杂交兰ChCAO基因克隆及其在叶艺品系中的表达特征

Molecular cloning and expression analysis of ChCAO gene in leaf art formation of Cymbidium hybrid

【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,对杂交兰CAO基因进行克隆及表达特征分析可为探究其在杂交兰叶艺形成中的调控作用奠定基础。【方法】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,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;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;并用VIGS技术对ChCAO进行沉默表达。【结果】ChCAO基因编码区长1608 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。【结论】克隆获得了杂交兰ChCAO基因,并对其功能进行了初步鉴定,为进一步研究杂交兰叶艺形成机理提供重要依据。

[Objective]Chlorophyllide a oxygenase(CAO)is a key enzyme in the formation of chlorophyll b.Cloning and expression analysis of CAO genes may be helpful in exploring their function in regulating leaf color variation of Cymbidium hybrid.[Methods]ChCAO gene was cloned from leaves of Cymbidium hybrid by RACE and RT-PCR.ChCAO was analyzed for structure characteristics,physicochemical properties,sequence alignment,and phylogenetic relationship.The expression of ChCAO in different tissues and leaves of K21-3 were analyzed by qRT-PCR.Meanwhile,ChCAO expression was silenced by VIGS technology.[Results]The results showed that ChCAO coding region was 1608 bp,encoding 535 amino acids.ChCAO was closely related to Cymbidium sinense,which belonged to genus Cymbidium.qRT-PCR assay showed that the expression of ChCAO was the highest in leaves and the lowest in roots.The expres-sion of ChCAO in K21 leaves was significantly higher than that of K21-3.The VIGS silencing vector of ChCAO gene was constructed and transferred to tobacco.The viral vector was successfully invaded the to-bacco,and the old leaves of tobacco were turned yellow,while chlorophyll content and expression of ChCAO gene were reduced.[Conclusion]The ChCAO gene was obtained and its function was preliminari-ly characterized,which are helpful for the understanding of the formation of leaf art in Cymbidium hybrid.