Cloning And Expression Analysis Of DoObgC Gene In Dendrobium Officinale

Dendrobium officinale is perennial herb and belongs to dendrobium(Orchidaceae),which could be nourishing Yin and clearing heat,engendering liquid and benefiting the stomach.The major function of Dendrobium officinale is curing calenture and body fluid deficiency,thirst and polydipsia,the lack of stomach yin,and so on.Modern pharmacology study suggests,Dendrobium officinale can improve the immunity of human body,specialize in anti-aging,anti-tumor,and regulate blood sugar level.Dendrobium officinale grabs attention of research scholars because of extensive phamaceutical values,more and more molecular biology study about Dendrobium officinale will be put in effect.On molecular level,studying Dendrobium officinale physiology and biochemistry,and analysis molecular mechanism,especially gene clone and functional analysis study progress develop rapidly.This article was taking Dendrobium officinale as the object of study,using the method of gene clone,and cloned full-length cDNA of DoObgC gene,then carried out bioinformatics analysis and molecular biology studys,preliminarily clarified gene structure and protein function of DoObgC.The main research results are as follows:1.Through the bioinformatics analysis of Dendrobium offcinale genome data,gene structure was analyzed and primers were designed.Sequence analysis indicate that full-length of DoObgC gene was 1993 bp,open reading frame was 1872 bp,5'-UTR and 3'-UTR were 6 bp and 115 bp respectively,which encoded 623 amino acids,molecular weight of DoObgC protein was 67.62 kDa,theoretical isoelectric point was 5.29,and DoObgC was hydrophilous and unstable protein.Secondary structure analysis showed that Alpha helix was 32.42%,Extended strand was 20.87%,Beta turn was 10.27%,Random coil was 36.44%.It predicted that Cys3-471,Cys10-163 and Cys402-571 were disulfide bond.Phylogenetic tree analysis demonstrated that Arabidopsis thaliana,Rice,and a few plants possess the sequences similar to DoObgC,Dendrobium officinale had the nearest genetic relationship with Elaeis guineensis and Musa acuminate.2.By means of Gateway clone method,finally DoObgC gene sequenc was cloned into pMDC83 and pEarly Gate101 successfully.Using Agrobacterium-mediated method,the recombinant,pMDC83-DoObgC,was transformed into tobacco.Fluorescence confocal assay indicated that green signals of DoObgC-GFP,like grain,overlapped with the red chlorophyll autofluorescence(CHL),suggested that DoObgC is specifically targeted to the chloroplast.DoObgC functioned in chloroplast.Then,pMDC83-DoObgC and pEarly Gate101-DoObgC were transformed into obg-t mutant.homozygous obgc-t mutants were obtained from T2 plants of transformed heterozygous obgc-t plants with 35S-DoObgC-GFP construct.The results revealed that the ratio of wild-type:heterozygous:homozygous plants was 48:80:47,conformed to Mendel's law of segregation,the overexpression of DoObgC suppressed the embryonic lethality caused by obgc-t mutation,demonstrating that DoObgC is a functional homolog of AtObgC.DoObgC,like AtObgC,may play a function in ribosome biogenesis of D.officinale chloroplasts.3.DoObgC?(1-160)protein prokaryotic expression analysis,pGEX-6p-1-DoObgC?(1-160)was transformed into BL21 and BL21(DE3)cell.After induced under ImM IPTG,30 ?for 5 hours,The size of DoObgC?(1-160)and GST proteins was respectively evaluated as?75 kDa and 25 kDa by using SDS-PAGE.Then under the treatment with 25 ?g/mL streptomycin,the GST-DoObgC?(1-160)expressed cells showed much higher survival percentage compared to the GST expressed cells,the result showed that the overexpression of GST-DoObgC?(1-160)exhibited substantial resistance to streptomycin.the cell densities of untreated and treated with streptomycin were measured by microplate spectrophotometer,the treatment of streptomycin had no effect during the exponential phase(0?8 h)of the expressed GST-DoObgC?(1-160)cells,DoObgC?(1-160)overexpressed cells exhibited mild resistance to streptomycin in the stationary phase,as compared with the control cells.These results suggested that the expression of DoObgC?(1-160)antagonize the growth-inhibitory effect of streptomycin in the early period of growth cycle,to maintain normal growth of the cell.4.The consequence analysis of real-time fluorescence quantification showed that,DoObgC gene expressed in all tissues,but the DoObgC gene was predominantly expressed in leaves,especially more abundant in new bud leaves,and were expressed very low in protocorms,roots,stems and flowers.We further analyzed the expression pattern of DoObgC in response to wounding stress.The results showed that DoObgC mRNA level reduced within 1 h after wounding.Notably at 3 h,transcript levels of DoObgC decreased to 53%compared with untreated plants.Then,the expression of DoObgC gene gradually increased at 6 h,and further surpassed the transcription level before wounding at 12 h,the result indicate that DoObgC play a role in response to wounding stress.Finally what we found was that illumination is more helpful to express DoObgC gene,compared with darkness.It demonstrated the expression of DoObgC gene was induced by illumination.