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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。这些结果表明,大白菜BrTCP24基因可能通过抑制细胞的生长参与调节植物器官大小的发育。
In recent years, due to changes in family structure, the market demand for small plant cabbage is increasingly urgent. Development of negative regulation of leaf size and development of genes related to cloning and function research help to speed up the small plant type cabbage breeding process. In this study, we isolated a member of TCP second subfamily named BrTCP24 from the leaf of Brassica rapa L.ssp.pekinensis inbred line Fukuyama by RT-PCR. No introns were found in the coding region of BrTCP24 gene. The open reading frame (ORF) was 1221 bp in length and predicted to encode 406 amino acids. The evolution analysis of BrTCP24 and Arabidopsis thaliana TCP family genes using MEGA4.0 software found that both BrTCP24 and AtTCP3 belong to one branch and have a relatively close phylogenetic relationship. Multiple sequence alignment analysis of BrTCP24 and AtTCP3 using DNAMAN software showed that the amino acid identities of the two proteins were 55.17% and 91.53% respectively in the conserved TCP domain. This evolutionary genetic relationship and sequence conservation suggest that both may have similar biological functions. Semi-quantitative RT-PCR analysis showed that BrTCP24 gene was expressed in roots, stems, rosette leaves, covered leaves, blooming flowers, fruit clip and flower buds 10 days after fertilization, of which the highest expression was in rosette leaves, The expression level of BrTCP24 was the lowest in 10 days after fertilization, and the expression level of BrTCP24 in the shortened stem was the lowest. In addition, the expression level of BrTCP24 did not change significantly within 12h after 5μmol NAA treatment. To further investigate the function of BrTCP24 gene, we constructed a sense expression vector (35S :: BrTCP24) transformed into Arabidopsis thaliana and transferred it into Arabidopsis thaliana. Seventeen transgenic Arabidopsis plants were obtained by kanamycin screening and genomic DNA PCR. Five of them were analyzed by RT-PCR and found that all of them can transcribe BrTCP24 gene. Fluorescence quantitative real-time PCR was used to detect the inserted copy number of some transgenic plants. The results showed that the BrTCP24 gene was inserted into Arabidopsis thaliana genome as a single copy. Further studies showed that overexpression of BrTCP24 gene resulted in a significant reduction of hypocotyls and leaf organs in transgenic Arabidopsis thaliana compared with wild type Arabidopsis thaliana. In addition, overexpression of BrTCP24 gene suppressed the expression of organ size-related genes ANT, AtEXP10, AtGRF5, AtGIF1 and CycD3; 1. These results indicate that BrTCP24 gene in Chinese cabbage may be involved in the regulation of plant organ size by inhibiting cell growth.