无核白葡萄脱水素等位基因的克隆及序列分析

以欧洲葡萄无核白品种(Vitis vinifera cv.Thompson Seedless)为研究材料,根据霞多丽品种脱水素基因序列设计引物并PCR扩增。PCR产物用地高辛标记并Southern杂交分析,发现无核白基因组含有2个不同的脱水素基因。设计并采用一种基于对琼脂糖凝胶梯状切割回收的方法克隆这2个不同脱水素基因的DNA片段,分别命名为DHN1-L和DHN1-S。进一步用反向PCR法获得这2个脱水素基因DNA片段的侧翼序列,获得基因DNA全长序列(NCBI登录号分别为EF371882和EF371881)。与已公布葡萄基因组序列比对,这2个基因位于第4号染色体相同位置,为等位基因,说明无核白品种脱水素基因为杂合体。2个脱水素基因所翻译蛋白在疏水性及三级结构方面有差异。无核白葡萄脱水素基因等位基因DNA序列的获得为进一步分析等位基因变异对植物抗逆性的影响提供了依据。

DNA sequencing of alleles of DHN1 in Vitis vinifera cv.Thompson Seedless and their bioinformatics analysis

According to the DHN1 sequence(NCBI accession number: AY706989) in Vitis vinifera cv.Chardonnay,primer pairs of VDE-F and VDE-R were designed for PCR amplification.Then the PCR amplicon was modified by digoxin for Southern blot assay.It was found that Vitis vinifera cv.Thompson Seedless bears two similar dehydrin genes.Then,DNA fragments of these two similar DHN1 genes were obtained by the ladder-like purification of agarose gel,and designated as DHN1-L and DHN1-S respectively.Flanking sequences were also obtained by the inverse PCR method(NCBI accession numbers :EF371882 and EF371881 respectively).BLAST search showed that they located at the same locus(alleles) of chromosome 4 in the grape genome.Bioinformatics analysis showed that there were some differences in hydrophobicity and tertiary structure of the two corresponding encoded proteins.These results would facilitate the ongoing differential functional relevancies of these two alleles in Thompson Seedless in response to the stresses.