丹参牛牻儿基牻牛儿基焦磷酸合酶基因的克隆与分析

Cloning and characterization of geranylgeranyl diphosphate synthase gene of Salvia miltiorrhia

目的:获得丹参牻牛儿基牻牛儿基焦磷酸合酶基因(GGPS),并进行生物信息学分析和初步的表达特性研究。方法:根据GGPS蛋白序列保守区域设计简并引物,利用同源扩增和cDNA末端快速扩增技术从丹参根中获得目的基因;利用BLAST进行序列比对,ORF F inder寻找开放阅读框,Prosite分析蛋白质的基本结构域,Target P 1.1分析信号肽序列,MEGA4.0比对已有GGPS蛋白序列,并构建进化树;用半定量RT-PCR检测其在丹参子叶期根、叶和花期根、茎、叶、花蕾、花中的表达情况;设计特异引物,从丹参基因组DNA中扩增,获得编码完整的GGPS基因,初步分析该基因结构。结果:得到1条长1298 bp的GGPS cDNA序列,其ORF框长1 095 bp,编码1段含有52个氨基酸质体定位信号肽的364个氨基酸序列,具有多聚异戊二烯基合成酶的特异序列,与已报道的GGPS基因有60%以上的同源性;RT-PCR半定量分析表明该基因在所分析的各组织中均有表达,尤以花期叶片中的表达最强;基因结构分析表明该基因有1个397 bp的内含子。结论:首次得到丹参牻牛儿基牻牛儿基焦磷酸合酶基因,为其功能研究提供了基础。

Objective： To obtain geranylgeranyl diphosphate synthase gene of Salvia miltiorrhiza, and conduct bioinfformatic and transcript expression analysis of the cloned SmGGPS1 gene. Method ： The degenerate primers were designed based on the conservative regions of GGPS protein sequences from public databases. The target gene was obtained from root of S. miltiorrhiza by use of homologous cDNA amplification and RACE technologies. The sequence alignment was performed using BLAST. The open reading frame was identified by use of the ORF Finder. The protein domains were defined by use of Prosite software and the signal peptide sequence was predicted by Target P1.1. MEGA4.0 was used to conduct multiple amino acid sequence alignment and construct the phylogenetic tree. Roots and leaves at the seedlings stage and roots, stems, leaves, buds and flowers in the flowering stage were sampled for transcript analysis. Semi-quantitative RT-PCR was used to detect the gene expression level. The complete gene of GGPS was obtained from S. miltiorrhiza genomic DNA by PCR using the cDNA-derived specific primer. The gene structure of GGPS was analyzed by comparison of the gcnomic DNA and its cDNA. Result： The obtained 1 298 bp SmGGPS1 cDNA sequence contains an 1095 bp ORF, encoding 364 amino acids. It is predicted that it has a plastid targeting signal peptide of approximately 52 amino acid at the N-terminal end. It is to believe that this is the polyprenyl synthetase signature, and nucleic acid sequence comparison revealed that SmGGPS1 ORF has more than 60% identity to the reported GGPS. RT-PCR semi-quantitative analysis showed that the gene expresses in the all tested tissues, and with much higher level of expression in the leaves in the flowering stage. SmGGPS1 has a 397 bp intron. Conclusion： For the first time the cloning of geranylgeranyl diphosphate synthase gene from S. miltiorrhiza was reported, and it provides a good basis for further functional study of SmGGPS1.