茶树丝氨酸羧肽酶基因的克隆及表达分析

近年来,人们发现丝氨酸羧肽酶类蛋白(SCPL)参与植物次生代谢产物的酰基转移过程,即具有转酰基功能。本研究采用RT-PCR技术,获得了3个茶树丝氨酸羧肽酶基因的全长序列;生物信息学分析表明,3个CsSCPL蛋白均包含了1个底物结合位点、3个催化作用保守区和多个N-糖基化位点,及其Ser-Asp-His三联体催化中心等SCPL家族的典型特征;进化树分析表明,3个CsSCPL可能具有酰基转移酶的功能。实时荧光定量PCR结果表明3个基因在芽叶茎根中都有表达,其中,CsSCPL1和CsSCPL3在叶中的相对表达量明显高于茎和根,而CsSCPL2则在根中高表达。本研究成功地将CsSCPL重组到表达载体pET32a(+)上进行原核表达,并对诱导时间及诱导温度进行了优化;经IPTG诱导、SDS-PAGE检测,目标蛋白条带分子量为70 kD,与预测大小相符。

The Gene Cloning and Expression Analysis of SCPL in Tea Plant (Camellia sinensis)

In recent years,serine carboxypeptidase-like proteins(SCPL) have been found that they are involved in plant secondary metabolites with the function of transferring acyl.The full-length cDNA of three CsSCPL were cloned from Camellia sinensis by RT-PCR technology.Bioinformatics analysis showed that the three CsSCPL proteins contained SCPL family's characteristic structures,such as one substrate binding and three catalysis conserved regions,a number of N-glycosylation sites and a conserved catalytic triad Ser-Asp-His amino acid active catalytic site and so on.The phylogeny analysis showed that CsSCPL probably possessed acyltransferase function.Quantitative RT-PCR analysis showed that the CsSCPL genes expressed in bud,leaf,stem and root.The relative expression of CsSCPL1 and CsSCPL3 in the leaves was significantly higher than that in stems and roots,while CsSCPL2 was highly expressed in the root.The CsSCPL genes were constructed into expression vector pET-32a(+) for over expression in prokaryotic cells and optimal inducing conditions including time,temperature were studied.The SDS-PAGE showed that recombinant proteins with formula weight 70 kD were induced successfully by IPTG,which coincided with the prediction.