Construction Of R. Glutinosa Transcriptome And Identification Of Continuous Cropping Obstacle-related Key Genes And Specific MiRNAs

Rehmannia glutinosa (Rehmannia 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 sells well both in the domestic market and abroad and is one of famous Four Huai Medicines. However, the land must be given up for the other purpose for a period of8-10years after one season of R. glutinosa cultivation because the problem of continuous cropping obstacles is rarely serious, which has become an urgent problem to be resolved in agriculture production. Researches on continuous cropping of R. glutinosa are mainly focused on identifying the causative factors for this yield decline, the physiological activity and autotoxicity of the root exudates. At present, the molecular basis of the species’ sensitivity to its own exudate remains unclear. In our study, R. glutinosa cultivar "Wen85-5" was planted as an experimental material, at the same time, whole plants, roots and leaves from normal growth (the first year planting, FP) as control samples and continuous cropping (second year planting, SP) as treatments. The samples were collected at the earlier tuberous root expansion stage of R. glutinosa. Exploring Solexa/Illumina sequencing technology, we established the transcriptomes of plants (including roots and leaves), roots and leaves from continuous cropping R. glutinosa. With Digital Gene Expression Profiling (DGE) analysis and bioinformatics technology, we analyzed root and leaf different expression gene profiles from SP and FP, collected candidates for continuous cropping-related genes. We compared differential miRNAs expression in SP and FP of R. glutinosa plants, and identified specific miRNAs and its targets which are related to R. glutinosa continuous cropping obstacles. Expression levels of some candidate key genes and miRNAs were massured by qRT-PCR. This preliminarily unraveled the molecular mechanism underline R. glutinosa continuous cropping obstacles. The main results are as follows:At first, using Solexa/Illumina sequencing technology, more than40million raw reads were generated from both roots and leaves of mixed FP and SP R. glutinosa. Based on the bioinformatics analysis with assembler microsofts,99,708(Root, R),94,544(Leaf, L) and87,665(including roots and leaves) transcriptomic sequences were obtained. Furthermore,50,653CDS (coding sequences) of R. glutinosa were found by Blast and ESTscan analysis. Nr and Swissprot annotation of87,665transcriptome sequences showed that about that of40thousand (40%) had homologous with other species.48,227(55.01%) and54,321(61.97%) sequences were classified by COG and GO functional annotation, respectively. Based on KEGG analysis,21,138sequences were involved in119metabolic pathways. With unknown R. glutinosa genome, our study provide with useful technology and information platform to further reveal molecular mechanism of R. glutinosa continuous cropping obstacles. Secondly, more than5million clean tags were obtained from continuous cropping (R2) and normal growth (R1) of R. glutinosa roots by constructing tag libraries. DGE analysis revealed that the transcript profile of continuous cropping R. glutinosa was considerably changed, as indicated by the different significant2,817(1,676up-and1,141down-regulation) expression in R2. Nr, GO and KEGG analysis showed that114of these different expression genes played key roles in continuous cropping obstacle-responsive processes. Based on qRT-PCR, expression patterns of16candidate genes were analyzed. The results suggested that Ca2+and MAPK signal pathways were involved in sense, transduction and magnification of R. glutinosa allelochemicals, disturbing synthesis and transport of auxin and zeatin, DNA replication, RNA synthesis, nucleotide synthesis, protein synthesis and transport, respiration, various nutrition metabolisms and other key pathways. Thus this bascially outlined the molecular mechanism of continuous cropping harm in R. glutinosa.Thirdly, we constructed tag libraries from continuous cropping (the second year crop, L2) and normal growth (the first year crop, L1) of R. glutinosa leaves, respectively. More than5million clean tags were both obtained in two samples. DGE analysis screened1,954different significant genes. Nr, GO and KEGG analysis showed that115different expression candidate genes could be relevant to continuous cropping obstacles in R. glutinosa. Expression profiles of10key genes were analyzed by qRT-PCR. The results suggested that important metabolism pathways, which included DNA replication, RNA synthesis, protein synthesis, photosynthesis, respiration and so on, were interfered in continuous cropping R. glutinosa as repressing and closing a series of key gene expression. This gave rise to harm events of continuous cropping R. glutinosa.Fourthly, the study sequenced R. glutinosa sRNAomes from SP and FP of R. glutinosa plants, respectively. Based on R. glutinosa transciptome data,22novel and448conserved miRNA families were identified. We contrasted the miRNA different profiles of SP and FP libraries and screened312differentially expressed miRNAs (including10novel and302conserved miRNAs) which were predicted to target1,379genes. Nr, GO and KEGG analysis showed the potential targets of these differentially expressed miRNAs involved in the main transcription regulation, plant development, signal transduction, stress response and other biological processes.16different expression miRNA patterns were analyzed by qRT-PCR method. The results elucidated that, miRNA expression levels were changed in continuous cropping R. glutinosa, appearing in specific responsive miRNAs. Comparison with tag libraries of root (R2and R1) and leaf (L2and LI), we found that expression levels of targets were accordance with that of the corresponding different expression miRNAs, i.e. these miRNAs expression levels were higher while that of its targets were lower, and vice verse. Many targets regulated essential metabolism pathways.To sum up, with the shortage of R. glutinosa genetic backgrounds, we firstly constructed transcriptome libraries of plants, roots and leaves of continuous cropping R. glutinosa by next generation sequencing technology (Solexa/Illumina sequencing). At the same time, analysis of different expression genes and-miRNAs profiles obtained candidates of R. glutinosa continuous cropping obstacle-responded genes and specific miRNAs. The data from different research layers and angles, an entirely proof chain come to light that continuous cropping were sensed and conduced by Ca2+and MAPK signal pathways, in turn, specific miRNAs synthesis were directed, gene normal expression programs changed, key metabolism pathways repressed, the symptom (especially the roots) appeared. The study basically described regulated mechanism of continuous cropping obstacles in R. glutinosa. The present results lay the foundations for forming causes R. glutinosa continuous cropping obstacles. It is helpful for disclosing its molecular mechanism and exploiting effective technologies of subtracting its harm. Furthermore, our studies are of theoretical and practical significance for further explaining R. glutinosa growth and development mechanism. These also provide valuable references for researches on continuous cropping obstacles for other plants and for the sustainable development of Chinese medicine resource.