马尾松GGPPS基因克隆及序列分析

Cloning and Bioinformatics Analysis of GGPPS of Pinus massoniana

牻牛儿基牻牛儿基焦磷酸合成酶(GGPPS)是萜烯类物质合成的关键酶之一,本研究通过同源序列检索分析设计引物,从马尾松幼苗针叶组织中克隆到其GGPPS基因,命名为Pma GGPPS,该基因c DNA长1 143bp,开放阅读区编码380个氨基酸。Pma GGPPS编码的蛋白质序列分析显示具有Trans IPPS结构域及FARM、SARM两个富含天冬氨酸区域,序列特点与GGPPS蛋白的酶学功能相符。同源性分析结果显示Pma GGPPS核酸序列与其他两种松属植物GGPPS基因同源性达到99%以上,Pma GGPPS编码蛋白与挪威云杉等植物中GGPPS蛋白同源性也在69%以上。对Pma GGPPS编码蛋白进行理化性质及结构的生物信息学分析,结果显示该蛋白为一种无信号肽、无跨膜区的碱性蛋白质,二级结构由16段α-螺旋组成,三级结构同源建模显示与欧薄荷的异聚牻牛儿基焦磷酸合酶大亚基同源性最高。系统进化树分析显示该蛋白与红豆杉、银杏亲缘关系较近。本研究中克隆得到Pma GGPPS基因为深入研究其在松脂合成的影响奠定了基础,同时根据GGPPS基因设计荧光定量引物并优化反应条件,为下一步研究基因表达水平与产脂力关系提供了理论依据和技术参考。

Geranylgeranyl diphosphate synthase （GGPPS） is the key synthase in terpenes synthesis. Its gene was cloned from the needles of Pinus massoniana seedlings by using specific primers based on the highly conserved se- quences, and it was named as PmaGGPPS. Sequence analysis indicated that its cDNA sequence were 1 143 bp with its open reading frame encoding 380 amino acid residues. PmaGGPPS encoding sequence was analyzed, the result showed it has a Trans IPPS domain and two aspartate-rich motif which were FARM and SARM respectively, and the protein sequence characteristics consistent with GGPPS protein enzyme function. The results of homology a- nalysis showed that PmaGGPPS nucleotide sequence had more than 99% sequence homology with the other two pine plants, and PmaGGPPS encoding protein had more than 69% homology with Picea abies and other plants. The physicochemical property and structure of PmaGGPPS encoding protein were analyzed by using bioinformatics tools. The result showed it was an basic protein with no signal peptide or transmembrane domain, and its seconda- ry structure was composed of 16 alpha helix segments. Tertiary structure modeling showed it was most similar to heterotetramerie geranyl pyrophosphate synthase from mint. Phylogenetic analysis showed that it had close relation with Taxus and Ginkgo. The cloning of PmaGGPPS gene provided effective resources to further research on pine resin synthesis. This design of qRT-PCR primers according to the GGPPS gene in this study could optimize the re-action conditions, and provide theoretical basis and technical reference for next-step study on the relations of gene expression level and 1 pine resin production.