摘要
目的:克隆通粳1号水稻乙醛脱氢酶(aldehyde dehydrogenase,ALDH)基因,并在大肠杆菌中进行体外表达。方法:提取通粳1号水稻幼根总RNA,RT-PCR法扩增ALDH基因开放阅读框架片段,双酶切后连接至pET5a表达质粒中,转化BL21(DE3)宿主菌,平板培养筛选,挑取阳性菌落并提取质粒,酶切、电泳鉴定插入片段并测定其序列。含重组质粒的BL21(DE3)进行常规LB扩大培养,用不同浓度的IPTG作用不同时间进行诱导表达,SDS-PAGE检测表达产物。结果:质粒中插入的ALDH片段为1668bp,含有完整的开放阅读框架,序列与GenBank比较无差异,插入方向正确无误,表达蛋白分子量为61.8kDa左右,符合预期值,IPTG最佳诱导时间为3h,0.1mmol/L～0.8mmol/L浓度的IPTG作用效果无显著差异。结论:该基因的成功克隆和表达为进一步研究水稻中ALDH的作用和应用生物工程法大量获得ALDH奠定基础。
Objective：To clone the gene of aldehyde dehydrogenase（ALDH） and expression in E.coli from rice of No.1 Tongjing.Method：Total RNA was extracted from young root of rice,and ORF fragment of ALDH was amplified using RT-PCR.The fragment was connected to expression plasmid pET5a after double cutting of restriction enzyme,and than transformed to BL21（DE3）,and cultured at plate medium for screening.Positive colony of E.coli was selected and the plasmid was extracted.Insert was confirmed by double cutting of restriction enzyme,electrophoresis and sequencing.BL21（DE3） containing recombinant plasmid was amplified cultured using LB medium as usual,and IPTG was used to induce expression at different concentration and time.SDS-PAGE was used to detect production of expression.Result：The length of inserted ALDH containing complete ORF was 1 668bp in plasmid,and the sequence was coincident compared to GenBank and direction of insert was correct.Molecular weight of expression was around 61.8kDa,and was consistent with expectation.The best time of induction by IPTG was 3h,and the level of expression did not change obviously when the concentration of IPTG varied from 0.1 mmol/L to 0.8 mmol/L.Conclusion：Succession of cloning and expression of ALDH established the base for research on effect of ALDH in rice and obtaining a great quantity of ALDH by means of bioengineering.
出处
《生物技术》
CAS
CSCD
北大核心
2010年第3期16-18,共3页
Biotechnology
