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以‘宝石无核’(Ruby Seedless)葡萄试管苗为材料,采用RT-PCR技术,克隆得到8个葡萄Sn RK2家族基因。序列分析发现,该家族基因蛋白结构在N端相对保守,而在C端极其特异;聚类结果表明葡萄Sn RK2基因家族可以分为3个亚家族;对这8个基因所编码的蛋白质进行分析发现,其富含酸性氨基酸,且均为亲水蛋白;基因组结构分析发现Vv Sn RK2.2和Vv Sn RK2.8含有10个外显子,其他6个均含9个外显子;对蛋白二级结构分析发现8个基因编码的蛋白主要以α–螺旋、β–转角和不规则卷曲为主;亚细胞定位预测,8个基因主要定位于细胞质中。顺式作用元件分析表明,除Vv Sn RK2.1、Vv Sn RK2.2、Vv Sn RK2.6外,其他基因顺式作用元件包含ABRE、DRE/CRT、LRTE中的一个或多个。定量PCR分析表明,Vv Sn RK2的表达存在组织差异性,Vv Sn RK2.7在根中表达水平最高,是叶片的3.8倍,Vv Sn RK2.8在茎中表达水平最高,是叶片的5.0倍。0~–4℃处理后,表达水平下调幅度最小的为Vv Sn RK2.2,Vv Sn RK2.7下调幅度较大,Vv Sn RK2.8的表达水平为0;30℃处理后Vv Sn RK2.2和Vv Sn RK2.5上调表达,分别为对照的3.8倍和3.6倍;Vv Sn RK2.1和Vv Sn RK2.2与盐胁迫调节紧密相关,Vv Sn RK2.5与干旱胁迫调节密切相关。
Using the grape seedlings of “Ruby Seedless” grape as materials, 8 grape Sn RK2 family genes were cloned by RT-PCR. Sequence analysis revealed that the protein structure of this family gene was relatively conserved at the N-terminus and very specific at the C-terminus. The clustering results indicated that the Sn RK2 gene family could be divided into three subfamilies. The proteins encoded by these eight genes were analyzed It was found that it was rich in acidic amino acids and both were hydrophilic proteins. The genome structure analysis showed that Vv Sn RK2.2 and Vv Sn RK2.8 contained 10 exons and the other 6 contained 9 exons. Secondary structure analysis found that the eight genes encoded mainly α-helix, β-turn and irregular curly predominant; subcellular localization predicted that eight genes located in the cytoplasm. Cis-acting element analysis showed that cis-acting elements of other genes included one or more of ABRE, DRE / CRT, and LRTE, except for VvSnRK2.1, VvSnRK2.2, and VvSnRK2.6. Quantitative PCR analysis showed that the expression of Vv Sn RK2 was differentially expressed. The highest expression level of Vv Sn RK2.7 in roots was 3.8 times that of leaves, and the highest expression level of Vv Sn RK2.8 in stems was 5.0 times that of leaves . After treated with 0 ~ -4 ℃, the expression of Vv Sn RK2.2 was the smallest, and the amplitude of Vv Sn RK2.7 was greatly decreased. The expression level of Vv Sn RK2.8 was 0; Vv Sn RK2 was treated at 30 ℃. 2 and Vv Sn RK2.5 were up-regulated by 3.8 and 3.6 fold respectively. Vv Sn RK2.1 and Vv Sn RK2.2 were closely related to salt stress regulation. Vv Sn RK2.5 was closely related to drought stress regulation.