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从韩国浦项科技大学(Pohang University of Science and Technology,POSTECH)水稻T-DNA插入突变体库PFG_3A-04871.R株系中筛选到一个NADK3(NAD Kinase 3)激酶(LOC_Os05g32210)基因OSNADK3表达缺失的突变杂合体。与其野生型Dongjing(Oryza sativa L.var.japonica cv Dongjing)比较,该OSNADK3基因突变杂合体表现出明显的植株矮小、花发育异常以及不育等特点。为明确水稻NADK3激酶的功能,我们利用RT-PCR的方法克隆了水稻(Dongjing)OSNADK3基因的编码区(CDS),将OSNADK3导入表达载体pCAMBIA1301::35S::GFP(pCG)中,构建了OSNADK3和绿色荧光蛋白(GFP)融合表达载体pCAMBIA1301::35S::NADK3::GFP(pCNG),该融合表达载体能够在拟南芥叶中表达,亚细胞组织定位分析表明,OSNADK3在拟南芥细胞质中表达,表明该酶是细胞质定位的酶类。然后,利用农杆菌介导的方法侵染水稻愈伤组织,获得了24株独立的转基因水稻植株。经PCR初步鉴定,目的片段OSNADK3已经成功整合到水稻基因组中,为进一步深入研究水稻OSNADK3的功能与作用机制奠定了基础。
A NADK3 (LOC_Os05g32210) gene was screened for deletion of OSNADK3 gene from rice T-DNA insertion mutant PFG_3A-04871.R strain of Pohang University of Science and Technology (POSTECH) Mutant heterozygotes. Compared with its wild-type Dongjing (Oryza sativa L.var.japonica cv Dongjing), this heterozygous mutant of OSNADK3 showed obvious plant dwarfism, abnormal flower development and sterility. To clarify the function of rice NADK3 kinase, we cloned the coding region of OSNADK3 gene in rice (Dongjing) by RT-PCR and introduced OSNADK3 into the expression vector pCAMBIA1301 :: 35S :: GFP (pCG) to construct OSNADK3 And green fluorescent protein (GFP) fusion expression vector pCAMBIA1301 :: 35S :: NADK3 :: GFP (pCNG), the fusion expression vector can be expressed in Arabidopsis leaves, subcellular localization analysis showed that OSNADK3 in Arabidopsis cytoplasm , Indicating that the enzyme is a cytoplasmic enzyme. Then, using Agrobacterium-mediated method to infect rice callus, 24 independent transgenic rice plants were obtained. Preliminary identification by PCR, the target fragment OSNADK3 has been successfully integrated into the rice genome, which laid the foundation for further study of the function and mechanism of OSNADK3 in rice.