菊花CmAP1基因克隆及其植物表达载体构建

摘要

[目的]克隆菊花CmAPETALA1基因(CmAP1)并构建植物表达载体,为该基因功能的遗传改良打下研究基础.[方法]采用同源克隆及RT-PCR从菊花叶片中克隆CmAP1基因,运用在线生物信息学分析工具对其核苷酸及氨基酸序列进行分析,利用实时荧光定量PCR对该基因的组织表达差异进行分析,并用双酶切法将目的基因与pCAMBIA1300载体连接,构建植物表达载体.[结果]克隆获得的CmAP1基因(GenBank登录号KU872999)编码区开放阅读框(ORF)长741 bp,编码246个氨基酸;CmAP1蛋白属亲水性蛋白,分子质量约28.3 kD、理论等电点(pI)为8.76,预测该蛋白内含10个磷酸化位点和2个潜在的N-糖基化位点;蛋白氨基酸序列比对和系统发育进化分析结果表明,CmAP1蛋白与CDM111蛋白的同源性达98%,属MADS-box基因家族A类基因的euAP1分支;实时荧光定量PCR分析结果表明,CmAP1基因在花蕾中表达量极高,在舌状花和筒状花中表达量较低,而在营养器官中仅痕量表达.将CmAP1基因连接到pCAMBIA1300载体上,可成功构建植物表达载体pCAMBIA1300-CmAP1.[结论]CmAP1基因在花的发育及形成过程中发挥重要作用,成功构建获得的植物表达载体pCAMBIA1300-CmAP1可为后期利用基因工程手段改变菊花花形态结构、调控花期及遗传改良打下基础.%[Objective]CmAPETALA1 gene(CmAP1) in Chrysanthemum morifolium was cloned and its plant expres-sion vector was established, in order to provide references for further study on genetic improvement of CmAP1 gene func-tion. [Method]CmAP1 gene was cloned from leaf of C. morifolium by method of homology cloning and RT-PCR. The nu-cleotide and amino acid sequence were analyzed by online bioinformatics tool. Expression difference of this gene was deter-mined by real-time fluorescence quantitative PCR. CmAP1 gene was linked to pCAMBIA1300 vector by double enzyme di-gestion to establish plant expression vector. [Result]CmAP1 gene(GenBank accession number:KU872999) was obtained by cloning. The open reading frame(ORF) of CmAP1 was 741 bp encoding 246 amino acids. CmAP1 protein was a hy-drophilic protein,and the molecular weight of this protein was about 28.3 kD. Its theoretical isoelectric point (pI) was 8.76. It was predicted that the protein contained ten phosphorylation sites and two potential N-glycosayation sites. Sequence and phylogenic analysis showed that CmAP1 protein was close to C. morifolium CDM111 protein with 98% similarity, which both belonged to euAP1 clade of A type gene in MADS-box gene family. Real fluorescence quantitative PCR analysis indicated that expression of CmAP1 gene was extremely high in buds. The expression level was low in tubiform and lingu-late florets and was in trace expression in vegetative organs. CmAP1 was linked to pCAMBIA1300 vector and the plant expression vector pCAMBIA1300-CmAP1 was established successfully. [Conclusion]CmAP1 gene plays an important role in growth and development of C. morifolium. The established plant expression vector pCAMBIA1300-CmAP1 is use-ful for further research on changing morphological structure of C. Morifolium by genetic engineering, regulation of flowering and genetic modification.