二穗短柄草细胞分裂素氧化酶BdCKX编码基因的克隆及功能分析

细胞分裂素是一类重要的植物激素,通过控制细胞分裂、诱导促进芽萌发生长等方面控制植物发育。细胞分裂素的代谢主要通过细胞分裂素氧化酶来进行,在作物性状改良和抗旱品种培育方面具有重要的研究价值。为研究麦类作物细胞分裂素氧化酶功能,本实验采用同源克隆的方法,在二穗短柄草中获得了一个与拟南芥细胞分裂素氧化酶基因At CKX3高度同源的候选基因,命名为Bd CKX。将该基因与p CXSN质粒连接构建植物超表达载体,并以农杆菌介导法转化二穗短柄草植物幼胚。所获得的阳性转基因植株生长迟缓,茎短,叶片小,地上部分变小。另外,组织表达谱分析表明Bd CKX在二穗短柄草生长发育的不同时期和不同组织中具有不同的表达模式,在生长40天的植株叶片中表达量有明显升高。由于细胞分裂素氧化酶编码基因在早熟禾亚科物种中较为保守,本研究为理解麦类作物的发育调控机制和遗传改良麦类作物生长提供必要的理论基础。

Cloning and functional analysis of a BdCKX gene in Brachypodium distachyon

As one of the most important plant hormones, cytokinin plays diverse roles during plant development including regulation of cell division, promotion of shoot growth etc. The metabolism of cytokinin is primarily dependent on functions of cytokinin oxidase encoding genes. To gain insightful understanding and take full advantage of cytokinin oxidase in the genetic improvement of crop growth traits, a homologous gene to the AtCKX3 (Arabidopsis Cytokinin Oxidase 3) gene in Brachypodium distachyon, BdCKX, was cloned. To elucidate its bioloical function, CDS sequence of the BdCKX gene was amplified and ligated with the pCXSN vector, which was then introduced to callus tissue of young embryo by Agrobacterium-mediated transformation for over-expression analysis. Positive transformants exhibited phenotypes including short stem, shrunk leaf, retarded development, and small overall shoot. It could be concluded that the elevated expression of BdCKX gene was able to interfere plant growth through the degradation of endogenous cytokinin levels. In addition, gene expression profile indicated that BdCKX was differentially expressed in various tissues at different developmental stages. In particular, expression level of BdCKX was markedly shifted in leaf of 40 days old seedling. Because cytokinin encoding genes were conserved among Pooideae species, our research may provide a theoretical base for the regulatory mechanism and genetic improvement of Triticeae crops.