米虾鞣化激素的表达特征分析及体外合成

Expresstion Characteristics Analysis and in vitro Synthesis of Bursicon from Caridina

[目的]观察米虾的胚胎发育过程。制备米虾发育过程中鞣化激素(Bursicon)的α和β这2个亚基基因蛋白并探讨其在不同组织中的表达特征。[方法]利用光学显微镜观察米虾的胚胎发育过程。根据转录组提示,PCR扩增2个亚基基因的编码区。目的片段经双酶切后分别连入载体p ET-28a-c(+)中,构建表达载体p ET-28a-α、p ET-28a-β。重组质粒转入Rosetta表达菌后用IPTG诱导其表达。采用实时荧光定量PCR检测bursicon-α和bursicon-β在米虾的眼柄、心、胸神经节和腹神经节4个组织中的表达差异。[结果]米虾胚胎发育阶段包括卵裂期、囊胚期、原肠期、前无节幼体期、后无节幼体期、复眼色素形成期以及蚤状幼体期共7个阶段。SDS-PAGE凝胶电泳检测发现体外原核系统能够成功表达Bursα和Bursβ,二者的相对分子质量约为15 k D。此外表达特征分析显示bursicon-α和bursicon-β在米虾腹神经节中的相对表达量最高,胸神经节中的相对表达量次之,在眼柄和心中的相对表达量较低。[结论]米虾中鞣化激素的2个亚基基因主要是在其腹神经节中表达,属于神经多肽激素,能够分泌到血淋巴中发挥调节作用;体外原核表达系统获得重组蛋白Bursα和Bursβ,为进一步研究其功能及应用于生产奠定了基础。

[Objective]To observe the process of embryo development in Caridina and express the proteins of bursicon-αand bursicon-β.Besides,to explore the expresstion characteristics of bursicon in different tissues from Caridina.[Method]The light microscopy was used to observe the embryo development.According to the transcriptome hints,the coding regions of bursicon-αand bursicon-βwere amplified with PCR.The PCR products were inserted into the pET-28a-c(+)vector after double digestion to construct expresstion vectors,pET-28a-αand pET-28a-β.The recombinant plasmids were transformed into Rosetta and expressed by induction with IPTG.The expression differences of bursicon-αand bursicon-βin four tissues of eyestalk,heart,thoracic ganglia and abdominal ganglia from Caridina were detected by the real-time quantitative PCR.[Result]The process of embryo development in Caridina included seven stages:cleavagestage,blastula,gastrula,egg-nauplius stage,egg-metanauplius stage,embryo with eye pigments formed stage and prehatching stage.Bursαand Bursβwere obtained successfully by prokaryotic expression analyzed by SDS-PAGE.The relative molecular mass of both bursicon subunits were 15 kD.In addition,the expresstion characteristics analysis showed that the relative expression levels of bursicon-αand bursicon-βwere reached to the highest level in abdominal ganglia and declined in thoracic ganglia.Nevertheless,low level relative expressions were clearly detectable in the eyestalk and heart.[Conclution]These findings indicated that bursicon-αand bursicon-βexpressed mainly in abdominal ganglia.Bursicon is a kind of neuropeptide hormone.It plays a regulatory role by releasing into hemolymph.The proteins of Bursαand Bursβobtained by prokaryotic expression laid a foundation of further study on the function.