阳春砂磷酸甲羟戊酸激酶的基因克隆与表达谱分析

Cloning and expression profiles analysis of PMK gene in Amomum villosum Lour.

目的：为了了解阳春砂磷酸甲羟戊酸激酶（PMK）的功能与表达特性，根据阳春砂的转录组注释设计引物，PCR 克隆阳春砂甲羟戊酸途径中的关键酶磷酸甲羟戊酸激酶基因的编码区 cDNA，使用生物信息学方法对其编码的蛋白进行相似性检索及同源序列比对，构建进化树，预测二级及三级结构并分析其功能，探索其各器官中的表达差异．结果：获得了全长1539 bp 的编码阳春砂 PMK 的 cDNA 序列，命名为 AvPMK （GenBank 登录号：KF569902），编码由512个氨基酸组成的磷酸甲羟戊酸激酶，该蛋白含有 GHMP 激酶超家族特异的 N －保守区域、C －保守区域和 ATP 结合位点 Gly-X-Gly-XX-Ala．AvPMK 与其他植物的 PMK 氨基酸相似性达61％～69％，进化树结果显示其与二穗短柄草（Brachypodium distachyon）等单子叶植物具有较近的亲缘关系．AvPMK 蛋白包含14个α-螺旋（Alpha helix），占37.1％；16个β链（Beta strand），占15.6％；32个无规则卷曲结构，占47.3％，无跨膜结构域，其3级结构含有一个催化反应的口袋状结构域．实时荧光定量 PCR 结果显示 AvPMK 基因在叶中表达量较高，茎、根中表达量相对较低．结论：首次从阳春砂仁中扩增磷酸甲羟戊酸激酶的基因片段，为分析其基因表达特性及其在砂仁萜类生物合成中的功能奠定基础．

Phosphomevalonate kinase is involved in an essential step in the so-called mevalonate path-way.In order to understand the function and expression of phosphomevalonate kinase in Amomum villo-sum Lour.,the full-length cDNA encoding phosphomevalonate kinase （PMK）from Amomum villosum Lour.was successfully cloned （designated as AvPMK,GenBank accession number KF569902）.It enco-ded a protein with 512 amino acids and the calculated size was found at 55.95 KDa.The deduced amino acids sequence of AvPMK exhibited specific recognition sequence of GHMP kinase super family：the N-terminal domain,the C-terminal domain and the ATP-binding sites （Gly-X-Gly-XX-Ala）.AvPMK had 61% ~69% amino acid sequence similarities to other plants,and the phylogenetic analysis showed that it was closely related to the monocotyledons,such as Brachypodium distachyon.In the secondary structure, the protein contained 14 alpha helixs （37.1%）,16 extended strands （15.6%）,32 random coils （47.3%）without transmembrane domain.The tertiary structure exhibited a pocket structure domain.＆nbsp;The q-PCR analysis showed that AvPMK was expressed highly in the leaf and lowly in the stem and root by realtime PCR analysis.Conclusion：The AvPMK gene was cloned from Amomum villosum Lour.for the first time,and the results laid a foundation for studying the gene expression pattern and regulatory func-tions of AvPMK in terpenoids biosynthesis.