Transcriptomic Analysis Of Dendrobium Huoshanense And Prliminary Research On Its GDP-mannose Pyrophosphorylase Gene

Objective: This paper aims to study GDP-mannose pyrophosphorylase gene(GMPP)which plays an important role in polysaccharide biosynthesis in Dendrobium huoshanense,and to serve as a stepping stone for a broader range regarding its whole polysaccharide biosynthetic pathway.Methods: The strategies picking D.huoshanense fresh plants as materials,comparing RNA extraction methods were optimized.Using agarose gel electrophoresis,nucleic acid trace quantitative instrument and specificity of PCR,total RNA quality resulted from different extraction methods were compared.Make use of Illumina platform to sequence different organs of D.huoshanense and yield sufficient transcritpomic data,from which biosynthetic information related to its useful secondary metabolites were carefully combed.Because D.huoshanense accumulates abundant polysaccharides in various organs,transcriptomic analysis was focused on polysaccharide biosynthetic pathway and GMPP gene was singled out as the target.Design specific primers and choose Ex taq enzyme to obtain GMPP gene by means of PCR augmentation and subsequent electrophoresis.TIANgel Mini Purification Kit was utilized to recover DNA from agarose gel.After adopting pGM-T Fast cloning kit to create recombinant plasmid,the plasmid containing GMPP was transformed into E.coli DH5ɑ by heat shock approach.By conducting colony PCR using GMPP specific primer pairs,the resulted colonies in LB culture medium with ampicillin were confirmed again.Plasmids were extracted with TIANpure Mini Plasmids Kit and then GMPP contained in the plasmids was sequenced to confirm consistency with D.huoshanense in silico data.To create GMPP entry clone,pDONR221 was adopted for BP reaction,followed by pDEST17 as expression cloning for LR reaction to construct recombinant protein expression vector.RT-PCR was also used to detect GMPP gene expression profile in D.huoshanense root,stem,leaf,flower.Results: By comparing the results of total RNA extraction methods for D.huoshanense,total RNA retrieved by MCHAN approach had high quality.Electrophoresis showed clear stripe in agarose gel without dispersion phenomenon and no impurity contamination.Through D.huoshanense transcriptomic analysis,17 cDNA libraries were built,study on gene information and the expresson profile related to different organs was now made possible.Seven important enzymes,including invertase,sucrose synthase,hexokinase,glucose phosphate isomerase,mannose phosphate isomerase,mannose phosphate enzyme and GDPmannose pyrophosphorylase in D.huoshanense,which are very likely involved in D.huoshanense polysaccharide biosynthesis were retrieved.As for D.huoshanense GMPP gene,its cDNA sequence is 1867 bp,containing 1245 bp open reading frame,coding 415 amino acids.Sequence analysis showed that D.huoshanense GMPP sequence shared 99 percent similarity with that of D.officinale and D.moniliforme.Evolutionary tree analysis showed that D.huoshanense,D.officinale and D.moniliforme have a close genetic relationship.qPCR test demonstrated that GMPP gene has highest expression in D.huoshanense stem,followed by the leaf,then the flower,with lowest expression in root.Meanwhile,D.huoshanense GMPP gene expression levels were found to be highly correlated with its polysaccharide contents in stem,leaf,flower and root.The research will play a positive role in promoting D.huoshanense polysaccharide biosynthetic research in the future.Conclusion: Through the optimization study of D.huoshanense RNA extraction method,a simple,economic and efficient total RNA extraction approach,namely MCHAN method was discovered.This solved the D.huoshanense RNA extraction problem due to its high polysaccharide and polyphenol contents,and thus provided a solid foundation for subsequent transcriptome sequencing.Transcriptomic data resulted from 17 D.huoshanense cDNA libraries showed an overall landscape regarding gene information as well as specific expression profile in different organs which would facilitate the study on biosynthetic mechanism of meaningful secondary metabolites.Cloning and constructing recombinant expression vector of D.huoshanense GMPP gene provided the basis for further function validation of GMPP gene.