水稻OSNADK3基因的克隆及其遗传转化

CLONING AND GENETIC TRANSFORMATION OF OSNADK3 GENE IN RICE

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 heterozygote of OSNADK3gene knockout mutant of rice was identified from the T-DNA insertion mutant line PFG_3A-04871.R,which was ordered from Pohang University of Science and Technology（POSTECH）.The heterozygous mutant with OSNADK3 gene knockout has distinct phenotypes from wild type Dongjing（Oryza sativa L.var.japonica cv Dongjing）,such as small plants,abnormal flowering development,sterile seeds,and so on.To get further insight on the function of OSNADK3（NAD Kinase 3）,the CDS fragment of OSNADK3gene was cloned from rice（cv Dongjing） by means of RT-PCR.Fusion expressing vector pCAMBIA1301：：35S：： NADK3：：GFP（pCNG） was constructed with the gene of OSNADK3 fusing in fragment with the pCAMBIA1301：：35S：：GFP（pCG）.A transient expression test showed that the vector pCNG could be expressed in Arabidopsis thalianaand OSNADK3 is a cytoplasmic enzyme according to the subcellular localization.At the same time,pCNGwas introduced into rice callus with agrobacterium-mediated method and 24 transgenic plants were obtained.The results provided a good prefoundation for further studies to understand the function and mechanism of OSNADK3 in stress response and development of rice.