叶绿素是植物重要的光合色素之一,其含量影响着植物最终合成的生物量或产量.CAO(Chlorophyllide a oxygenas)是叶绿素生物合成的关键限速酶之一,主要参与叶绿素b的形成.为探究杧果CAO基因的生物学功能,本研究利用RACE技术,从杧果'金煌'品种中克隆得到MiCAO基因并对其蛋白序列进行相应的生物信息学分析、构建其植物超表达载体进行亚细胞定位和基因功能验证.本试验克隆了杧果MiCAO基因,该基因全长cDNA序列为2 151 bp,开放阅读框为1 608 bp,编码535个氨基酸.进化树分析表明,MiCAO蛋白与双子叶植物的开心果PvCAO蛋白的亲缘关系最近.利用植物瞬时表达技术转化烟草,对MiCAO蛋白进行亚细胞定位显示MiCAO蛋白定位于细胞质和细胞核中;对MiCAO进行基因功能验证,结果表明转基因株系表型与野生型无明显差异,推测MiCAO基因可能不是调控叶绿素生物合成途径中的主效基因,为研究杧果叶绿素生物合成提供了一定的理论参考依据.
Abstract
Chlorophyll is one of the important photosynthetic pigments of plants,and its content affects the final biomass or yield of plants.CAO(chlorophyllide a oxygenas)is one of the key rate limiting enzymes in chlorophyll biosynthesis,mainly involved in the formation of chlorophyll b.In order to explore the biological function of mango CAO gene,using RACE technology,MiCAO gene was cloned from mango'Jinhuang'variety and its protein sequence was analyzed by bioinformatics,construct its plant overexpression vector for subcellular localization and gene function verification.In this experiment,MiCAO was cloned.The full-length cDNA sequence of this gene is 2 151 bp,and the open reading frame is 1 608 bp,and it encodes 535 amino acids.The phylogenetic tree analysis showed that MiCAO protein was closely related to PvCAO protein in pistachios of dicotyledons.Technology of tobacco transformation by plant instant genetics,the subcellular localization showed that MiCAO was localized in the cytoplasm and nucleus of tobacco plants.The gene function verification of MiCAO showed that there was no significant difference between the phenotype of the transgenic strain and that of the wild type.It was speculated that MiCAO gene might not be the main gene regulating the pathway of chlorophyll biosynthesis,which provided a theoretical reference for the study of mango chlorophyll biosynthesis.
关键词
杧果/叶绿素酸酯a加氧酶,MiCAO/基因克隆/载体构建
Key words
Mangifera indica/Chlorophyllate a oxygenase/MiCAO/Gene cloning/Vector construction
维普
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