摘要
克隆新疆野生橡胶草顺式异戊烯转移酶基因,对其进行生物信息学分析,构建该基因的原核表达载体,在大肠杆菌中诱导表达融合蛋白,为进一步研究该基因的功能和植物体内天然橡胶合成分子机制奠定基础。采用同源克隆法结合RT-PCR技术获得目的基因,测序正确后将该基因片段连接到原核表达载体pET-30a中,转化大肠杆菌BL21并诱导表达。结果表明:该基因开放阅读框为927bp,编码308个氨基酸,推测的蛋白分子质量为34.9ku,理论等电点为8.74;具有cis-异戊烯基转移酶的5个高度保守区,属于cis-IPPS superfamily蛋白家族;系统进化树表明,橡胶草顺式异戊烯基转移酶与短角蒲公英的亲缘关系最近;经IPTG诱导和SDS-PAGE检测,所构建的原核表达载体表达的融合蛋白与预期蛋白相符合。可见,利用RT-PCR技术从橡胶草叶片中克隆得到顺式异戊烯转移酶基因,成功构建原核表达载体,并使其在大肠杆菌中得到表达。
Cloning,sequence analysis of the cis-prenyltransferase gene from Taraxacum kok-saghyz Rodin,and expression of recombinant proteins in Escherichia coli were conducted to further research the gene＇s function and the molecular mechanism of Natural Rubber Biosynthesis in the plant.The TkCPT1 gene was amplified by homologous sequence cloning method and RT-PCR,then the cloned genes of TkCPT1 were inserted into vector pET-30 a.The recombinant plasmids pET-30a-TkCPT1 were expressed in the prokaryotic expression system after its transformation into E.coli BL21.The TkCPT1 genes＇ s ORF of 927 bp and encodes 308 amino acids.The putative protein of the gene had an isoelectric point of 8.74 and a calculated molecular weight of 34.9ku.The protein contained five highly conserved regions,belonged to the cis-IPPS superfamily protein family;phylogenetic analysis showed that the TkCPT1 had nearest relationship with Taraxacum brevicorniculatumcis-prenyltransferase.Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG.In this study,the cis-prenyltransferase gene was obtained from Taraxacum koksaghyz Rodin by RT-PCR,and the prokaryotic expression vector for this gene was constructed,andthen successfully induced to express by IPTG in BL21.
出处
《西北农业学报》
CAS
CSCD
北大核心
2015年第8期84-91,共8页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家自然科学基金(31360060)
关键词
橡胶草
顺式异戊烯转移酶
序列分析
原核表达
Taraxacum kok-saghyz Rodin
Cis-prenyltransferase
Sequence analysis
Prokaryotic expression
