红花1-脱氧-D-木酮糖-5-磷酸合成酶基因CtDXS1的克隆及表达分析

Cloning and Expression Analysis of CtDXS1 Encoding 1-Deoxy-D-Xylulose-5-Phosphate Synthase Gene in Carthamus tinctorius L.

1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose-5-phosphate synthase,DXS)是2-C-甲基-D-赤藓糖醇-4-磷酸(2-C-methyl-D-erythritol-4-phosphate,MEP)途径中的第一个关键酶,在植物萜类化合物生物合成中起着重要作用。为了明确红花DXS基因的序列特征和表达特点,结合红花转录组数据,以豫红花1号为试验材料,首次克隆得到1个红花DXS基因的全长cDNA序列,命名为CtDXS1,并对其进行生物信息学分析和基因表达特点分析。结果表明,红花CtDXS1基因的开放阅读框(ORF)长2136 bp,编码711个氨基酸,其蛋白质分子质量是76503.10 u,蛋白质保守区预测表明,CtDXS1具有典型的转酮醇酶家族功能结构域。系统进化分析显示,CtDXS1与来自黄花蒿的DXS亲缘关系最近,属于DXS基因家族的Ⅰ类基因。组织特异性表达分析表明,CtDXS1基因在苞片中表达量最高,其次是叶和茎,在其他组织部位中表达量较低;不同花色红花基因表达定量分析表明,CtDXS1基因在黄色红花中表达量较高;干旱、低温胁迫能够诱导CtDXS1基因表达上调;茉莉酸甲酯(MeJA)能诱导CtDXS1基因的表达,而赤霉素(GA3)和脱落酸(ABA)对CtDXS1基因表达有一定的抑制作用。构建原核表达载体pET28-CtDXS1并转化至大肠杆菌Transetta(DE3)进行原核表达,成功表达了CtDXS1重组蛋白。

1-deoxy-D-xylulose-5-phosphate synthase(DXS)is the first key enzyme of 2-C-Methyl-D-erythritol-4-phosphate(MEP)synthetic pathway,which plays an important role in plant terpenoid biosynthesis.In order to clarify the sequence characteristics and expression characteristics of DXS in Carthamus tinctorius L.,a full length cDNA sequence of CtDXS1 gene was cloned by reverse transcription PCR from Carthamus tinctorius L.cultivar‘Yuhonghua No.1’based on the transcriptome data.Meanwhile,bioinformatics analysis and gene expression characteristics analysis were carried out.The open reading frame(ORF)of CtDXS1 gene was 2136 bp,encoding a protein of 711 amino acids with a calculated molecular mass(MW)of 76503.10 u.Protein conserved domain analysis indicated that CtDXS1 contained the conserved domain of transketolase.The phylogenetic analysis indicated that CtDXS1 protein had the highest level of homology with DXS protein from Artemisia annua,and belonged to DXS cladeⅠ.Tissue-specific expression analysis showed that CtDXS1 gene had the highest expression level in bracts,followed by leaves and stems,and lower expression in other tissues.Quantitative analysis of gene expression of different color safflower showed that the expression of CtDXS1 gene was higher in yellow safflower.Drought and low temperature stress induced the CtDXS1 gene expression.The expression of CtDXS1 also induced by MeJA,but GA3 and ABA could inhibite the transcription of CtDXS1.Prokaryotic expression vector pET28a-CtDXS1 was constructed,and transformed into E.coli Transetta(DE3),and CtDXS1 recombinant protein was successfully expressed.