Idenfication Of The Genes Specificly Responding To Consecutive Monoculture In The Roots Of Rehmannia Glutinosa L.

Rehmannia glutinosa L.,a perennial herbaceous plant of Sreophulariaceae,which is commonly used in medicinal materials in our country,has wide application in clinic.Therefore,it has large market demand.Nevertheless,the actual production of Rehmannia frequently received consecutive monoculture problems.Consecutive monoculture problems caused poor plant growth,yield and quality decreased obviously,and total crop failure,and moreover the inhibitive effect will sustain eight years.Consecutive monoculture problems have seriously affected the sustainable production of Rehmanniae.It becomes a major issue of Chinese local medicine agriculture and regional economic development.In order to further study the specific molecular mechanism of Rehmannia glutinosa consecutive monoculture problems emergence,this research set different abiotic stress(continuous cropping,drought,waterlogging stress,salt stress,exogenous phenolic acids(certain plant allelochemicals))processing,the first cropping as control,using digital gene expression profile update sequencing(RNA-SEQ)technology to detect differential gene expression,build Rehmannia digital gene expression patterns under different stress treatments.In this study,by eliminating the commoly expressed genes in different stresses,a Digital Gene Expression Profiling of Rehmannia genes specific response to consecutive monoculture was obtained.These genes was analyzed by Agri GO and be screened for specific molecular marker of candidate genes,Unigene33449_All was projected using the ORF Finder.Main results are as follows:281,603,980?286,921,117?297,077,935?294,993,230?289,115,190 and 301,565,012 Total Base Pairs were obtained in in the first harvest control(x1),continuous cropping(x2),ferulic acid(x3),salt stress(x4),drought and waterlogging stress(x6)samples using high-throughput sequencing,after the removal of impurities treatment,5747020 strip,5855533 strip,6062815 strip,6020270 strip,5900310 strip,and 6154388 strip clean reads were obtained,of which 96.74 to 98.81 clean reads.Adaptor sequence is the primary contaminant reads.More than 80% clean reads can be annotated according to the transcriptome.The uniqueness with unique match by clean reads(51.02%-54.56%)was far more than that with multi-position matched(29.28%-32.70%).The distribution of reads in each part of the gene was relatively uniform,m RNA was interrupted in very good level in the process of preparing library.So it suggested little self-degradation of m RNA and nonhomogenous fragmentation,the number of genes tend to be saturated.According to standards of P-value<0.005,FDR?0.001 and ?log2 Ratio?? 1 P-value<0.005 and FDR<0.01,2502,1956,2672,2672 and 7249 significant differentially expressed genes were screened out respectively from the first harvest control(x1),continuous cropping(x2),ferulic acid(x3),salt stress(x4),drought and waterlogging stress(x6)samples.Gene differential expression profiling was built in Rehmannia under different stress treatments.The data were being analyzed using GO function classification and KEGG pathway analysisGO and KEGG significant enrichment analysis showed that all of five stress treatments affected many of the same or similar physiological and biochemical processes in the the Rehmannia body,the genes response to the stress in the GO enrichment term and KEGG pathways had obvious overlap,it verified that the networks in Rehmannia responding to adversity stress is crosstalking.Some genes were down-regulated in Rehmannia glutinosa under stressed,such as the genes related to primary metabolism(nucleic acid synthesis,protein synthesis,synthetic sugars)and secondary metabolism(alkaloid synthesis),and then the activity of transcellular pathway and transduction was affected.It caused chaos of regulation and supervision of biological processes,biological processes of photosynthesis,the growth and development,and likely damaged in plastid,thylakoid,membrane organelles and mitochondria.Of course,it also activated plant response mechanism to strees.By eliminating the jointly expressed genes in different stress common and leaving the genes pecifically expressed in consecutive monoculture problems,510 significantly differentially expressed genes were identified.These genes were analyzed by Agri GO.According to standards of FDR?0.001 and ?log2Ratio??4,and refer to the analysis results of Nr,GO and KEGG function,57 specific molecular marker of candidate genes were identified,of which 35 was raised in continuous cropping and 22 was reduced.Two molecular markers specific in continuous cropping obstacle,CL4723.Contig2_All and Unigene33449_All,were identified by q RT-PCR.Unigene33449_All was taken ORF projections Using ORF Finder,as a result,got two ORF area,and its deduced amino acid sequence.According to the amino acid sequence,Unigene33449_All was predicted using NCBI CCD tools,it might belong to the SDR protein family,has NADP activity.Unigene33449_All was predicted using blastp,it also might had a higher similarity with multidrug MFS transporter.Two prediction results suggested that Unigene33449_All Combined with NAD(P)to obtain energy,then,It involved in the biological process of extracellular active transportas a transport protein.