参与家蚕免疫反应的clip丝氨酸蛋白酶家族基因分析

An Analysis on clip Serine Protease Family Genes Involved in Innate Immunity of Bombyx mori

丝氨酸蛋白酶在昆虫的新陈代谢和生长发育等多种生理过程中起重要作用,特别是含clip结构域的丝氨酸蛋白酶与昆虫的免疫级联激活途径密切相关。为探索家蚕clip丝氨酸蛋白酶在家蚕先天免疫反应信号通路中的作用,对家蚕(Bombyxmori)与烟草天蛾(Manduca sexta)中鉴定获得的含有clip结构域的丝氨酸蛋白酶进行序列比对和系统进化分析,发现BmSP78与MsPAP-1、BmSP127与MsHP8、BmSP124与MsHP1、BmSP125与MsHP17位于同一进化支上,4对clip丝氨酸蛋白酶的氨基酸序列相似度分别为70%、68%、81%和57%,另外的8个蛋白酶BmSP23、BmSP111、BmSP95、BmSP135、BmSP129、BmSP137、BmSP91和BmSP99构成了家蚕特有的一群clip丝氨酸蛋白酶。用革兰阴性细菌沙雷氏菌和革兰阳性细菌黑胸败血菌分别注射感染家蚕5龄第4天幼虫后,通过半定量RT-PCR检测分析clip丝氨酸蛋白酶基因在不同感染时间点的表达特征:病原菌诱导后蚕体中有11个clip丝氨酸蛋白酶基因mRNA转录水平有明显变化,其中BmSP102和BmSP96于黑胸败血菌诱导12 h后在血细胞中明显上调表达,而BmSP125在沙雷氏菌和黑胸败血菌感染12 h后的脂肪体中均明显上调表达。研究结果为建立家蚕clip丝氨酸蛋白酶可能参与的免疫级联反应路径提供了重要线索。

Serine proteases are involved in various physiological processes of insect,such as metabolism,growth and development.Especially,serine proteases with clip domain are closely related with the activation of immune cascade pathways of insect.In order to understand the roles of serine proteases in the signal pathways of innate immune reaction in silkworm（Bombyx mori）,we performed sequence alignment and phylogenetic analysis to clip serine proteases identified in B.mori and Manduca sexta.The result showed that BmSP78 and MsPAP-1,BmSP127 and MsHP8,BmSP124 and MsHP1,BmSP125 and MsHP17 were located on the same phyletic clade respectively.Amino acid sequences of these four pairs of clip serine proteases were 70%,68%,81% and 57% identical respectively.In addition,the other 8 proteases,namely BmSP23,BmSP111,BmSP95,BmSP135,BmSP129,BmSP137,BmSP91 and BmSP99,formed a species-specific clade of silkworm clip serine proteases.After day 4 silkworm larvae of the 5th instar were infected through injection with Gram-negative Serratia marcescens or Gram-positive Bacillus bombysepticus,semi-quantitative RT-PCR assay revealed that clip serine protease genes had the following expression patterns at different time points： mRNA transcriptional levels of 11 clip serine protease genes in silkworm body showed obvious change after bacterium challenge.Among them,BmSP102 and BmSP96 genes were obviously up-regulated in the hemocytes at 12 hours post-infection with B.bombyseptieus,and BmSP125 gene was obviously up-regulated in the fat body at 12 hours post infection with S.marcescens or B.bombyseptieus.These results provide important clues for establishing the immune cascade pathway with involvement of clip serine proteases in B.mori.