CAJ | 학술논문

应用RACE技术克隆了青虾(Macrobrachium nipponense)的Hc基因全长cDNA序列,并对该基因序列特征进行了分析。青虾Hc基因cDNA全长2235 bp,包括10 bp的5′末端非翻译区(UTR),2074 bp的开放阅读框(ORF),151 bp的3′UTR,开放阅读框编码688个氨基酸。蛋白相似度比对显示,青虾血蓝蛋白含有典型的6个保守的铜离子结合位点。系统进化树分析表明,青虾Hc与脊尾白虾(Exopalaemon carinicauda) Hc聚在一起,具有最近的亲缘关系。荧光定量PCR检测显示, Hc基因在青虾不同组织中均有表达,其表达量在肝胰腺中最高；使用荧光定量PCR检测青虾Hc基因在低氧胁迫和复氧条件下在肝胰腺中的mRNA时空表达情况,结果显示,经低氧和复氧刺激后,与对照组相比, Hc 在肝胰腺中的表达量分别在低氧胁迫12 h、24 h 和复氧6 h出现了3次明显上调,由此推测Hc基因参与低氧应激分子过程。此外,本研究通过血蓝蛋白的分离纯化技术制备了多克隆抗体,本研究结果可为更进一步的了解青虾血蓝蛋白的生理功能提供理论支撑。
응용RACE기술극륭료청하(Macrobrachium nipponense)적Hc기인전장cDNA서렬,병대해기인서렬특정진행료분석。청하Hc기인cDNA전장2235 bp,포괄10 bp적5′말단비번역구(UTR),2074 bp적개방열독광(ORF),151 bp적3′UTR,개방열독광편마688개안기산。단백상사도비대현시,청하혈람단백함유전형적6개보수적동리자결합위점。계통진화수분석표명,청하Hc여척미백하(Exopalaemon carinicauda) Hc취재일기,구유최근적친연관계。형광정량PCR검측현시, Hc기인재청하불동조직중균유표체,기표체량재간이선중최고；사용형광정량PCR검측청하Hc기인재저양협박화복양조건하재간이선중적mRNA시공표체정황,결과현시,경저양화복양자격후,여대조조상비, Hc 재간이선중적표체량분별재저양협박12 h、24 h 화복양6 h출현료3차명현상조,유차추측Hc기인삼여저양응격분자과정。차외,본연구통과혈람단백적분리순화기술제비료다극륭항체,본연구결과가위경진일보적료해청하혈람단백적생리공능제공이론지탱。
Hemocyanin is a copper-binding protein that plays a crucial role in the physiological processes of crustacean. In this study, the cDNA-encoding hemocyanin subunit (MnHc) from the oriental river prawn, Macrobrachium nippo-nense, was cloned using expressed sequence tag (EST) analysis and a rapid amplification of cDNA ends (RACE) ap-proach. The full-length cDNA of MnHc was 2 235 bp, consisting of a 5′untranslated region of 10 bp, a 3′untranslated region of 151 bp, and an open reading frame of 2 064 bp. The deduced protein had 688 amino acid residues with a mo-lecular mass of 79.16 kD. Based on a protein similarity comparison, the two copper-binding sites and six histidine resi-dues necessary for stabilization of oxygen-binding were highly conserved. Phylogenetic tree analysis indicated that M. nipponense has the closest relationship with Exopalaemon carinicauda. Quantitative real-time RT-PCR analysis showed that the MnHc gene was expressed in hemocytes, the hepatopancreas, muscles, testis and intestines, with the highest level of expression in the hepatopancreas and the lowest in muscles. After environmental hypoxia and reoxygenation challenge, the relative expression level of MnHc in the hepatopancreas was significantly higher compared with the con-trol group at 12 h, 24 h post-hypoxia and 6 h reoxygenation, followed by a gradual recovery at 24 h reoxygenation. The highest titer of antiserum was up to 1︰120 000, as revealed by ELISA assay. The specificity of antibodies against hemocyanin was verified by westernblotting. The study indicates that the expression levels of MnHc in the hepatopan-creasmay be affected by environmental dissolve oxygen, and hemocyanin may potentially be involved in the hypoxia stress response of M. nipponense.