The Screening And Functional Analysis Of IncRNAs Responding To Consecutive Monoculture Stress In Roots Of Rehmannia Glutinosa L.

Rehmannia glutinosa L.belonging to perennial herbaceous plants of scrophulariaceae,well known as one of the famous Four Huai Medicine,has a long cultivation history and high medicinal value.However,R.glutinosa has serious consecutive monoculture problems in production,which seriously affected its production and quality.Currently,it has become a difficult problem to be solved urgently for the sustainable development of Chinese herbal medicine.Previous studies found that some special lncRNAs(long non-coding RNA),which transcribed like mRNAs(mlncRNAs)are involved in the perception and response to consecutive monoculture stress in R.glutinosa.However,majority of lncRNAs are transcribed through transcripts with "non-polyA" during growth and development of plants.Conventional high-throughput sequencing methods have no thus ability that captured lncRNAs.To more completely explore the role of lncRNAs in the formation of consecutive monoculture problems and elucidate the corresponding molecular regulation network,in this study root transcript libraries without polyA(non-polyA)derived from normal growth(first year planting,FP)and consecutive monoculture(second year planting,SP)R.glutinosa were constructed to identify the candidate lncRNAs of R.glutinosa.Simultaneously,their categories and roles in R.glutinosa were in detail analyzed.The full length of representative lncRNAs transcripts and their promoter sequences were cloned by RACE and genome walking technology to confirm the reality of lncRNAs in R.glutinosa.Based on above,the differential expression profile of IncRNAs in FP and SP were constructed to screen the specific lncRNAs responding to consecutive monoculture practice.The expression patterns of lncRNAs and target genes that were further identified and analyzed through qRT-PCR.The detail analysis of above data on different levels revealed that IncRNAs played a important role in consecutive monoculture formation.The main results are as follows:1.Construction of "non-polyA" transcript libraries for FP and SP.Transcripts with polyA and rRNAs were removed from total transcripts,and only transcripts without polyA were retained.On basis of the"non-polyA" libraries was constructed by high throughput sequencing technology.As results,the two libraries generated 34,427,634 and 47,558,986 raw reads,respectively.Reads from two libraries were assembly together by Trinity software,leading to produce 88,951 transcript sequences with the average length of 594 bp.To remove coding sequences in "non-polyA" transcript library,the potential CDS region located in all transcripts were predicted using BLASTX and ESTscan software,and 25,944 sequences were identified containing obvious coding region or partial region.At the same time,a training datasets that consist of the coding sequences from R.glutinosa mRNA transcripts were used to identify the possible coding sequences presented lncRNAs,finally leading to removing of 6,302 potential coding sequences.In addition,BLASTN software was used to identify housekeeping ncRNAs based on Rfam database,and 1,179 sequences have similar alignment with ncRNAs in Rfam databases.After removing sequences with coding region and housekeeping ncRNAs,a set of 55,526 transcripts were finally remained to be as R.glutinosa candidate IncRNAs.The construction of R.glutinosa lnRNAs candidate sequences sets provides important basis for the roles of lncRNAs in R.glutinosa.2.IncRNAs classification and function identification in R.glutinosa.Based on the present genetic information in R.glutinosa including mRNA transcripts and sRNAs degradome,R.glutinosa lncRNAs were preliminarily classified according to their functions by the bioinformatics methods.Meanwhile,the functions of lncRNAs were identified on the post-transcriptional level.According to lncRNAs functional roles that regulated the target genes,R.glutinosa lncRNAs were divided into 3 types.(1)317 IncRNAs were identified as the precursors of miRNAs(miRNAs-derived lncRNAs)by using miREAP and related bioinformatics software.(2)Targets with coding or non-coding of the conserved miRNAs of R.glutinosa were identified based on mRNAs coding transcriptome and lncRNAs database,respectively.155 lncRNAs were identified as competitive lncRNAs(If mRNAs and lncRNAs can be the targets by same miRNAs,and this lncRNAs were called as competitive lncRNAs).(3)Through analyzing the free energy of lncRNAs and target genes,12,961 lncRNAs were identified as directly regulated lncRNAs.As R.glutinosa genome is limited,remaining 42,093 transcrips were considered as other types of lncRNAs,whose function still not unknown till this moment.Based on the previous degradome and transcriptome,92 miRNAs-derived lncRNAs were identified to target towards 135 genes.At the same time,114 lncRNAs were found to have obvious competition with 214 target genes for 95 miRNAs.In addition,12,961 mRNAs genes were identified to be directly regulated by lncRNAs.The functional analysis of target genes for lncRNAs showed that lncRNAs involved the multiple celluar process of R.glutinosa development.3.Cloning of full-length transcripts and corresponding genes sequences for representative lncRNAs.To confirm the reality of candidate lncRNAs and functional characteristics,the full-length of 8 lncRNAs transcripts randomly selected from lncRNAs candidate sets were obtained by RACE technology.ORF finder software was used to predict coding regions of these full length sequences for further confirming property of lncRNAs without coding regions.The results indicated that the ORF lengths of the lncRNAs were all less than 300 bp,which satisfied the typical characteristics of non-coding RNA.To analyze the promoter of lncRNAs and further explore the regulation of lncRNAs transcription,the flanking region of four lncRNAs sequences were extended through genome walking technology,and four lncRNAs of full-length were obtained.On basis of this,the promoter elements of four lncRNAs were predicted.The results indicated promoters of lncRNAs contained stress-related elements including AP2、SP1、TFIID and so on.After the comparison between transcripts and genes,we found that there was no intron presented in the four lncRNAs.The full length clone and analyses of four lncRNAs provide important bases for further revealing the functions of these lncRNAs on either transcript level or genome level.4.Identification and functional analysis of key lncRNAs responding to SP R.glutinosa.Through the differential expression analysis of lncRNAs in SP compared to FP in R.glutinosa,we obtained 764 differential expression genes.Of which,490 lncRNAs were significantly up-regulated and 274 lncRNAs were significantly down-regulated in SP compared to FP R.glutinosa.Specifically,there were 14 miRNAs-derived lncRNAs that included 11 up-regulated and 3 down-regulated in SP R.glutinosa.Moreover,32 competitive lncRNAs that differentially expressed in SP compared to FP R.glutinosa,consisting of 19 up-regulated and 13 down-regulated lncRNAs in SP.In addition,we identified 223 directly regulated lncRNAs were differentially expressed in SP compared to FP,including 160 up-regulated and 63 down-regulated lncRNAs.In order to confirm the regulated function of the differentially expressed lncRNAs,we analyzed 12 key lncRNAs and their targets regulatory patterns by qRT-PCR.The results showed that lncRNAs were deeply involved in the formation and generating of consecultive monoculture of R.glutinosa.In other words,the up-regulated lncRNAs in SP R.glutinosa promoted or inhibited the expression of key target mRNAs so that changed gene expression.The roots development were thus controlled and leaded to the formation of concecultive monoculture of R.glutinosa.In conclusion,this study firstly constructed R.glutinosa non-polyA transcript libraries,and identified the candidate lncRNAs sequence sets,which enriched the genetic information data of R.glutinosa.Moreover,this study screened key lncRNAs responding to consecutive monoculture practice,and investigated its possible roles in consecutive monoculture formation.More importantly,this study provides a important theoretical basis for revealing molecular mechanism of consecutive monoculture formation in R.glutinosa and developing effective technology to solve consectutive monoculture problems.