家蚕丝氨酸蛋白酶BmSP141的表达特征及对饥饿的响应

Characterization and Expression Analysis of Serine Protease BmSP141 from the Silkworm(Bombyx mori ) in Response to Starvation

【目的】分析家蚕(Bombyx mori)丝氨酸蛋白酶基因BmSP141序列信息,明确其时空表达模式,结合饥饿与重新喂食处理对其表达的影响,探究该基因在家蚕中的功能。【方法】对家蚕丝氨酸蛋白酶基因BmSP141进行T-A克隆,得到其编码区核苷酸序列;应用生物信息学在线网站对该基因编码区推导的氨基酸序列、分子量、结构域等信息进行分析;利用Clustal X1.8和MEGA5.02软件对BmSP141与其他物种的丝氨酸蛋白酶进行多序列比对和系统发生树分析;构建原核表达载体p28-BmSP141,并转化到Rosetta(DE3)菌株中诱导表达,利用镍柱亲和层析纯化重组蛋白。利用半定量RT-PCR和实时荧光定量PCR方法对其组织和时期表达特征进行分析;采用蛋白质免疫印迹(Western blot)技术,分别分析BmSP141蛋白在家蚕5龄第3天各组织的表达情况和5龄幼虫中肠的表达变化;通过免疫荧光定位分析BmSP141蛋白在4龄幼虫中肠的分布情况;利用实时荧光定量PCR和Western blot方法对饥饿处理和再喂食后BmSP141的表达情况进行分析。【结果】BmSP141编码含有292个氨基酸的蛋白,其中第1—17位氨基酸为信号肽,其成熟体蛋白预测分子量为25.9 k D,等电点为7.8。同源序列比对表明,BmSP141与烟芽夜蛾丝氨酸蛋白酶和蓓带夜蛾丝氨酸蛋白酶序列同源性较高,分别达到62%和63%。这些同源序列具有保守的催化三联体,与活性相关的基序也很保守,但与胰凝乳蛋白酶保守的底物特异性位点相比,BmSP141的底物特异性位点发生改变。在16和37℃条件下诱导后,重组蛋白均以包涵体形式表达,经镍柱亲和层析纯化后得到了较纯的重组蛋白,并用该蛋白制备了效价较高的多克隆抗体。组织和时期表达特征分析表明,该基因主要在家蚕幼虫的中肠组织特异性表达,且在幼虫起蚕到食桑期表达逐渐增加,但眠期表达下降,蛹期和成虫期表达水平较低。Western blot分析也表明,BmSP141蛋白仅在家蚕中肠组织表达,且从5龄起蚕到上蔟表达量先增加后降低。免疫荧光定位结果表明,BmSP141定位于中肠上皮细胞的细胞质中,进一步证实BmSP141在中肠特异表达。在转录和翻译水平,BmSP141饥饿处理后表达量显著下调,而重新喂食后其表达量上调,表明BmSP141的表达受到消化道食物的诱导。【结论】家蚕丝氨酸蛋白酶BmSP141在中肠组织表达,在幼虫期食桑期高表达,蛹期和成虫期表达水平较低,受消化道食物的诱导而上调表达,推测该基因可能参与家蚕中肠的消化过程。

【Objective】The objective of this study is to analyze the sequence of BmSP141, clear its spatial and temporal expression patterns, and explore its potential function by starvation and re-feeding in silkworm（Bombyx mori）. 【Method】The nucleotide sequence of serine protease gene BmSP141 coding region was obtained by TA cloning. Biology online software was used to analyze the amino acid sequence, molecular weight, functional domain and other information of the coding region of this gene.Sequence alignment and phylogenetic tree analysis of BmSP141 with serine proteases from other species were done by Clustal X1.8 and MEGA5.02 software. Prokaryotic expression vector p28-BmSP141 was constructed and introduced into Rosetta（DE3） to express the target protein, which was purified by nickel affinity chromatography. The expression profile of BmS P141 at m RNA and protein levels in different tissues and development stages were analyzed by semi-quantitative reverse transcription PCR（RT-PCR）, quantitative real-time PCR（q PCR） and Western blot. Immunofluorescence localization was used to detect the location of BmSP141 protein in the 4th instar larvae. The impacts of starvation and re-feeding on the expression of BmSP141 m RNA and protein were detected by q PCR and Western blot. 【Result】The BmSP141 encoded a 292 amino acid protein with a 17-residues signal peptide. The predicted molecular mass and isoelectric point of BmS P141 mature protein are 25.9 k D and 7.8, respectively. Multi-sequence alignment showed that BmS P141 share a highly sequence identity（62% and 63%） with the serine proteases of Heliothis virescens and Mamestra configurata, respectively. These homologs have conserved catalytic triad and similar serine protease catalytic motifs, but the substrate specificity pocket sites of BmS P141 are relatively different compared with the conversed pocket sites of chymotrypsin. Furthermore, the recombinant protein BmSP141 was expressed as inclusion body at 37℃, and purified by nickel affinity chromatography. Then, the purified protein was used to generate the BmSP141-specific polyclonal antibodies. RT-PCR analysis of BmSP141 revealed that BmSP141 m RNA was mainly expressed in the larval midgut and highly expressed at the larval feeding stage. Whereas its expression level was lower in the pupae and moth than in the larvae. Western blot analysis showed that BmS P141 protein was only expressed in the midgut and the expression level was increased first and then decreased from 5th instar to wandering stage. Immunohistochemical analysis further confirmed that BmSP141 protein was predominately present in the cytoplasm of midgut epithelium cell. The expression of BmS P141 m RNA and protein was down-regulated after starvation but up-regulated by re-feeding. As a result, the expression of BmSP141 was induced by the food in the midgut.【Conclusion】BmS P141 was highly expressed in the midgut of larval feeding stage and lower expressed in the pupae and moth stages than in the larvae stage. The expression of BmSP141 m RNA and protein was down-regulated after starvation but up-regulated by re-feeding, which suggested that this gene might be involved in food protein digestion in the midgut during larval development.