金银花糖基转移酶基因LjUGT71E1的克隆及原核表达

Gene Cloning and Prokaryotic Expression of LjUGT71E1 from Lonicera japonica Thunb.

糖基化广泛存在于生物体中,具有非常重要的作用,而糖基转移酶则是催化糖基化反应的关键酶。本研究根据转录组数据库,设计引物从金银花(Lonicera japonica Thunb.)中克隆得到一个糖基转移酶编码基因LjUGT71E1,序列分析表明该基因的CDS为1 503 bp,理论可编码氨基酸数目为501,等电点(p I)预测为5.09,理论分子量为55 579.71 Da。多重序列比对表明LjUGT71E1与其它植物UGT71家族成员在序列上保守性很高,并且其C端具有催化植物次生代谢产物糖基化的糖基转移酶所具有的保守的PSPG-box基序。系统发生分析表明LjUGT71E1与人参(Panax ginseng)和甜叶菊(Stevia rebaudiana)的UGT71E亚家族成员聚为一支。荧光定量PCR分析表明,该基因在金银花的五个花期均有表达,在幼蕾时期相对表达量达到峰值。进一步构建了LjUGT71E1基因的原核表达载体并且在大肠杆菌中诱导表达得到重组蛋白,为后续的功能研究提供了基础。

Glycosylation is widely present in all living organisms, which plays important role in metabolism. Glycosyltransferase is the key enzyme for catalyzing glycosylation reactions. In the present study, we cloned a glycosyltransferase gene LjUGT71E1 from Lonicera japonica Thunb.. The sequence analysis showed that the CDS of the gene was 1 503 bp and the number of amino acids could be 501. The isoelectric point （pI） is predicted to be 5.09 and the theoretical molecular weight is 55 579.71 Da. Multiple sequence alignment indicated that LjUGT71E1 was highly conserved with UGT71 family members from other plants and had a conserved PSPG-box motif at the C-terminus, which was important for catalytic activity in plant secondary metabolism. Phylogenetic analysis showed that LjUGT71E1 was clustered with UGT71E subfamily of Panax ginseng and Stevia rebaudiana. qRT-PCR analysis showed that LjUGT71E1 was expressed in the five flowering stages of honeysuckle, and the relative expression reached the peak at the early bud stage. The recombinant prokaryotic expression vector of LjUGT71E1 gene was further constructed and recombinant protein was expressed in Escherichia coli, which provided the basis for further study.