茶尺蠖丝氨酸蛋白酶基因EoSP1的克隆、时空表达及对饥饿的表达响应

张新; 陈成聪; 杜琴; 李喜旺; 孙晓玲

doi:10.13305/j.cnki.jts.2019.06.006

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茶叶科学 ›› 2019, Vol. 39 ›› Issue (6) : 669-680. DOI: 10.13305/j.cnki.jts.2019.06.006

茶尺蠖丝氨酸蛋白酶基因EoSP1的克隆、时空表达及对饥饿的表达响应

张新^1,2, 陈成聪³, 杜琴^1,2, 李喜旺^1,2, 孙晓玲^1,2,*

作者信息 +

Cloning and Expression Analysis of Serine Protease EoSP1 in Tea Geometrid (Ectropis obliqua) and Its Response to Starvation

ZHANG Xin^1,2, Chen Chengcong³, DU Qin^1,2, LI Xiwang^1,2, SUN Xiaoling^1,2,*

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文章历史 +

摘要

丝氨酸蛋白酶是鳞翅目昆虫消化系统中一类重要的蛋白酶。本研究从茶尺蠖（Ectropis obliqua）中克隆到一条丝氨酸蛋白酶基因EoSP1并分析了其结构特征和表达特性。EoSP1基因序列全长858 bp,编码285个氨基酸,预测蛋白分子量为29.53 kDa,等电点为5.44。经与其他丝氨酸蛋白酶比对,EoSP1含有保守的丝氨酸蛋白酶催化位点（H₉₅,A₁₆₁和S₃₂₈）及蛋白互作结构域,与蓓带夜蛾（Mamestra configurata）中SPs亲缘关系较近。进一步获得了与EoSP1-GST融合蛋白大小接近的目的蛋白。qRT-PCR分析发现,EoSP1在幼虫期的表达显著高于成虫、蛹和卵期,并在幼虫中肠中特异性表达;饥饿处理后EoSP1表达下降,恢复饲喂后表达量接近对照组。以上结果为茶尺蠖消化酶功能解析及抗虫新靶点的筛选提供了依据。

Abstract

Serine protease plays an important role in the digestion process of Lepidoptera insects. In this study, we cloned a serine protease encoding gene EoSP1 from Ectropis obliqua and analyzed its basic characteristics and expression patterns. The coding sequence of EoSP1 is 858 bp, encoding 285 amino acid residues with deduced molecular weight of 29.53 kDa and isoelectric point of 5.44. Compared with other serine proteases, EoSP1 contains conserved serine protease catalytic sites (H₉₅, A₁₆₁ and S₃₂₈) and protein interaction domains, and shows the closest relationship with SPs from Mamestra configurata. Further, EoSP1-GST fusion protein similar to the predicted size was purified from E. coli cells. qRT-PCR analysis showed that the expression level of EoSP1 was much higher in larvae than that in adults, pupae and eggs, and expressed in midgut of larvae specifically. EoSP1 was down-regulated by starvation treatment, and the expression level was change back to that of control group after re-feeding. The above results provide a basis for the function analysis of digestive enzyme and screening of new insect-resistance targets in Ectropis obliqua.

导出引用

张新, 陈成聪, 杜琴, 李喜旺, 孙晓玲. 茶尺蠖丝氨酸蛋白酶基因EoSP1的克隆、时空表达及对饥饿的表达响应[J]. 茶叶科学. 2019, 39(6): 669-680 https://doi.org/10.13305/j.cnki.jts.2019.06.006

ZHANG Xin, Chen Chengcong, DU Qin, LI Xiwang, SUN Xiaoling. Cloning and Expression Analysis of Serine Protease EoSP1 in Tea Geometrid (Ectropis obliqua) and Its Response to Starvation[J]. Journal of Tea Science. 2019, 39(6): 669-680 https://doi.org/10.13305/j.cnki.jts.2019.06.006

中图分类号： S435.711 Q52

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