罗氏沼虾几丁质酶3B基因的克隆及其在蜕皮周期中的表达

Cloning and expression analysis of chitinase-3B from giant freshwater prawn(Macrobrachium rosenbergii) during molting cycle

罗氏沼虾的生长发育及繁殖都与蜕皮紧密相关。几丁质酶是甲壳动物在蜕皮过程中最重要的酶之一。本研究对罗氏沼虾蜕皮周期外观特征和腹肢刚毛进行了观察描述,克隆并分析了罗氏沼虾的几丁质酶Ⅲ基因(MrChi3B),制备了几丁质酶Ⅲ蛋白的多克隆抗体,研究了其在蜕皮周期中的表达。罗氏沼虾几丁质酶基因cDNA的开放阅读框为1 143 bp,编码380个氨基酸,大小为41.91 ku。通过氨基酸序列比对发现,MrChi3B与日本沼虾几丁质酶基因(Chi3B)的同源性最高,为94%。MrChi3B含有一个糖苷键水解酶家族18的催化域。实时定量PCR (qRT-PCR)和蛋白印迹法(WB)检测结果显示,MrChi3B在罗氏沼虾多个组织中均有表达,但是不同组织中的表达有差异。在蜕皮期之后,其在胃、表皮和肌肉中的表达量显著上升。在胃中其表达量在蜕皮间期达到最高;在表皮和肌肉中其表达量在蜕皮后期达到最高;肠中的表达量在蜕皮前期和蜕皮期有所上升;眼柄期的表达量在所有蜕皮期都偏低。本研究结果为进一步研究几丁质酶的功能提供参考依据。

Giant freshwater prawn(Macrobrachium rosenbergii)is an important cultured species in China.Molting is closely related to the growth and breeding of M.rosenbergii.Chitinase,a member of the glycoside hydrolase family 18,plays vital physiological roles in crustaceans,including molting,digestion,and immunity.The molting cycle of M.rosenbergii consisted of five stages,named postmolt(A),postmolt(B),intermolt(C),premolt(D),and molt(E).To understand the characteristics and molecular dynamics of chitinase in the molting cycle of M.rosenbergii,the chitinase gene of M.rosenbergii(MrChi3B)was cloned.Thereafter,polyclonal antibody against the chitinase was generated in rabbit.The cDNA of MrChi3B spans 1 143 bp that encodes a protein of 380 amino acids(aa)with a predicted molecular weight of 41.91 ku.Phylogenetic analysis revealed that chitinase contains the GH18 family catalytic domain,and it was closely related to chitinase of M.nipponense with 94%homology at the aa level.The expression profile of MrChi3B in six different tissues(eyestalk,gill,stomach,muscles,endocuticle,intestine)and at different stages of the molting of M.rosenbergii was investigated by quantitative real-time PCR(qRT-PCR)and Western blot(WB)analysis.The results showed that the MrChi3B was ubiquitously expressed in all the tested tissues,whereas its expression was significantly increased in stomach,skin,and muscle at A,B stages.At the stage C,highest expression of MrChi3B was observed in the stomach.At the stages A,B,the expression of MrChi3B in the endocuticle and muscle reached the highest.The expression of MrChi3B in the intestine has increased during the stage D and E.Relative lower expression of MrChi3B was observed in the eyestalk at all the stages.The results will shed a new light on the further study of the function of MrChi3B from M.rosenbergii.