crassostrea gigas. Based on phylogenetic analysis, 5 representative genes, Cgsi3, Cgsi5, Cgsi6, Cgsi16 and Cgsi19, were selected for gene expression and functional assessment. They were confirmed by cDNA cloning to represent expressible real genes and expressed in digestive gland, gills, adductor muscle, gonad, mantle, and hemocytes, with the highest level of expression being detected in the digestive gland (P < 0.05). In the larval oysters, none of the 5 selected genes was detected in zygote, and the expression pattern in the remaining developmental stages differed with analyzed genes. The expression level of CgSi3 increased significantly in the pediveliger stage, and then decreased. The expression level of CgSi6 increased significantly from the umbo larvae. In oysters challenged with Pathogen associated molecular patterns (PAMPs), lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and Polyinosinic-polycytidylic acid (Poly (I:C)), gene expression pattern also differed with genes and PAMP. In oysters injected with LPS and PGN, only the expression level of CgSi6 significantly changed, while in those injected with Poly (I:C) and GLU the expression level of CgSi3 significantly changed. This study revealed the gene expansion and functional differentiation of the Family I84 family protease inhibitors C. gigas. The results also indicated the functional potentials of Family I84 protease inhibitors in host defense of the Pacific oyster.