Cloning Of Circadian Clock-related Genes In Dendrobium Huoshanense And Their Relations With Polysaccharides Content

Dendrobium huoshanense is perennial herbaceous plant,which belongs to Dendrobium genus,Orchidaceae family,with high medicinal and ornamental values.In the present research,conditions of in vitro culture were optimized for the development of the protocorm-like bodies(PLBs)and in vitro plantlets.In different photoperiod conditions,various developmental partens and the structure and morphology of the cultures were observed,and the contents of polysaccharides in PLBs and in vitro plantlets were measured.Two genes,one for the central oscillator of circadian clock,CIRCADIAN CLOCK-ASSOCIATED 1(CCA1),and an unknown gene MYB1Rl,potential for stress tolerant in other plants,were isolated from PLBs and in vitro plantlets,and their expressions were analyzed via qPCR assay in different photoperiod and other culture conditions.Finally,combination of different photoperiod conditions,conditions of in vitro culture,propagation rate,polysaccharides contents and gene expression as well as their relationships were studied,providing theoretical basis for experimental biology and molecular biology to discover the metabolic mechanisms of polysaccharides in D.huoshanense.1.Propagation rates of D.huoshanense in relation to different photoperiodsPLBs were cultured in MS medium supplemented with 0.1mg/L NAA,0.05mg/L KT and 50 g/L potato juice;in vitro plantlets were cultured in MS medium supplemented with 0.5mg/L IBA,0.5mg/L NAA and 200 g/L banana juice;the proliferation rate of PLBs and in vitro plantlets under different photoperiod treatments was compared.The results showed that different photoperiod conditions influenced the proliferation rate of PLBs to a certain extent.The proliferation rate of PLBs was lowest under dark condition;the proliferation rate peaked and reached similar level in the photoperiod conditions of 24h,18h,12h and 6h light treatments after 50d culture.Among all treatments,18h light group showed the highest proliferation rate after 50d culture;meanwhile,24h light group showed the highest proliferation rate in every culture stages and reached near the peak after 40d culture.Different photoperiod conditions had less effect on the proliferation rate of in vitro plantlets than on PLBs.After 10d culture,the proliferation rate of 12h/12h L/D group kept ahead and maintained the highest level.However,the proliferation rate of PLBs was generally much higher than that of the in vitro plantlets.2.Determination of the polysaccharides content under different photoperiod treatmentsPLBs were cultured in MS medium supplemented with O.lmg/L NAA,0.05mg/L KT and 50 g/L potato juice;in vitro plantlets were cultured in MS medium supplemented with 0.5mg/L IBA and 0.5mg/L NAA;the polysaccharides content of PLBs and in vitro plantlets under different photoperiod treatments was compared.The results showed that polysaccharides content of the cultures under the 6h light or full darkness condition firstly reached highest level and then reduced gradually.In all photoperiod treated gourps,polysaccharides content of PLBs from 6h light group reached the highest level after 10d culture.After 30d culture,both PLBs and in vitro plantlets with 24h light treatment accumulated highest polysaccharides and then reduced gradually,among which polysaccharides content of PLBs from 24h light group showed the highest value after 30d culture.After 40d culture,the cultures of different photoperiod treatments all showed reduced polysaccharides content to the mean level that indicated that the cultures in the depleted medium(40d-50d)were unable to support accumulation of polysaccharides but only maintain the growth and morphological development.Different photoperiod conditions had more effect on the polysaccharides content of in vitro plantlets than on PLBs.After 10d culture,there was a positive correlation between the increase of illumination time and the polysaccharide contents.The polysaccharide contents of in vitro plantlets were higher than that of the PLBs in the corresponding periods,but PLBs accumulated polysaccharides earlier than that of the in vitro plantlets.3.Cloning and bioinformatics analysis of CCA1 and MYB1R1 genesPLBs and in vitro plantlets were used to isolate genes by means of homology cloning and RACE techniques.Full-length cDNA sequences were obtained for two genes:CCA1 was 1652 bp(Genbank:KX710175),and MYB1R1 was 1332 bp(Genbank:KX710176),and both of the genes contained an open reading frames(ORF)which encoded 532 and 427 amino acids respectively.Bioinformatics analysis of CCA1 and MYB1R1 showed that CCA1 was an alkaline protein while MYB1R1 was an acidic protein;both of them had no signal peptides,not belonging to secreted proteins;there was myb conserved domain respectively in both CCA1 and MYB1R1,which proved that both CCA1 and MYB1R1 were belonged to MYB transcription factor gene family.4.Gene expression analysis of CCA1 and MYBIR1The expressions of CCA1 and MYB1R1 were detected in different photoperiodic stages of PLBs and in vitro plantlets with Actin gene as the internal reference gene.In the present study,the 12h light group,similar to the natural circadian cycle,was set as the reference group,and qPCR analysis was conducted on the plant materials after Id,10d,20d,30d,40d,and 50d culture.The results showed that CCA1 and MYB1R1 gene had similar expression trend in PLBs and in vitro plantlets.CCA1 expression showed earlier fluctuations than MYB1R1 gene,and their expression patterns were generally synchronized in in vitro plantlets.The expression patterns of CCA1 and MYB1R1 were commonly different in PLBs from that of the in vitro plantlets,and they both showed higher expression levels in PLBs than that of the in vitro plantlets.Based on the gene expression data in a macro-time scale,it was inferred that MYB1R1 acted the part of circadian rhythm regulations,or that MYB1R1 was the directly regulated gene by the central oscillator of the circadian clock.When cultured for 30d,the materials in different photoperiod treatment groups were sampled every 6h,with the 12h light group as the reference group,the expression patterns of CCA1 and MYB1R1 were observed during 24h.The results showed that CCA1 shifted from the 12h/12h photoperiod,and varied oscillations and frequncy appeared,and MYB1R1 was more close to the control(12h/12h photoperiod)than that of CCA1 gene.In the current experimental conditions,MYB1R1 also showed varied expression oscillation and frequency,and CCA1 and MYB1R1 showed similar expression patterns.Based on the gene expression data in a micro-time scale,it was inferred that MYB1R1 gene acted or as a part of the circadian rhythm regulators,or as a directly regulated gene by the central oscillator of the circadian clock.With 18SrRNA as the reference gene,with the treatments of semi-submersion and drought,MYB1R1 showed significant expression level changes,and it also showed a high expression level in the dark condition.These results pointed to that MYB1R1 was related to light/dark conditioning and might be involved in drought stress signal transduction.In summary,the present thesis revealed that CCA1 as the typical MYB gene was affected by photoperiods but played a role in the polysaccharides metabolism and also function in propagation rate,dry matter contents of D.huoshanense.PLBs were found an idial explants for regulation of the photoperiod-induced polysaccharide content.CCA1 showed earlier up-regulated expression in PLBs than in in vitro plantlets.The up-regulated expression of CCA1 tended to result in improved morphological and physiological conditions.The expression ranges of CCA1 and MYB1R1 were both larger in PLBS than in in vitro plantlets,while their expression patterns were similar.On the 24h scale when cultured for 30d,CCA1 and MYB1R1 showed time deviation and expression imbalance in deviated half-light conditions.MYB1R1 gene acted the part of circadian rhythm regulators and might be involved in drought stress signal transduction.The current article first provides the expression data of MYB1R1 gene involved in photoperiod and drought stress in Orchidaceae plants.5.Co-purification of RNA and polysaccharidesPLBs and in vitro plantlets were used to isolate RNAs,and the waste during RNA extraction was used as materials to extract polysaccharides.The results showed that the developed co-purification method not only provided high quality RNA for gene expression assay,but also supplied a steady relation with the results of polysaccharides purification via the method used conventionally,which made an in situ and real-time analysis of gene expression and polysaccharides be possible.In conclusion,the present research improved the in vitro culture conditions for D.huoshanense,and revealed the rule of the polysaccharides accumulation in the in vitro cultured plant materials.Two circadian clock related genes were cloned,the bioinformatics of the genes were analyzed,and the expression of the genes were investigated in both a macro-and a micro-time scale.The mechanisms for polysaccharides accumulation in the in vitro culture conditions were discussed with the propagation and dry matter proportion as the background.At the same time,a co-purification of RNA and polysaccharides technique was developed and this will significantly improve the efficiency of the molecular research in D.huoshanense.Finally,combination of different photoperiod conditions,conditions of in vitro culture,the propagation rate,polysaccharides contents and gene expression as well as their relationships were analyzed,which provided a theoretical basis of experimental biology and molecular biology to undersand the metabolic mechanism of polysaccharides in D.huoshanense.