巨峰葡萄Rs基因的克隆鉴定、序列分析与时空表达特征研究
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摘要
探究白藜芦醇合酶(Resveratrol synthase,Rs)在巨峰葡萄果实发育中的潜在作用。对其序列进行结构及功能分析,并鉴定其在果实不同发育时期的时空表达特异性。采用生物信息学分析及qRT-PCR的方法,对巨峰葡萄Rs基因序列进行分析,并对其结构和亚细胞定位进行预测,以及分析果实不同时期不同组织的表达情况。克隆得到的Rs基因cDNA全长1 539 bp,开放阅读框(ORF)为1 179 bp,编码392个氨基酸,预测蛋白分子质量42.88 ku,理论等电点(pI)为6.09,且该基因含有查耳酮和二苯乙烯合酶活性位点以及完整的芪合酶家族特征位点;蛋白互作预测发现,Rs和OMT2.1具有互作,后者可催化白藜芦醇生物合成紫檀芪;亚细胞预测结果表明,Rs基因主要在细胞质和叶绿体中表达;启动子分析发现,Rs基因表达可能受光照、MYB、真菌和激素的调控并存在一定的组织特异性。qRT-PCR表达分析显示,葡萄Rs基因在果皮和果肉中不同时期均有表达,但在花后25 d的青皮中表达量最高。结合Rs基因在白藜芦醇积累的作用,可以推测可能在果实发育前期的果皮中白藜芦醇有较高积累。巨峰葡萄Rs基因在进化过程中具有较高的保守性,其表达可能受环境、真菌和激素的调控且与OMT2.1具有互作效应。而且在不同的组织和时期存在一定的特异性。
To explore the potential role of Resveratrol synthase(Rs)in fruit development of Vitis vinifera cv. Kyoho,the structure and function of Rs sequence were analyzed,and the spatio-temporal expression specificity at different developmental stages of fruit was identified. The bioinformatics analysis and qRT-PCR method were used to analyze the sequence of Rs gene in Kyoho grape. The structure and subcellular localization of Rs gene were predicted,and its expression in different tissues of fruit at different developing stages was analyzed. The full-length cDNA of Rs gene was 1 539 bp,with an open reading frame(ORF)of 1 179 bp,encoding 392 amino acids. The predicted molecular weight was 42.88 ku,and the theoretical pI was 6.09. The gene contained chalcone,stilbene synthase activity sites and complete stilbene synthase family characteristic sites. The protein interaction predicted that Rs interacts with OMT2.1,which catalyzes resveratrol biosynthesis of Pterotilbene. The results of subcellular prediction showed that the Rs gene mainly expressed in the cytoplasm and chloroplast. The promoter analysis showed that the expression of Rs gene might be regulated by light,MYB,fungi and hormones,presenting a certain tissue specificity. qRT-PCR analysis showed that the Rs gene of grape expressed at different stages of the peel and flesh,but the expression level was highest in the 25 th day after anthesis. Combined with the role of Rs gene accumulation in resveratrol,it could be inferred that there might be a high accumulation of resveratrol in the fruit peel during the early period of fruit development. The Rs gene of Kyoho grape had a high conservation during evolution,and its expression might be regulated by environment,fungi and hormone. It had the interaction with OMT2.1 and had a certain specificity in different tissues and development periods.
To explore the potential role of Resveratrol synthase(Rs)in fruit development of Vitis vinifera cv. Kyoho,the structure and function of Rs sequence were analyzed,and the spatio-temporal expression specificity at different developmental stages of fruit was identified. The bioinformatics analysis and qRT-PCR method were used to analyze the sequence of Rs gene in Kyoho grape. The structure and subcellular localization of Rs gene were predicted,and its expression in different tissues of fruit at different developing stages was analyzed. The full-length cDNA of Rs gene was 1 539 bp,with an open reading frame(ORF)of 1 179 bp,encoding 392 amino acids. The predicted molecular weight was 42.88 ku,and the theoretical pI was 6.09. The gene contained chalcone,stilbene synthase activity sites and complete stilbene synthase family characteristic sites. The protein interaction predicted that Rs interacts with OMT2.1,which catalyzes resveratrol biosynthesis of Pterotilbene. The results of subcellular prediction showed that the Rs gene mainly expressed in the cytoplasm and chloroplast. The promoter analysis showed that the expression of Rs gene might be regulated by light,MYB,fungi and hormones,presenting a certain tissue specificity. qRT-PCR analysis showed that the Rs gene of grape expressed at different stages of the peel and flesh,but the expression level was highest in the 25 th day after anthesis. Combined with the role of Rs gene accumulation in resveratrol,it could be inferred that there might be a high accumulation of resveratrol in the fruit peel during the early period of fruit development. The Rs gene of Kyoho grape had a high conservation during evolution,and its expression might be regulated by environment,fungi and hormone. It had the interaction with OMT2.1 and had a certain specificity in different tissues and development periods.
引文
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