‘辽宁2号’DELLA蛋白编码基因的克隆及表达分析

Cloning and expression analysis of JrGAI gene in DELLA protein from 'Liaoning 2'

以‘辽宁2号’核桃的叶片为试材,通过同源克隆和RACE技术克隆DELLA家族蛋白的编码基因,并进行了同源性分析和氨基酸序列的多重比对;通过实时荧光定量PCR分析了该基因在‘辽宁2号’核桃中的组织特异性表达情况。主要研究结果如下:获得了DELLA蛋白编码基因的cDNA全长,命名为JrGAI(Genebank:JF766606),cDNA全长为2 361bp,包含1 842bp编码区序列,推测其编码的蛋白包含613个氨基酸,分子量为66.78kDa。同源性分析及氨基酸序列的多重比对结果表明该蛋白与其他物种的DELLA蛋白高度相似,并且具有完整的DELLA、TVHYN、VHIID、RVER和SAW等结构域;实时荧光定量PCR结果显示JrGAI基因在‘辽宁2号’核桃的叶芽、混合花芽、叶片、雄花、茎段和果实中普遍表达,并且在休眠期组织中具有较高的表达量,其中以休眠期的混合花芽中的表达量最高。

The leaves of the dwarf walnut cultivar ‘Liaoning 2＇ were used for cloning the gene encoding DELLA protein by in this research homologous cloning and RACE techniques. Homology analysis and multiple sequences alignment analysis of the amino acid sequences were carried out with bioinformatics technology. The expression pattern of JrGAI gene in different tissues of dwarf walnuts was analyzed with real-time quantitative PCR detection. The main results are as follows： The full-length cDNA was isolated from the ‘Liaoning 2’ cultivar and was named as JrGAI （Genebank.JF766606）. It was 2 361 bp long including 1 842 bp of ORF. The protein it encoded is deduced as 66.78 kDa and contains 613 amino acid residues. Accoriding to the results of homology analysis and multiple alignment analysis of the amino acid sequences, the protein that the gene encoded is highly similar to the DELLA protein in other species,having complete domains like DELLA, TVHYN, VHIID, RVER and SAW. The real-time quantitative PCR analysis showed the JrGAI gene was always expressed in leaves, leaf buds,shoot tips,internodes, mixed floral buds, male flowers and fruits of ‘Liaoning 2＇cultivars. Mean-while,JrGAI gene was highly expressed in tissues of dormacy period,and it had the highest expression in mixed floral buds in dormancy.