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

Abstract

[ 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 prop- erties , 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 1 608 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 ChCA O gene were reduced. [ Conclusion ] The ChCAO gene was obtained and its function was preliminarily characterized , which are helpful for the understanding of the formation of leaf art in Cymbidium hybrid.

Keywords

• cymbidium hybrid
• chcao gene
• gene cloning
• expression analysis