中文摘要：

昆虫主要依靠先天免疫反应来抵御外源异物的入侵,而与血淋巴黑化及抗菌肽合成等过程密切相关的丝氨酸蛋白酶激活级联反应在其中起着重要作用。为阐明丝氨酸蛋白酶在菜粉蝶Pieris rapae免疫中的作用,本文通过简并引物RT-PCR克隆获得了菜粉蝶丝氨酸蛋白酶家族基因Pr-SP1的cDNA片段,并利用RACE法扩增获得其全序列。该cDNA序列长1489bp,其中开放阅读框长1059bp,共编码353个氨基酸残基。Pr-SP1含一长度为20个氨基酸残基的信号肽序列,其蛋白理论分子量为36.85kDa,理论等电点为6.41。多序列比对结果表明,Pr-SP1与其他昆虫的同源蛋白基因序列上存在较高一致性,在N端有一个发夹结构域,而C端是一个具有催化活性的结构域。实时荧光定量RT-PCR及免疫印迹结果表明,蛹期Pr-SP1主要在颗粒血细胞内进行转录,其蛋白产物主要定位在血浆;Pr-SP1在不同虫态及虫龄都有转录,其蛋白产物在不同虫态及虫龄都有表达,其中5龄幼虫最高,卵期最低;Pr-SP1的转录水平及其蛋白产物的表达水平均会被大肠杆菌Escherichia coli、藤黄微球菌Micrococcus luteus和巴斯德毕赤酵母Pichia pastoris诱导。根据这些结果分析认为,Pr-SP1属于Sptzle蛋白酶前体激活酶,并参与菜粉蝶的先天免疫反应。

英文摘要：

Insects mainly rely on innate immune responses to resist foreign invasion. Activation of the serine proteinase cascades,involved in hemolymph melanization and antimicrobial peptide synthesis,plays an important role. To define the role of serine proteinases in the innate immune responses of Pieris rapae,a cDNA fragment of a serine proteinase gene,Pr-SP1 screened by RT-PCR using degenerate primer,was sorted out in this article. The full-length cDNA of Pr-SP1 was cloned using RACE strategy. The cDNA length was found to be 1 489 bp,including a 1 059 bp open reading frame,which encodes 353 amino acids with a 20-amino-acid signal peptide. The predicted protein molecular mass and pI are 36.85 kDa and 6.41,respectively. Multiple sequence alignment result revealed that Pr-SP1 shares high identities with its homologs in other insect species,which also possesses a clip and active catalytic domain at N-terminus and C-terminus,respectively. Real-time quantitative PCR and Western blotting results indicated that Pr-SP1 was primarily transcribed in granulocytes in the pupal stage and the protein product of Pr-SP1 was detected mainly in plasma. Pr-SP1 was transcribed and its protein product expressed in different developmental stages and instars. The highest and lowest transcript and expression levels appeared in 5th instar larvae and egg stages,respectively. The transcript level of Pr-SP1 and expression level of Pr-SP1＇s protein product could be induced by Escherichia coli,Micrococcus luteus and Pichia pastoris. According to these results,Pr-SP1 could be considered as a candidate of Sptzle processing enzyme,which may participate in the innate immune responses in P. rapae.