刺五加甲羟戊酸焦磷酸脱羧酶基因的克隆与表达分析

利用RACE技术克隆刺五加甲羟戊酸焦磷酸脱羧酶(mevalonate diphosphate decarboxylase,MDD)基因的全长cDNA序列,运用生物信息学方法对该基因进行分析,并通过RT-PCR法检测MDD在刺五加不同生长发育时期和不同器官中的表达情况。结果表明:(1)刺五加MDD基因cDNA序列全长1 769bp(GenBank登录号为JQ905594),开放阅读框全长1 263bp,编码420个氨基酸残基,包含GHMP激酶超家族的特异性识别序列;刺五加MDD蛋白的二级结构中含有161个α螺旋,占38.33%;68个延伸链,占16.19%;19个β折叠,占4.52%;172个无规则卷曲,占40.95%;刺五加MDD蛋白无跨膜区域,定位于膜外。(2)刺五加MDD基因在不同生长发育时期和器官中均有表达,但表达量具有显著差异(P<0.05)。在整个生长期中,MDD的表达呈现高-低-高-低的变化趋势,第一个表达高峰出现在萌芽期至叶片完全展开时,第二个高峰出现在果实体积快速增长期,最高表达量(叶片完全展开期)为最低表达量(叶片衰老期)的4.51倍;不同器官中,幼茎的表达量最高,为最低表达量(叶片)的7.22倍,但叶片、叶柄和根中的表达量差异不显著。研究结果为阐明刺五加皂苷的生物合成及对其进行表达调控奠定了基础。

Cloning and Expression Analysis of Mevalonate Diphosphate Decarboxylasa Gene in Eleutherococcus senticosus

Rapid amplification of cDNA ends (RACE) method was used to clone the full length of mevalonate diphosphate decarboxylase (MDD) gene in Eleutherococcus senticosus.Then,the gene was analyzed by bioinformatic method and its expression patterns in different organs and different developmental periods were tested by RT-PCR.The results were as follows:(1)The full length of MDD cDNA sequence was 1 769 bp containing a 1 263 bp ORF that encoded 420 amino acids.The deduced amino acids sequence of MDD exhibited specific recognition sequence of GHMP kinase super family,and the accession number of MDD gene was JQ905594.In the secondary structure,the protein contains 161 alpha helixs,takes up 38.33%,68 extended strands,takes up 16.19%,19 beta turns,takes up 4.52%,172 random coils,takes up 40.95%.Without transmembrane domain,MDD was located in the outer membrane region.(2)RT-PCR results showed that MDD gene expressed in different developmental periods and different organs of E.senticosus,and the gene expression significantly differed among the periods or organs (P<0.05).The expression pattern of MDD,was likely to firstly raise in the period from bud germination to fully expanding of leaves,then decrease to the lowest content,and finally raise to another peak level when the fruits grow with a high speed.The highest expression (in fully expanding leaves) was 4.51 fold to the lowest expression (in aging leaves).The highest gene expression was detected in young shoots,which was 7.22 fold to the lowest level in leaves.However,no significant difference was detected among leaves,petioles and roots.The results had profound effect on the better understanding of the biosynthesis and regulation of E.senticosus saponins.