四川农业大学学报 ›› 2019, Vol. 37 ›› Issue (04): 475-480.doi: 10.16036/j.issn.1000-2650.2019.04.007

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油茶CoFAD2-1基因的克隆、亚细胞定位及组织表达

陈潇潇1, 罗红艳1, 顾真琪2, 陈世品1, 曹光球1, 曹世江1,*   

  1. 1.福建农林大学林学院,福州 350002;
    2.福建农林大学植物保护学院,福州 350002
  • 收稿日期:2018-01-28 出版日期:2019-08-28 发布日期:2021-01-31
  • 作者简介:陈潇潇,硕士研究生。*责任作者:曹世江,博士,讲师,主要从事林木遗传育种研究,E-mail:csjiang1123@126.com。
  • 基金资助:
    国家“十二五”支撑计划项目(2014BAD15B00)

Cloning Subcellular Localization and Tissues Expression of CoFAD2-1 Gene from Camellia oleifera

CHEN Xiaoxiao1, LUO Hongyan1, GU Zhenqi2, CHEN Shipin1, CAO Guangqiu1, CAO Shijiang1,*   

  1. 1. College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
    2. College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China
  • Received:2018-01-28 Online:2019-08-28 Published:2021-01-31

摘要: 【目的】克隆油茶CoFAD2-1基因,并进行亚细胞定位和组织表达研究。【方法】采用CTAB法提取油茶4种组织中的总RNA;运用RT-PCR方法,设计基因特异性引物,从油茶未成熟胚中克隆CoFAD2-1基因;用生物信息学手段分析了CoFAD2-1基因的结构和进化关系;利用实时荧光定量PCR技术研究CoFAD2-1基因在不同组织的表达;同时运用烟草瞬时表达技术对该基因进行亚细胞定位分析。【结果】该基因编码区全长为1 149 bp,共编码382个氨基酸,具有FAD2基因家族保守的3个组氨酸簇。系统树分析显示该基因与种子特异表达的FAD2基因聚在一起,组织表达分析也表明CoFAD2在种子中特异表达,转化烟草叶片验证CoFAD2-1蛋白定位于内质网膜上。【结论】CoFAD2-1基因在种子中特异性表达,且定位在内质网膜上。

关键词: 油茶, CoFAD2-1, 基因克隆, 表达分析, 亚细胞定位

Abstract: 【Objective】 The aim of this study was to clone CoFAD2-1 gene, and to investigate the subcellular localization and tissues expression. 【Method】 The total RNA of Camellia Oleifera Abel. was extracted by CTAB method. RT-PCR was used to clone the CoFAD2-1 gene with gene specific primers from the immature embryo of Camellia Oleifera Abel. The structure and evolution of CoFAD2-1 gene was analyzed by bioinformatics. Meanwhile, real-time PCR was used to check CoFAD2-1 gene expression profile in different tissues and transient tobacco expression system was employed to reveal its subcellular localization. 【Result】 CoFAD2-1 gene was 1 149 bp long in open reading frame. It encoded382 amino acids and contained three conserved His box of CoFAD2-1. Phylogenetic tree analysis showed that the gene was clustered in the same class with seed specific expression FAD2 gene. Tissue expression analysis also suggested that the specific expression of CoFAD2 was found in the embryo and the gene was verified to localize in the endoplasmic reticulum membrane using tobacco leaf. 【Conclusion】 The results indicate that CoFAD2-1 gene is seed-specificity expressed and localized in the endoplasmic reticulum membrane.

Key words: Camellia oleifera Abel, CoFAD2-1, gene cloning, expression analysis, subcellular localization

中图分类号: 

  • S794.4