Molecular Cloning And Expression Analysis Of DXS?DXR?HDR In Dendrobium Officinale Kimuraet Migo

Dendrobium officinale Kimuraet Migo is one of rare medicinal Dendrobium,with the effects of tonifying stomach and promoting fluid,nourishing yin and clearing heat.Alkaloid is one of the main medicinal components of D.officinale.The alkaloids in D.officinale mostly belongs to the sesquiterpenoids alkaloids or terpenoids indole alkaloids,which are originated from the terpenoids pathway(MVA,MEP).1-deoxy-D-xylulose-5-phosphate synthase(DXS),1-deoxy-D-xylulose-5-phosphate reductoisomerases(DXR)and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphatereductase(HDR)are all the key enzymes of MEP pathway.However,the research about DXS?DXR?HDR and its function in D.officinale is still unknown.In this experiment,some novel genes encoding DXS?DXR?HDR were isolated and characterized from D.officinale,all these were in order to built the foundation for subsequent in-depth studys of their relationships with the terpenoids pathway of D.officinale.The main results of this study were as follows:(1)RT-PCR and RACE technologies were used to clone the full length cDNA of DoDXS?DoDXR?DoHDR,the accession numbers are KF803334?KF412827?KC344827,respectively.(2)Tissue specific expression analysis showed that the DoDXS ?DoDXR ?DoHDR expressed in all tissues of D.officinale.DoDXS had the highest expression in the roots,which was 3.86 folds than in the leaves;DoDXR had a high expression in stems,which was 2.26 folds than in the protocorm.While,the DoHDR had a high expression in the leaves,was 2.72 folds than in the protocorm.(3)After the treatment with methyl jasmonate(MeJA),the expressions of DoDXS reached the peak at 48 h,which was 5.17 folds than the control group.While the expressions of DoDXR and DoHDR could not be induced by MeJA.By the treatment of salicylic acid(SA),the expressions of DoDXS ? DoDXR ? DoHDR were significantly up-regulated at 4h,the peak was 5.46?5.25?3.36 folds than the control group,respectively.Treated by Abscisic acid(ABA),the expressions of DoDXS ? DoDXR ? DoHDR were up-regulated,the peak was 2.82?4.49?6.37 folds than the control group,respectively.Treated by Brassinolide(BR),The expressions of DoDXS ? DoDXR ? DoHDR were significantly up-regulated within a short time,and reached the peak at 24 h.(4)The eukaryotic expression vectors-pCambia1301-DoDXS,pCambia1301-DoDXR and pCambia1301-DoHDR were constructed.They were used to analyze the subcellularlocalization in Nicotiana tabacum and over-expression of genetic transformation in Arabidopsis thaliana.The results showed that the all proteins including DoDXS?DoDXR and DoHDR were located in the chloroplast by a laser scanning confocal microscope.The results of Quantitative PCR showed that DoDXS was over-expressed in DoDXS-T2 generation transgenic Arabidopsis thaliana plants.Another 30 lines of DoDXR-T1 generation transgenic Arabidopsis thaliana plants were obtained.(5)In order to find the role and contribution of the MVA and MEP pathway in the alkaloids biosynthesis of D.officinale,MVA pathway inhibitor Mevinolin and MEP pathway inhibitor Fosmidomycin were used to treat the plants.The results show that the inhibition rate of mevinolin on the biosynthesis of total alkaloids was 26.27%,while the inhibition rate of fosmidomycin was 33.62%.It was suggested that the MEP pathway may play a dominant role in the biosynthesis of alkaloids in D.officinale.Through the detection of enzyme activity,we assume there is interactive using regulation between the two pathway.