CAJ | 학술논문

[目的]研究蜡梅香基因SAMT的cDNA克隆及在大肠杆菌中的表达。【方法]以蜡梅花瓣总RNA为模板进行RT—PCR，扩增得到与预期大小相f司的PCR产物带，回收RT—PCR产物，将其与PMD18．T载体进行T—A克隆连接并导入大肠杆菌DH5，经菌落PCR和双酶切鉴定，筛选带有目的基因的重组质粒并进行测序。[结果]测序证实成功克隆得到蜡梅花香基因SAMTcDNA的ORF片段，其长度为1196bp，编码380个氨基酸残基，与已报导的蜡梅SAMT（ABU88887）的同源性为99．2％将SAMT基因亚克隆到原核表达载体PGEX4T—i中，转化大肠杆菌DH5d获得其原核表达菌株pGSAMT；采用O．01mol／L的IPTG进行诱导表达，经SDS—PAGE分析，SAMT的融合表达蛋白分子量约为66kDa，与预期的26KDa的GST带和42-3KDa的蜡梅SAMT基因编码蛋白构成的融合蛋白大小接近。[结论]该研究成功克隆并表达了蜡梅花香基因SAMT。
[목적]연구사매향기인SAMT적cDNA극륭급재대장간균중적표체。【방법]이사매화판총RNA위모판진행RT—PCR，확증득도여예기대소상f사적PCR산물대，회수RT—PCR산물，장기여PMD18．T재체진행T—A극륭련접병도입대장간균DH5，경균락PCR화쌍매절감정，사선대유목적기인적중조질립병진행측서。[결과]측서증실성공극륭득도사매화향기인SAMTcDNA적ORF편단，기장도위1196bp，편마380개안기산잔기，여이보도적사매SAMT（ABU88887）적동원성위99．2％장SAMT기인아극륭도원핵표체재체PGEX4T—i중，전화대장간균DH5d획득기원핵표체균주pGSAMT；채용O．01mol／L적IPTG진행유도표체，경SDS—PAGE분석，SAMT적융합표체단백분자량약위66kDa，여예기적26KDa적GST대화42-3KDa적사매SAMT기인편마단백구성적융합단백대소접근。[결론]해연구성공극륭병표체료사매화향기인SAMT。
[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable size. The RT-PCR products were reclaimed and transformed into E. coli DH 5o together with the PMD18-T vector after ligating by T-A cloning. Identified by colony PCR and EcoRI and Notl digestion, the recombinant plasmid with target gene was screened out and conducted the sequence analysis. [Result] Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned from Chimonanthus praecox gene, with the length of 1 196 bp and encoding 380 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Chimonanthus praecox （ABU88887）. The SAMT cDNA fragment was sub-cloned into the prokaryotic expression vector PGEX1-4T-1, and the obtained re- combinant plasmid was named PGSAMT. After inducting by 0.01mol/L IPTG, the re- sult of the SDS-PAGE analysis showed that the molecular weight of the fusion ex- pression SAMT protein was about 66 kDa, which was close to the predicted fusion protein derived from the 26 kDa GST band and 42.3 kDa SAMT gene of Chimo- nanthus praecox encoded protein. [Conclusion] This study successfully cloned and expressed the SAMT gene of Chimonanthus praecox.