Journal of Nuclear Agricultural Sciences ›› 2021, Vol. 35 ›› Issue (11): 2482-2492.DOI: 10.11869/j.issn.100-8551.2021.11.2482

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

Cloning and Expression Analysis of Three Orange Genes in Kurodagosun Carrot

WANG Yinggang(), XIONG Aisheng, XU Zhisheng*()   

  1. College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095
  • Received:2020-08-26 Accepted:2020-11-26 Online:2021-11-10 Published:2021-09-18
  • Contact: XU Zhisheng

黑田五寸胡萝卜中3个Orange基因的克隆与表达分析

王迎港(), 熊爱生, 徐志胜*()   

  1. 南京农业大学园艺学院,江苏 南京 210095
  • 通讯作者: 徐志胜
  • 作者简介:王迎港,男,主要从事胡萝卜类胡萝卜素基因功能研究。E-mail: 2019804220@stu.njau.edu.cn
  • 基金资助:
    国家自然科学基金资助项目(31872098);江苏高校优势学科建设项目(PAPD);国家自然科学基金资助项目(31501775)

Abstract:

Orange (Or) protein plays an important role in the biosynthesis and accumulation of carotenoids in plants. Three Or genes were identified across the whole carrot genome. To analyze the relationship between these three Or genes and carotenoids biosynthesis, the three Or genes were cloned with the orange carrot Kurodagosun and analyzed by bioinformatics and qRT-PCR. Sequence analysis showed that the ORFs of DcOr1, DcOr2, and DcOr3 were 933 bp, 858 bp, and 939 bp in length, respectively, encode 310, 285, and 312 amino acids, respectively. The predicted amino acid sequence of each Or contains 4 highly conserved cysteine-rich regions. DcOr1 and DcOr3 are both composed of 8 exons and 7 introns, while DcOr2 is composed of 7 exons and 6 introns. The phylogenetic tree analysis suggested that DcOr1 and DcOr2 showed the closest evolutionary relationship with each other, and DcOr3 showed the closest evolutionary relationship with Or protein (XP_031112030.1) from Ipomoea triloba. qRT-PCR results indicated that the three DcOr genes were expressed in carrot roots at 30 d, 60 d, 90 d, and 120 d. DcOr2 and DcOr3 showed the lowest and the highest relative expression levels at all stages, respectively, whereas DcOr3 showed the highest transcript levels at 90-day-old stage. And the trend of relative expression of DcOr3 is close to that of DcPSY1 and DcPSY2. It was speculated that DcOr3 was involved in carotenoid biosynthesis. This study provides a theoretical basis for exploring the mechanism of carotenoid biosynthesis during the growth and development of carrot.

Key words: Orange protein, carotenoids, gene cloning, expression analysis, carrot

摘要:

在植物中Orange (Or) 蛋白对类胡萝卜素的生物合成和积累具有重要的作用。对胡萝卜全基因组分析后鉴定到3个Or基因,为分析3个Or基因与类胡萝卜素合成的相关性,以橙色胡萝卜品种黑田五寸为研究材料,克隆得到3个Or基因,并对其进行生物信息学分析及实时荧光定量(qRT-PCR)分析。序列分析表明,DcOr1、DcOr2和DcOr3的开放阅读框(ORF)全长分别为933、858 和939 bp,分别编码310、285和312个氨基酸;推测的氨基酸序列含有4个高度保守的富含半胱氨酸区域。DcOr1和DcOr3均由8个外显子和7个内含子组成,DcOr2由7个外显子和6个内含子组成。进化树分析显示,DcOr1与DcOr2进化关系最近,而DcOr3与三裂叶薯Or蛋白(XP_031112030.1)进化关系最近。qRT-PCR结果表明,3个DcOr基因在30、60、90和120 d的胡萝卜根中均有表达,其中DcOr2的相对表达量最低,DcOr3在各个阶段的相对表达量都为最高,在90 d时达到峰值,且相对表达量趋势与DcPSY1和DcPSY2最接近,推测DcOr3与胡萝卜类胡萝卜素合成最相关。本研究为探究胡萝卜生长发育过程中类胡萝卜素的生物合成机制提供了一定的理论依据。

关键词: Orange蛋白, 类胡萝卜素, 基因克隆, 表达分析, 胡萝卜