建兰花色调控关键基因的克隆与表达

Cloning and expression of key regulatory gene of flower color formation in cymbidium ensifolium

对建兰花色形成的关键调控基因(CHS)编码区片段克隆至pET28a质粒,转化至大肠杆菌菌株BL21(DE3)。SDS-PAGE检测表达产物,针对IPTG浓度、温度与时间等因素,优化其最佳诱导表达条件。结果显示:(1)总RNA提取质量高,经反转录成cDNA,设计引物通过PCR扩增,琼脂糖电泳显示片段为1200 bp;(2)对转化子进行菌落PCR与质粒双酶切鉴定,片段大小均为1200 bp与预计相符,经测序获得片段插入方向正确,未发生突变,可用于蛋白诱导表达;(3)诱导表达优化显示,温度为37℃、IPTG浓度1 mM及诱导时间6 h是重组子(pET28-CeCHS1)在大肠杆菌BL21(DE3)菌株中诱导表达的最佳条件。

In this study,the coding region of a key regulatory gene(CHS)was cloned into pET28a plasmid and transformed into Escherichia coli strain BL21(DE3).The expression products was detected by SDS-PAGE,and the inductive expression conditions of protein were optimized according to the factors such as IPTG concentration,temperature and time.The results showed that:(1)The total RNA was extracted with high quality and reverse-transcripted into cDNA,which were amplified by PCR.The agarose electrophoresis showed a fragment of 1200 bp.(2)The fragment of 1200 bp was identified by colony PCR and digestion again,which was consistent with the prediction.Sequencing confirmed that the insertion direction of the fragment was correct without mutation,also indicated that the recombinant could be used for inducible expression.(3)The optimal conditions for the expression of pET28-CeCHS1 in Escherichia coli BL21(DE3)were induced at 37℃,IPTG concentration of 1 mM and induction time of 6 h.