杂交兰ChCAO基因的克隆及其在叶艺品系中的表达特征
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福建省农业科学院作物研究所

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S682.31? ???????????

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福建省自然科学基金项目(2023J01199);福建省农业科学院英才项目(YC2021013);福建省农业科学院科技创新平台专项(CXPT202102);福建省农业科学院创新团队建设项目(CXTD2021010-2)


Molecular Cloning,Expression Analysis of ChCAO Gene in leaf art strain of Cymbidium hybrid
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    摘要:

    【目的】叶绿素酸酯a加氧酶(CAO)是叶绿素b形成过程中的关键酶,为探究CAO基因在杂交兰叶艺形成中的调控作用,并为进一步研究杂交兰叶艺形成机理提供重要依据。【方法】该研究以杂交兰‘紫妍氏’(K21)及其叶艺品系‘中透紫妍氏’(K21-3)为试验材料,运用RT-PCR和RACE技术从叶片中克隆获得ChCAO基因;对ChCAO进行结构特征、理化性质、序列比对以及系统进化关系等分析;并利用qRT-PCR法对ChCAO在不同组织及叶艺品系叶片中的表达特性进行分析。【结果】结果表明,ChCAO基因编码区长1 608 bp,编码535个氨基酸,ChCAO与墨兰CAO亲缘关系最近,并与其他兰科植物CAO聚为一类。qRT-PCR结果显示,ChCAO基因表达具有组织特异性,在叶中相对表达量最高,根中相对表达量最低;此外,ChCAO在K21绿叶和K21-3绿叶区域叶片中的相对表达量显著高于K21-3叶艺区域叶片中的相对表达量。构建该基因的VIGS沉默载体转化烟草,发现沉默ChCAO后烟草叶片呈黄化状态,叶片中的叶绿素含量及ChCAO基因的相对表达量也显著降低,由此推测ChCAO基因的沉默表达可能导致叶绿素含量降低,叶片黄化,初步明确了杂交兰ChCAO基因的功能。

    Abstract:

    【Objective】 Chlorophyllide a oxygenase (CAO) is a key enzyme in the formation of chlorophyll b. To investigate the regulation function of the CAO gene in the leaf color variation of Cymbidium hybrid, and to provide an important basis for studying the formation mechanism of leaf art. 【Methods】 ChCAO gene was cloned from leaves of Cymbidium hybrid by RACE and RT-PCR methods. ChCAO was analyzed for structure characteristics, physicochemical properties, sequence alignment and phylogenetic relationship. The expression characteristics of ChCAO in different tissues and leaves of K21-3 were analyzed by qRT-PCR. 【Results】The results showed that ChCAO coding region was 1 608 bp long, which encoding 535 amino acids. ChCAO was closely related to Cymbidium sinense, which also belonged to genus Cymbidium. The qRT-PCR results reflected the tissue-specific in the expression of ChCAO, with the highest relative expression in leaves and the lowest in roots. Among the different varieties, the expression level of ChCAO in K21 leaves were significantly higher than that of K21-3. The VIGS silencing vector of this gene was constructed and transferred to tobacco, it was found that the viral vector successfully invaded the body of tobacco, the old leaves of tobacco were in a yellow state, and the chlorophyll content in the old leaves and the relative expression of the ChCAO gene were also significantly reduced. It is speculated that the silent expression of ChCAO gene may lead to the reduction of chlorophyll content and the yellowing of leaves, which preliminarily clarifies the function of the ChCAO gene in Cymbidium hybrid.

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  • 收稿日期:2023-05-29
  • 最后修改日期:2023-10-30
  • 录用日期:2023-11-17
  • 在线发布日期: 2024-01-19
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