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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 the geranylgeranyl pyrophosphate synthase gene (GGPS) of Salvia miltiorrhiza.METHODS: Bioinformatics analysis and primary expression characterization were performed. Methods: Degenerate primers were designed according to the conserved regions of GGPS protein sequences. The target genes were obtained from Salvia miltiorrhiza roots by homologous amplification and rapid amplification of cDNA ends. Sequence alignment was performed by BLAST. ORF F inder was used to search for open reading frames. Prosite analysis The basic domain of protein, Target P 1.1 analysis of signal peptide sequence, MEGA4.0 Alignment of existing GGPS protein sequences and the construction of phylogenetic tree; Semi-quantitative RT-PCR detection of roots, leaves and florescence roots and stems of Salvia miltiorrhiza , Leaves, buds and flowers. The specific primers were designed and amplified from Salvia miltiorrhiza genome DNA to obtain the complete GGPS gene. The gene structure was initially analyzed. Results: One GGPS cDNA sequence with a length of 1298 bp was obtained. The ORF of the GGPS cDNA was 1 095 bp, encoding a 364 amino acid sequence of 52 amino acid plastid targeting signal peptide, a polygluorene synthase Specific sequence, and reported more than 60% homology GGPS gene; semi-quantitative RT-PCR analysis showed that the gene expression in all tissues analyzed, especially in the flowering leaves the strongest expression; gene structure Analysis showed that the gene has a 397 bp intron. Conclusion: The geranylgeranyl pyrophosphate synthase gene of Salvia miltiorrhiza for the first time provides the basis for its function study.