甜菜NADH-NR gDNA全长序列的克隆及酶切鉴定

Cloning of NADH-NR gDNA Full-length Sequence and Enzyme Digestion

为了研究甜菜NADH-NR gDNA的序列特征,从分子水平上为甜菜硝酸盐累积差异机理的研究做铺垫;利用二倍体甜菜品种‘甜研7号’,采用分步克隆方法,分别获得甜菜NADH-NR基因组DNA的3’端序列和5’端序列以及中间序列,再以3’端和5’端序列设计特异引物,克隆获得甜菜NADH-NR基因组DNA的全序列;结果表明,甜菜NADH-NR gDNA全长序列6346bp,经与先前获得的甜菜NADH-NR cDNA序列比对,甜菜NADH-NR gDNA含有3个内含子,4个外显子。内含子大小分别为197、1088、2343bp。甜菜NADH-NR gDNA的外显子序列与菠菜的同源性达到78%,但内含子序列在进化期间发生了较大分化,甜菜NADH-NR gDNA的内含子相对菠菜要大,两者也不处于同样的位点,甜菜NADH-NR gDNA的内含子靠近5'端较早出现,每个内含子大小均是菠菜的1.10～1.71倍。本研究首次从甜菜中克隆获得NADH-NR gDNA全长序列,揭示了其序列特征并将其与其他作物进行了比较分析,为进一步利用该基因开展甜菜硝酸盐含量的基因调控研究奠定了基础。

In order to study the sequence characteristics of sugar beet NADH-NR gDNA,and to pave the way for mechanism research of sugar beet nitrate accumulation differences on molecular levels,in the study,diploid sugar beet variety Ty7 was used,and by stepwise cloning method,3' end region,5' end region and middle sequence of sugar beet NADH-NR genomic DNA were respectively obtained;then specific primer was designed according to 3' end and 5' end region;the DNA complete genomic sequence of sugar beet NADH-NR was cloned.The results showed that the full-length sequence of NADH-NR gDNA was 6346 bp long.The results of sequence comparisons with sugar beet NADH-NR cDNA cloned previously showed that sugar beet NADH-NR gDNA has 3 introns,4 exons.The sizes of the introns were 197 bp,1088 bp and 2343 bp.Sugar beet NADH-NR gDNA exon sequence is 78% homologous to spinach,but intron sequence differentiated greatly during the evolution.The intron of sugar beet NADH-NR gDNA is bigger than that of spinach,and they were not at the same site;sugar beet NADH-NR gDNA introns appeared earlier near 5' end,and the size of each intron is 1.10-1.71 times of that of spinach.For the first time,the full-length sequence of NADH-NR gDNA was cloned from sugar beet in the study,and its sequence characteristics was revealed,and then made the comparison with those of other crops,thus laid the foundation for further study on gene regulation of sugar beet nitrate content on the base of it.