通育粳1号水稻乙醇脱氢酶基因克隆与原核表达

Cloning and Prokaryotic Expression of ADH in E.coli from Rice of No.1 Tongyujing

克隆通育粳1号水稻乙醇脱氢酶(alcohol dehydrogenase,ADH)基因,并在原核系统中进行体外表达。取通育粳1号水稻幼根,提取总RNA,RT-PCR法扩增ADH基因开放阅读框架片段,双酶切后连接至pGEX-4T-1表达质粒中。将质粒转化至BL21(DE3)宿主菌中,平皿培养,挑取阳性菌落培养,提取重组质粒,酶切、电泳鉴定插入片段并测定其序列。pGEX-4T-1-ADH/BL21进行常规LB扩大培养,IPTG(1 mmol/L)作用2、3和4 h诱导表达,SDS-PAGE检测表达产物。结果显示,插入质粒中的ADH片段序列和方向正确无误,表达蛋白分子量符合预期值42 kD,表达至最大值的诱导时间为3 h。因此,该基因的成功克隆和表达为进一步研究水稻中ADH的作用和应用生物工程法大量获得ADH奠定基础。

It was to clone of alcohol dehydrogenase（ADH） and expression in E.coli from rice of No.1 Tongyujing.Total RNA was extracted from young root of rice,and ORF fragment of ADH was amplified using RT-PCR.The fragment was connected to expression plasmid pGEX-4T-1 after double cutting using restriction enzyme,and then transformed into E.coli of BL21（DE3）,and was cultured at plate medium.Positive colony of E.coli was selected and cultured,and plasmid was extracted.Insert fragment was confirmed by cutting plasmid using restriction enzyme and electrophoresis,and sequencing was performed.pGEX-4T-1-ADH/BL21 was amplified cultured using LB medium as usual,and IPTG was used to induce expression for 2,3,and 4 h.SDS-PAGE was used to detect production of expression.The results showed sequence and direction of insert fragment of ADH was correct,and molecular weight of expression was consistent with expectation of 42 kD.The time of induction to the highest level of production of expression was 3 h.Succession of cloning and expression of ADH established the base for research on effect of ADH in rice and obtaining a great quantity of ADH by means of bioengineering.