薄壳山核桃MADS-box基因CiMADS9的克隆与功能分析

以薄壳山核桃（Carya illinoinensis）‘马罕’雄花为材料，利用获得的MADS-box基因保守片段设计特异引物，通过RACE技术克隆MADS-box家族基因的cDNA序列，命名为CiMADS9。该基因长为1 077 bp，开放阅读框（ORF）768 bp，编码255个氨基酸残基。生物信息学分析表明该基因具有典型的MADS-box结构域和半保守的K区，是MIKC型MADS-box基因。聚类分析分析表明该基因属于AGL15亚家族。实时荧光定量PCR结果表明，CiMADS9在生殖器官（雄花、雌花、幼果）中的表达量高于营养器官（叶、枝条）中的，并且在雄花中的表达量最大。将目的基因通过农杆菌介导法转化拟南芥，获得了转基因植株。与对照相比，转基因拟南芥中过量表达该基因使植株开花延期，基生叶增加。

Cloning and Functional Analysis of MADS-box CiMADS9 Gene from Carya illinoinensis

A MADS-box gene，named CiMADS9，was isolated from the male flowers of Carya illinoinensis using the gene specific primers obtained according to a MADS conserved fragment sequence by the method of RACE technology. The gene was 1 077 bp which has an ORF of 768 bp coding 255 amino acids．Multiple sequence comparison revealed that CiMADS9 was the typical MIKC-type MADS-box genes with the MADS-box domain and K semi-conservative region. Phylogenetic analysis indicated that CiMADS9 belonged to the AGL15 group of MADS-box gene family. qRT-PCR indicated that the expression level of reproductive organs（male flower，female flower，and young fruit）were striking higher than those of vegetative organs（leaf and branch），and the expression level of male flower was the highest among all the organs. The overexpression vector of CiMADS9 was constructed and transferred into Arabidopsis thaliana，and then resistant plants were obtained. RT-PCR analysis showed that CiMADS9were expressed in all these lines. Compared with the wide type，transgenic Arabidopsis thaliana overexpressing CiMADS9 gene delayed flowering time and increased the number of leaf.