芍药FPPS基因的克隆及生物信息学分析

发布时间：2018-04-17 23:16

本文选题：芍药 + 法呢基焦磷酸合酶　；参考：《中草药》2016年04期

【摘要】：目的克隆芍药萜类物质生物合成关键酶法呢基焦磷酸合酶(farnesyl pyrophosphate synthase,FPPS)c DNA全长序列并对其进行生物信息学分析。方法根据芍药转录组测序数据,设计一对特异性PCR引物,应用RT-PCR技术扩增出芍药FPPS基因目标条带并克隆至p MD18-T载体上,菌落PCR和质粒PCR鉴定出阳性重组子后进行序列测定及序列同源性搜索、分子系统进化树构建、结构域搜索及3D结构预测等生物信息学分析。结果测序结果表明芍药FPPS基因全长1 315bp,含60 bp的5’-UTR、1 050 bp的CDS和205 bp的3’-UTR,共编码349个氨基酸,Gen Bank登录号为KP708571;Blastn和Blastp分析结果显示芍药FPPS(KP708571)及其编码的蛋白(AKJ26301)在核苷酸水平和氨基酸水平上与多种植物的FPPS基因和FPPS蛋白具同源性;分子进化树分析结果表明芍药FPPS蛋白与其他物种FPPS蛋白的亲缘关系相对较远;预测芍药FPPS蛋白相对分子质量为40 200,等电点为5.33,是一个定位于胞液、不含跨膜结构域、不含信号肽分子的亲水性、稳定蛋白;发现芍药FPPS含有底物结合位点、底物-Mg2+结合位点、催化位点、富含天冬氨酸位点1及富含天冬氨酸位点2共7个保守结构域;3D结构中α-螺旋所占比例高且α-螺旋之间由β-转角(loop)相连接;中央腔(central cavity)由大约10个核心α-螺旋排列而成。结论首次从芍药中克隆了FPPS基因并对其进行了初步的生物信息学分析。
[Abstract]:Objective to clone and analyze the full-length sequence of farnesyl pyrophosphate synthase (pyrophosphate synthase) DNA from paeonia lactiflora terpene biosynthesis by bioinformatics.Methods A pair of specific PCR primers were designed according to the sequence data of Paeonia lactiflora transcriptome. The target bands of FPPS gene of Paeonia lactiflora were amplified by RT-PCR and cloned into p MD18-T vector.The positive recombinant was identified by colony PCR and plasmid PCR, and then sequenced and homologous search, molecular phylogenetic tree construction, domain search and 3D structure prediction were used for bioinformatics analysis.Results the sequencing results showed that the FPPS gene of Paeonia lactiflora was 1 315 BP in length, containing 60 BP CDS of 5tr 1 050 BP and 205 BP of 3G UTR. the total encode 349 amino acid genin Bank accession number was KP708571Blastn and Blastp analysis showed that Paeonia lactiflora FPPS K708571) and its encoded protein AKJ26301).The nucleotide level and amino acid level were homologous to FPPS gene and FPPS protein of many plants.Molecular phylogenetic tree analysis showed that the relationship between Paeonia lactiflora FPPS protein and other species FPPS protein was relatively distant, and predicted that the relative molecular weight and isoelectric point of Paeonia lactiflora FPPS protein were 40 200 and 5.33 respectively, which were located in cytosol and had no transmembrane domain.It was found that Paeonia lactiflora FPPS contained substrate binding site, substrate mg 2 binding site and catalytic site.Aspartic acid rich site 1 and aspartic acid rich site 2 have a high proportion of 伪 -helix in 3D structure of 7 conserved domains, and 伪 -helix is connected by 尾 -rotation loop, and central cavityof central cavity is arranged by about 10 core 伪 -helix.Conclusion FPPS gene was cloned from Paeonia lactiflora for the first time and its bioinformatics analysis was carried out.
【作者单位】：河南科技大学农学院;河南科技大学林学院;
【基金】：国家自然科学基金资助项目(U1204323) 河南省科技厅国际合作项目(134300510052)
【分类号】：S567.239

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