| Rehmannia glutinosa(ehmannia glutinosa Libosch.)is a large quantity of traditional Chinese medicine and perennial herbaceous plant,classified under the family Scrophulariaceae.The species has been extensively cultivated for a long history.Because of its higher production and superior medical value,R glutinosa,as one of Four Famous Huai Medicines,sells well both in the domestic market and abroad.However,some problems need to be addressed in breading and production of R.glutinosa.Firstly,unclearness of genetic background and species deterioration has seriously restricted the selection and cultivation of fine varieties of R.glutinosa.Secondly,tuberous root of R.glutinosa is the most crucial medicinal parts,the yield and quality of which directly determined medical ingredient and economic values,but the molecular mechanisms of formation and enlargement of tuberous root remained unclear.Thirdly,the developments of tuberous root were complexly regulated,in which miRNAs played an important regulator,but the mechanism of miRNA-mediated gene regulation have never investigated during the process of tuberous formation of R.glutinosa.To address these issues,R.glutinosa cultivar "Wen 85-5" was planted as an experimental material,and by performing Solexa/Illumina sequencing technology we established the high-capacity transcriptomes of R.glutinosa.With EST-SSR teanology,we screened polymorphic molecular markers and analysed genetic relationships of germplasm.The tuberous(P1)and fibrous root(P2)were collected at the earlier tuberous root expansion stage of R glutinosa,respectively.With Digital Gene Expression Profiling(DGE)analysis and iTRAQ quantitative proteomics,we analyzed different expression genes and proteins from PI and P2,collected candidates for tuberous formation-related genes and proteins.We compared differential miRNAs expression in P1 and P2 of R.glutinosa plants,and identified specific miRNAs and their targets which are closely related to R.gludtinosa tuberous root formation by degradome sequencing.The main results are as follows:1.This study constructs a high-capacity transcriptome library.Based on bioinformatics analysis with assembler(SOAP software),99,708 transcriptomic sequences in roots,94,544 in leaves and 94,479 in root and leaves were obtained.On this basis,transcriptome library of R.glutinosa tuberous root by 454,all R.glutinosa EST sequences by traditional Sanger sequencing technology and transcriptome library constructed in this study by Solexa sequencing technology were assembled together by a cross-platform joint optimization,a total of 87,665 transcripts were obtained.Furthermore,50,653 CDS(coding sequences)of R.glutinosa were found by the Blast and ESTscan analysis.The result of 87,665 transcriptome sequences annotated by Nr and Swissprot date showed that nearly 40 thousand sequences had homologous with other species.54,321 sequences were annotated by AmiGO.Based on KEGG analysis,21,138 sequences were involved in 119 metabolic pathways.On the basis of RepBase analysis,564 retrotransposons sequences were collected from R.glutinosa transcriptome,and we considered that active expression of these retrotransposons may be one of the major causes of unstable genetic variation of vegetative propagule generation.In this study,we firstly acquired the greatest quality,the minimum redundancy and the largest flux transcriptome library on R glutinosa,and provided an important information platform for the medicinal components synthetization and growth mechanism of R.glutinosa.2.This research constructed an amplification system and polymorphism verification system for R glutinosa EST-SSR molecular markers.We exploited 1,018 EST-SSR locus that were equal or more than 18 bp using MIS A software,of which 300 EST-SSR markers(>20 bp)were selected to confirm their authenticity in R,glutinosa by PCR,and the result showed there were 180 locus were amplified steadily with expectant bands.We had widely collected 36 R.glutinosa germplasm,of which 12 species were selected to identify polymorphism in 180 available SSR locus,and the results showed that 80 polymorphic EST-SSR markers with a higher polymorphism.Genetic relationships among 36 R.glutinosa species were analyzed by randomly selecting 5 polymorphic EST-SSR which had been shown a high polymorphism.Our studies revealed that genetic distance between cultivated and wild species of R.glutinosa were longer,and complicated relationship between cultivated species were detected,the same to wild species.In the present study,we firstly identified generic relationship among different R.glutinosa species by SSR molecular marker,because the result is significantly superior to other molecular markers in precision and resolution of identification.The polymorphic EST-SSR selected in this study prepared important molecular basis for genetic relationship identification and cluster analysis between R.glutinosa germplasm resources,and provided an important foundation for molecular assisted breeding methods in breeding many new higher heterosis R.glutinosa cultivars.3.We constructed DGE libraries and iTRAQ library from earlier tuberous root(P1)and fibrous root(P2)of R.glutinosa root,respectively.We analyzed differently expressed genes(DEG)profiles and differently expressed proteins(DEP)profiles from initiated tuberous root(PI)and fibrous root(P2),collecting candidate genes and proteins for tuberous root formation and development,isobaric tags of relative and absolute quantization(iTRAQ).The results showed that 1,556 differently expressed genes(DEG)and 288 differently expressed proteins(DEP)were obtained from P1 and P2,which involved in metabolism,transcription,protein synthesis,transport and other cellular process.By analysis DEG and DEP dates,we found that R,glutinosa tuberous root development were controlled by different hormones,of which IAA,CK,ABA promoted the formation and development of tuberous root,but GA inhibited the transition of fibrous root to tuberous root.IAA and CK could make a contribution for rapid swelling of tuberous root by prompting cell division and expansion,in which uniquitin may play key roles.We hypothesize that Ca2+ not only participated in formation and development of R.glutinosa tuberous root,but light signal.4.This study for R.glutinosa sRNAomes from P1 and P2 of R.glutinosa plants,respectively.Based on R glutinosa transciptome data,513 miRNAs families were identified.By contrasted the miRNA different profiles of P1 and P2,145 differentially expressed miRNAs were obtained.Moreover,dengradome sequence was performed to identify target genes of these differently expressed miRNAs basing on mixed sample of P1 and P2,and the results showed 36 reliable targets were obtained.Through detail functional analysis of these targets,we find that miR160,miR167,miR165,miR5153 and miR5225 were significantly up-regulated in fibrous root(P2),but they inhibited fibrous further enlargement by degrading their targets,of which miR160 and miR167 inhibited hormone response of fibrous root by degrading auxin response factor(ARF)mRNA,miR165 prevented proliferation of cambium cells of fibrous by degrading HD-ZIP III mRNA which has important role in cell division,miR5153 could not make cell maintain the function of division by reducing the expressing of NAP1 gene,miR5225 affected calcium signaling by degrading ACA(autoinhibited calcium ATPase)gene.By contrast,miR171 was up-regulated in tuberous root,and it prevented tuberous root producing lateral root and recognizing GA signal by inhibiting the expression of SCL gene.We hypothesize that miRNAs may play essential roles in root development by controlling some targeted genes expression that was closely related to root architecture.In a word,under lacking the genome information and genetic resources of Rehmannia,we constructed the transcriptome library of R.glutinosa by using Solexa sequencing technology,and optimally assembled the greatest quality and largest flux transcriptome library sequences on R.glutinosa combining with different sequencing platforms for the first time.In addition,our study identified the first R.glutinosa EST-SSR markers by performing biological method and we acquired the polymorphism marker library of R.glutinosa by using the existing varieties.Then clustering analysis was firstly performed between the existing main cultivated and wild R glutinosa gemplasm resources.The results will elaborate the genetic relationships among the germplasm resources of R.glutinosa.Accelerating the application of SSR molecular markers and combining ability testing laid the groundwork for breeding R glutinosa varieties with higher heterotic,high-yielding and high quality.At the same time,analysis of differently expressed genes,differently expression protein and miRNAs profiles were to obtain candidates genes and specific miRNAs during R.glutinosa tuberous formation.Under different layers and angles,an entirely proof come to light that environmental factor induced Ca2+ signaling and the balance of the metabolism of different hormone was broke,and then enlargement of R glutinosa root cell was activated.In addition,specific miRNAs of tuberous root and fibrous root can decide the direction of root development.These works lay a solid foundation for parsing molecular mechanism of R.glutinosa development and synthesis mechanism of medicinal ingredients,... |
PDF Full Text Request