Molecular Cloning And Preliminary Functional Characterization Of DoJAZ And DoJAR1 From Dendrobium Officinale

Dendrobium officinale Kimura et Migo has been a precious resource for traditional Chinese medicine since ancient times.It belongs to the genus Orchidaceae in taxonomy.It has the functions of nourishing yin and nourishing,clearing the eye and eyesight.The main active ingredients in D.officinale,are polysaccharides and alkaloids.However,the low content of polysaccharides and alkaloids has always been a key problem affecting the medicinal value of Dendrobium officinale.Current studies have shown that jasmonic acid,as a plant growth hormone,can effectively enhance the pharmacodynamic components of medicinal plant by exogenous induction.In this study,two major genes DoJAZ and DoJAR1,which were on the signal transduction pathway of jasmonic acid from were cloned.Their expression patterns were analyzed by Real-Time Fluorescent Quantitative PCR,and the interaction between DoJAZ and DoMYC2 was analyzed by yeast two-hybrid method.The aim of this study was to investigate the role of DoJAZ and DoJAR1 in jasmonic acid signal transduction pathway and their effects on secondary metabolism of Dendrobium officinale.The regulation mechanism of jasmonic acid in secondary metabolism of Dendrobium officinale was preliminarily elucidated in this experiment.It lays a theoretical foundation for further analysis of secondary metabolic pathway of Dendrobium officinale and for enhancing its medicinal value.The main results were as follows:1.The full-length cDNA of DoJAZ(accession number:MF680547)and DoJAR1 was successfully cloned from Dendrobium officinale by RACE and RT-PCR.Tissue-specific analysis showed that DoJAZ and DoJAR1 were expressed in various tissue parts of Dendrobium officinale and there were difference in expression.2.The protocorms of Dendrobium officinale were treated with three hormones(MeJA,SA,ABA)and heavy metal elicitors(AgNO3).The expression levels of DoJAZ and DoJAR1 were detected by qRT-PCR at different time points.After analysis,it was found that both DoJAZ and DoJAR1 were induced by MeJA,SA,ABA and AgNO3,and the expression levels increased first and then decreased.3.The DoJAZ and DoJARl proteins were successfully induced in E.coli by constructing pET-28a-DoJAZ and pET-28a-DoJAR1 prokaryotic expression vectors.The pCAMBIA1304-DoJAZ and pCAMBIA1304-DoJAR1 eukaryotic expression vectors were successfully constructed,and Arabidopsis thaliana was transformed by Agrobacterium mediated transformation,and T3 generation over-transgenic Arabidopsis thaliana was screened.Compared with the wild type,the root length of DoJAZ transgenic Arabidopsis thaliana was significantly higher than that of wild type.The root length of DoJAR1 transgenic Arabidopsis thaliana had no significant change compared with wild type.qRT-PCR analysis showed that the expression of scorpion-related genes in DoJAZ transgenic Arabidopsis thaliana was significantly lower than that in wild-type Arabidopsis thaliana.The expression of scorpion-related genes in DoJAR1 transgenic Arabidopsis thaliana was significantly higher than that in wild-type Arabidopsis thaliana.4.Yeast vectors pGBKT7-DoJAZ and pGADT7-DoMYC2 were successfully constructed.Yeast two-hybrid analysis showed that there was an interaction between DoJAZ and DoMYC2.