Clonging of BrcCUC3 from Brassica rapa ssp. chinensis and Its Effect on Leaf Morphology and Inflorescence Structure in Arabidopsis thaliana | 白菜NAC转录因子BrcCUC3基因克隆及其对叶缘和主枝发育的影响

Chinese. 植物NAC(矮牵牛NAM基因、拟南芥ATAF1/2和CUC2基因)转录因子CUP-SHAPED COTYLEDON(CUC)亚家族成员在植物茎尖分生组织形态建成、器官边界分离、叶发育方面发挥着重要作用。采用基因同源克隆的方法获得了白菜(Brassica rapa ssp. chinensis)基因BrcCUC3，并转化拟南芥(Arabidopsis thaliana)做了初步的功能鉴定。研究结果表明，白菜BrcCUC3的编码区长1 008 bp，编码335个氨基酸，基因结构分析显示BrcCUC3包含3个外显子、2个内含子，内含子剪接位点符合GT-AG规则。氨基酸序列分析表明，BrcCUC3蛋白具有典型的植物NAC domain结构域。 BrcCUC3编码区氨基酸序列与其它植物的CUC3蛋白有很高的一致性，尤其与甘蓝(Brasica oleracea)、萝卜(Raphanus sativus)和拟南芥CUC3蛋白高度一致，一致性分别达到98%，97%和83%。在不同物种CUC3的系统进化树上，BrcCUC3归属于双子叶植物分支的十字花科亚组，由不同植物20条CUC3编码区氨基酸序列所建立的系统进化树与真实的植物进化基本一致。荧光定量PCR分析结果表明，BrcCUC3在白菜叶深裂株系叶片中的表达量比叶全缘叶片中的高。利用根癌农杆菌(Agrobacterium tumefaciens)浸花法转化拟南芥，获得了转BrcCUC3基因的拟南芥植株。过表达BrcCUC3的转基因拟南芥呈现叶缘出现裂刻、主枝增加的新表型。初步说明该基因参与叶形和主枝的发育调控，为揭示白菜叶形发育分子调控机制和通过基因工程创制植物叶形新种质提供分子依据。

English. CUP-SHAPED COTYLEDON(CUC), members of the NAC family of transcription factors (such as NAM in Petunia hybrida, ATAF1/2 and CUC2 in Arabidopsis thaliana) play an important role in shoot apical meristem (SAM) construction，organ separation and leaf development. BrcCUC3 was cloned from the non-heading Chinese cabbag(Brassica rapa ssp. chinensis) by the method of homologous sequence amplification and transformed into Arabidopsis thaliana for functional identification. The coding sequence of BrcCUC3 was 1 008 bp in length and predicted to encode a typical NAC-domain transcription factor with the protein of 335 amino acids. The corresponding genomic DNA contained three exons and two introns. It was in conformity with the principles of GT-AG. The amino acid sequence analysis showed that it contained a conserved NAC-domain. Phylogenetic analysis revealed that amino acid sequences of BrcCUC3 were high similar to previously described CUC3 proteins from other plant species, especially shared 98%, 97% and 83% identity with cabbage(B. oleracea), radish(Raphanus sativus) and A. thaliana proteins, respectively. In the CUC3 phylogenetic tree of multiple species, BrcCUC3 protein belongs to the Brassicaceae sub-branche of dicot branche. According to the 20 CUC3 amino acid sequences of plants, the phylogenetic tree showed that it indeed reflected the evolution of plants. Real-time PCR was performed to observe the expression profiling of BrcCUC3, the results showed that BrcCUC3 was higher expressed in the different stage leaves of lines with lobed leaf than that in lines with entire leaf. The transformation of BrcCUC3 gene into A. thaliana was carried out by floral dipping with the Agrobacterium tumefaciens. Compared with wild-type plants, overexpression of BrcCUC3 cDNA under the control of a CaMV 35S promoter in A. thaliana caused serrated leaves and modified inflorescence structure with increased apical inflorescence. All the results suggested that BrcCUC3 involves in the regulation of leaf margin and braches development, which can provide clues for studying the molecular mechanism of leaf development in Chinese cabbage and creating new germplasm by gene engineering.