家蚕丝氨酸蛋白酶基因BmSP25转录分析及其免疫响应

发布时间：2018-11-19 13:57

【摘要】：【目的】分析家蚕丝氨酸蛋白酶(SP)基因序列BmSP25及转录情况,明确其表达规律对防御家蚕核型多角体病毒(BmNPV)入侵的免疫应答机制,为揭示BmSPs在家蚕免疫应答方面的功能作用提供理论依据。【方法】克隆BmSP25基因序列,对该基因编码蛋白的氨基酸序列、分子量、结构域等进行生物信息学分析;利用Gene Doc和MEGA5.0对BmSP25氨基酸序列进行多序列比对及系统发育进化树分析,采用半定量RT-PCR对家蚕不同组织和发育时期的BmSP25基因转录情况进行分析,并以实时荧光定量PCR检测BmSP25基因在BmNPV感染家蚕中肠组织中的转录水平。【结果】BmSP25基因的ORF全长885 bp,编码294个氨基酸,其中第1~17位氨基酸为信号肽,去信号肽的分子量为29.1 kD,理论等电点为7.8。BmSP25蛋白由4个α螺旋、15个β折叠和一些无规则卷曲构成;其氨基酸序列同源性比对分析发现,BmSP25(BGIBMGA008514-PA)与蓓带夜蛾SP序列(Gen Bank登录号ADM35105)的同源性最高,为62.1%。BmSP25基因在家蚕中肠组织中特异表达,且在整个幼虫时期呈持续性表达。BmSP25基因在家蚕感染BmNPV后发生明显变化,至感染6 h时呈下调趋势,而在感染3、12和24 h时均呈明显上调表达。【结论】BmSP25在防御BmNPV入侵家蚕的免疫应答过程中发挥重要作用。鉴于昆虫SP具有高度保守的底物特异性位点,因此可利用底物类似物、基因定点突变等方式来预防农林害虫。
[Abstract]:[objective] to analyze the sequence and transcription of serine protease (SP) gene in Bombyx mori (Bombyx mori), and to clarify the immune response mechanism of its expression to (BmNPV) invasion of Bombyx mori nuclear polyhedrosis virus. [methods] the sequence of BmSP25 gene was cloned, and the amino acid sequence, molecular weight and domain of the protein were analyzed by bioinformatics. Gene Doc and MEGA5.0 were used to analyze the amino acid sequences of BmSP25 and phylogenetic tree, and semi-quantitative RT-PCR was used to analyze the transcription of BmSP25 gene in different tissues and developmental stages of Bombyx mori (Bombyx mori). The transcriptional level of BmSP25 gene in the midgut of silkworm infected with BmNPV was detected by real-time fluorescence quantitative PCR. [results] the total length of ORF of BmSP25 gene encoded 294 amino acids, in which the first 17 amino acid was a signal peptide. The molecular weight of the de-signalling peptide is 29.1 kD,. The theoretical isoelectric point of 7.8.BmSP25 protein is composed of four 伪 helix, 15 尾 folds and some irregular curls. The homology analysis of amino acid sequence showed that BmSP25 (BGIBMGA008514-PA) had the highest homology with SP (Gen Bank accession number ADM35105), which indicated that 62.1%.BmSP25 gene was specifically expressed in the midgut of silkworm, Bombyx mori (Bombyx mori). The expression of BmSP25 gene in silkworm was significantly changed after infection of BmNPV, and decreased at 6 h after infection. The expression of BmSP25 was upregulated significantly at 12 and 24 h after infection. [conclusion] BmSP25 plays an important role in the immune response to BmNPV invasion of Bombyx mori (Bombyx mori). In view of the highly conserved substrate-specific sites of insect SP, substrates analogue and gene site-directed mutation can be used to prevent agricultural and forestry pests.
【作者单位】：云南省农业科学院蚕桑蜜蜂研究所;
【基金】：国家蚕桑产业技术体系建设专项项目(CARS-22) 云南省现代农业蚕桑产业技术体系建设项目(2013KJTX006) 云南农业科学院蚕桑蜜蜂研究所青年创新基金项目(QC2015004)
【分类号】：S884.51

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