猕猴桃类胡萝卜素裂解双加氧酶基因AcCCD的克隆与表达分析

Cloning and Expression Analysis of Carotenoid Cleavage Dioxygenase Gene AcCCD in Kiwifruit

【目的】对猕猴桃类胡萝卜素裂解双加氧酶(CCD)基因进行克隆,并进行理化和表达分析。【方法】从猕猴桃基因数据库中鉴定CCD候选基因,克隆3条目的基因全长,并利用荧光定量PCR技术分析所选基因在猕猴桃果实发育期及不同组织的表达量。【结果】克隆得到3个类胡萝卜素裂解双加氧酶(CCD)基因,命名为AcCCD1.1、AcCCD1.2和AcCCD7。其开放阅读框(ORF)分别为1 890、1 632和1 818 bp,编码629、543和605个氨基酸。经预测,3种AcCCD蛋白均定位于细胞质,不具有信号肽,不属于分泌蛋白。经分析,AcCCD1.1、AcCCD1.2蛋白属于CCD1蛋白家族,AcCCD7蛋白属于CCD7家族。系统进化树显示,3个基因编码的蛋白与野茶树等植物蛋白相似性高。对AcCCD基因qRT-PCR,结果表明AcCCD1.1在根中表达量高,叶中最低;AcCCD1.2在花、果实发育后期表达量高。AcCCD7表达量总体较低,根中表达量高于在其茎、叶、花和果的表达量。【结论】推测AcCCD1基因对于猕猴桃果实颜色以及香味的形成有重要联系。AcCCD7基因对于猕猴桃根系生长上具有重要调控作用。

【Objective】The carotenoid cleavage dioxygenase(CCD)gene was cloned and analyzed for physicochemical properties and expression.【Method】The CCD candidate genes were identified from the kiwifruit gene database,the full length of three genes were cloned,and the expression levels of these genes in different tissues and fruits of kiwifruit were analyzed by fluorescence quantitative PCR.【Result】Three CCD genes were cloned and named AcCCD1.1,AcCCD1.2 and AcCCD7.Their open reading frames(ORF)were 1890,1632 and 1818 bp,encoding 629,543 and 605 amino acids,respectively.Predic-tion of subcellular location showed that the three AcCCD proteins were located in the cytoplasm and do not have signaling peptides,suggesting they did not belong to secreted proteins.According to the analy-sis,AcCCD1.1 and AcCCD1.2 proteins belong to the CCD1 protein family,while AcCCD7 proteins be-long to the CCD7 family.The phylogenetic tree showed that these were highly similar to plant proteins such as the wild tea tree.The results of qRT-PCR showed that AcCCD1.1 was highly expressed in roots and minimally in leaves,while AcCCD1.2 was highly expressed in late flower and fruit development.AcCCD7 has generally low expression but is higher in roots compared to stems,leaves,flowers and fruits.【Conclusion】It is speculated that AcCCD1 gene is important for the formation of kiwi fruit color and flavor.AcCCD7 gene plays an important role in regulating the root growth of kiwifruit.