大白菜BrTCP24基因的克隆与功能分析  

Cloning and Functional Analysis of BrTCP24 Gene in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

作　　者：王凤德[1] 谭婷婷[1] 张一卉[1] 李景娟[1] 李化银[1] 李利斌[1] 刘立锋[1] 高建伟[1] 

机构地区：[1]山东省农业科学院蔬菜研究所,山东省设施蔬菜生物学重点实验室/国家蔬菜改良中心山东分中心,济南250100

出　　处：《农业生物技术学报》2013年第8期911-919,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31101553);国家高技术研究发展计划(863)项目(No.2012AA100103009);山东省优秀中青年科学家科研奖励基金项目(No.BS2010SW027);山东省良种工程项目(No.2011lzgcshucaizy;No.2012lzgcshucaizy)

摘　　要：近年来,由于家庭结构的变化,市场对小株型大白菜的需求日益迫切。开展负调控叶球大小发育相关基因的克隆与功能研究有助于加快小株型大白菜品种的选育进程。本研究利用RT-PCR方法从大白菜(Brassica rapa L.ssp.pekinensis)自交系福山包头叶片中分离了一个TCP第二亚族成员基因,命名为BrTCP24。BrTCP24基因编码区内无内含子,开放阅读框(ORF)全长1221bp,预测编码406个氨基酸。利用MEGA4.0软件将BrTCP24与拟南芥(Arabidopsis thaliana)TCP家族基因进行进化分析发现,BrTCP24与AtTCP3同属于一个分支,在进化上具有较近的亲缘关系。用DNAMAN软件对BrTCP24和AtTCP3蛋白进行多序列联配分析发现,二者的氨基酸一致性可达到55.17%,在保守的TCP结构域其一致性可高达91.53%。这种进化上的亲缘关系和序列上的保守性,暗示二者可能具有类似的生物学功能。通过半定量RT-PCR分析发现,BrTCP24基因在大白菜的根、茎、莲座叶、包叶、盛开的花、受精后10d的果夹和花蕾中均有表达,其中莲座叶中表达量最高,根、包叶、盛开的花、受精后10d的果夹和花蕾中的表达量次之,短缩茎中表达量最低;另外,在5μmolNAA处理的12h内BrTCP24的表达水平未发生明显变化。为进一步研究BrTCP24基因的功能,我们构建了转化拟南芥的正义表达载体(35S::BrTCP24),并转入拟南芥中。通过卡那霉素筛选以及基因组DNAPCR鉴定,共得到17株转基因拟南芥植株。通过对其中5株的RT-PCR分析发现,它们都能够转录表达BrTCP24基因。利用荧光实时定量PCR方法对部分转基因植株的插入拷贝数进行检测发现,BrTCP24基因以单拷贝形式插入拟南芥基因组。进一步研究发现,与野生型拟南芥相比,过量表达BrTCP24基因可导致转基因拟南芥的下胚轴和叶器官明显减小。此外,过量表达BrTCP24基因抑制了与器官大小相关基因ANT、AtEXP10、AtGRF5、AtGIF1和CycD3;1的表达。这些�It is very important to isolate and characterize the genes responsible for negative control of the leaf heading growth of Chinese cabbage, which can be help to speed up breeding progress of the small heading Chinese cabbage varieties that meet the current market demands. Here, a full-length TCP cDNA named BrTCP24, which belongs to the second subgroups of TCP domain family, was isolated from leaves of Chinese cabbage inbred line Fushanbaotou （Brassica rapa L. ssp. pekinensis）. The full-length cDNA of BrTCP24 consisted of 1 221 nucleotides, and was predicted to code a 406-amino acid polypeptide. In addition, there were no introns in BrTCP24 gene. The phylogenetic analysis about BrTCP24 and TCP families in Arabidopsis was carried out using the software of MEGA4.0. The result indicated that BrTCP24 gene and Arabidopsis AtTCP3 gene would belong to the same branch, which suggested they had closely genetic relationship. The alignment of predicted amino acid sequences of BrTCP24 and Arabidopsis AtTCP3 indicated that there was 55.15% identity between them. Additionally, these two proteins contained conserved TCP domain and had 91.53% identity in this domain. These results suggested that BrTCP24 and Arabidopsis AtTCP3 would have similar biological functions. The semiquantitive RT-PCR indicated that BrTCP24 gene was expressed in roots, dwarf stems, rosette leaves, folding leaves, flowers, siliques and bud flowers examined in Chinese cabbage. Among them, rosette leaves had the highest mRNA level, followed by roots, folding leaves, flowers, siliques and bud flowers, while dwarf stems had the 1owest mRNA 1eve1. Interestingly, the expression level of BrTCP24 didn’t effect by 5 μmol NAA treatment in 12 h period. To test the function of BrTCP24, we then engineered Arabidopsis plants that would over-express BrTCP24 ectopically, driven by CaMV 35S promoter, and obtained 17 transgenic lines by Kanamycin and PCR screening. Using RTPCR method, we randomly detected 5 transgenic lines and found all of them could express the BrTCP

关 键 词：大白菜 BrTCP24基因 器官大小 

分 类 号：S634.1[农业科学—蔬菜学]