滇水金凤4CL基因的克隆及表达分析

Cloning and Expression of 4CLs in Impatiens uliginosa

【目的】4-香豆酰-CoA连接酶(4CL)作为苯丙烷类代谢途径的关键酶之一,对花青素合成起着重要作用。探究滇水金凤4CL基因(命名为Iu4CL)对滇水金凤花色调控的分子机理,为其花色调控及花色育种提供参考依据。【方法】以滇水金凤为材料,采用RT-PCR技术分离克隆Iu4CL1、Iu4CL2、Iu4CL3和Iu4CL4基因,并对其生物信息学进行分析;通过qRT-PCR技术对4CL基因在4种不同花色(白色、粉色、红色和深红色)及其4个不同花发育时期(花苞期S1、始花期S2、盛花期S3和谢花期S4)中的表达情况进行分析。【结果】Iu4CL1、Iu4CL2、Iu4CL3和Iu4CL4的cDNA全长分别为1620、1653、1698、1638 bp,分别编码539、550、565、545个氨基酸;其中Iu4CL1和Iu4CL2分别含有2个和4个内含子,Iu4CL3和Iu4CL4没有内含子。生物信息学分析表明,Iu4CL1、Iu4CL2和Iu4CL4为稳定蛋白,Iu4CL3为不稳定蛋白;4个基因均为无信号肽疏水性蛋白;Iu4CL2有3个跨膜结构,其余3个基因均不存在跨膜结构;Iu4CL基因4个拷贝均属于AMP结合酶超级家族和腺苷酸形成域I类超级家族。同源性分析表明,Iu4CL基因的4个拷贝均与喜马拉雅凤仙花的同源性最高;且Iu4CL1和Iu4CL2处于同一个大的分支中,而Iu4CL3和Iu4CL4处于另一个大的分支中,推测可能为旁系同源。qRT-PCR分析表明,Iu4CL基因的4个拷贝在4种不同花色和4个不同花发育时期的滇水金凤花器官中均有表达,其中Iu4CL1和Iu4CL3基因在白色花器官S3(盛花期)阶段表达量最高;Iu4CL2基因在红色花器官S3(盛花期)阶段表达量达到顶峰;Iu4CL4基因在深红色花器官S3(盛花期)阶段表达量最高。【结论】Iu4CL基因可能在滇水金凤花青素生物合成中发挥重要作用。

【Objective】Genes of the enzyme in the phenylpropane metabolic pathway associated with anthocyanin synthesis of Impatiens uliginosa were studied for breeding purposes.【Method】RT-PCR was employed to isolate and clone Iu4CL1,Iu4CL2,Iu4CL3,and Iu4CL4 for bioinformatic analysis.Expressions of the genes in the flowers of different colors(i.e.,white,pink,red,and deep red)at the budding(S1),initial flowering(S2),blooming(S3),and withering(S4)stages of I.uliginosa were determined using qRT-PCR.【Result】Iu4CL1,Iu4CL2,Iu4CL3,and Iu4CL4 had the cDNA lengths of 1620 bp,1653 bp,1698 bp,and 1638 bp encoding 539,550,565,and 545 amino acids,respectively.Iu4CL1 contained 2 introns and Iu4CL2,4,but Iu4CL3 and Iu4CL4 had none.All 4 genes were signal peptide-free hydrophobic proteins,but only Iu4CL3 was unsterilized.Iu4CL2 had 3 transmembrane structures,but the other 3 had none.All of them belonged to the AMP-binding enzyme superfamily and the adenylate-forming domain class I superfamily with the highest homology with I.uliginosa.Iu4CL1 and Iu4CL2 were in one large branch,while Iu4CL3 and Iu4CL4 in another,suggesting a paraphyletic homology.The expressions of Iu4CL1 and Iu4CL3 peaked in S3 on the white flowers,Iu4CL2 on red flowers,and Iu4CL4 on dark red I.uliginosa.【Conclusion】It was evident that Iu4CLs played a crucial role in the anthocyanin biosynthesis of I.uliginosa.