| Abstract:Due to its efficacy in regulating immune, anti-tumor, anti-aging and hpyerglycemic, Rehmannia glutinosa L. is a very important crop of Chinese herbal medicines. Its production of roots and tubers, however, is suffering from continuous cropping obstacles seriously. At the same fields, its growth may return to normal after an interval of 8-10 years, this is dramatically hampered the production of R. glutinosa. The mechanism underline R. glutinosa continuous cropping obstacles are still unknown. To study on the function of the non-coding RNA in continuous cropping response, by using high-throughput sequencing technology, we constructed the transcriptome libraries of R. glutinosa, cultivar "Wen 85-5". From public databases we download all R. glutinosa EST sequence(NCBI), and integrated different database from different sequencing platforms, and produced a comprehensive transcriptome. Based on the transcriptome, we screened and identified a set of mRNA-like non-coding RNAs (mlncRNAs). By using DGE(Digital Gene Expression Profiling), we found that some mlncRNAs were differentially expressed in continuous cropping plants. Analysis of these mlncRNAs the temporal and spatial expression by qRT-PCR, showed that mlncRNAs were involved in the response to continuous cropping R. glutinosa. The main results are as follows:1. Construction of the high-capacity R. glutinosa transcriptome. By using Solexa sequencing technology we obtained 99,708 transcriptome sequences from the root, 94,544 from the leaves. Splicing the root and leaf transcriptome sequences we obtained 87,665 "All-Unigenes". Based on this, jointing optimization assembled EST sequences from NCBI(including Sanger and 454 sequencing data), going through a series of redundant processing, low-abundance fragments, we produced a total of 87,665 transcripts(Reh-Unigenes), the longest fragments up to 8,009 bp and the shortest fragment 204 bp, N50 reach 635 bp. In the case of the R. glutinosa genome unknown, this study constructed firstly a R. glutinosa transcriptiome library with the highest quality, lowest redundancy and high-through. This is a useful information platform for many studies of R. glutinosa such as medicinal ingredients, the mechanism of growth and development, responsive mechanism to biotic and abiotic including continuous cropping obstacles.2. Identification of R. glutinosa non-coding RNA (mRNA-like the RNA mlncRNA). By using bioinformatics methods, from Reh-Unigene transcriptome library we screened more than 10,000 mlncRNA gene sequences, the longest sequence of 2,417 bp,118 sequences more than 1,000 bp. Detail analysis found that there were 7 conservative mlncRNAs,48 housekeeping non-coding RNAs. The remaining sequences belonged to the R. glutinosa-specific mlncRNA. Family analysis showed that mlncRNAs were shortage of conservative property between different genes, beyond of coding capacity of mRNA family. The identification of Reh-mlncRNAs paves the way to reveal its’possible function in responding to continuous cropping obstacles in R. glutinosa.3. Different expression of Reh-mlncRNAs in first planting and replanting plants. To study different expression of Reh-mlncRNAs, we constructed 4 DGE libraries of first planting(1) and replanting (2) roots(R) and leaves(L), i.e. R1, R2, L1 and L2. Based on the DGE libraries, differently expressed mlncRNAs between first planting and replanting R. glutinosa were screened. The results showed that in leaves, the express up-regulated mlncRNA genes were 144 in the replanting and down-regulated were 161. Meanwhile in roots, genes up-regulated were 330 and down-regulated 164 mlncRNA genes. A large number of mlncRNA gene express levels were changed dramatically in the replanting, suggesting that mlncRNAs play an important role in forming continuous cropping obstacles of R. glutinosa.4. The temporal and spatial expression of the key mlncRNAs in response to continuous cropping. In order to detail expression difference of specific Reh-mlncRNAs in the replanted R. glutinosa,14 genes of mlncRNA were chosen and their temporal and spatial expression patterns were analyzed by qRT-PCR. Results indicated that there were three different expression patterns in the first cropped and replanted R. glutinosa. A), mlncRNAs expression exhibited significant higher in first cropping than in replanting during the entire development process. B), only during the roots elongation and expansion stages, mlncRNAs expression were significantly higher in replanted plants. C), there were no significant difference in the each period of growth, but this kind of regulation mechanism could be more complicated. Three kinds of expression patterns have a significant common characteristic, i.e. all of the peak value of expression patterns appeared root expansion period. In addition to this, type B accounted for most proportion of mlncRNA genes. The most of expression patterns could be consistent with the process of continuous cropping obstacle. The result indicated that mlncRNAs took part in the formation of continuous cropping obstacle of the R. glutinosa.New ncRNA discovery and function analysis are the hotspot of modern life science, and mlncRNA belongs to a new type of non-coding RNA. Here by constructing high-capacity R. glutinosa transcriptome, and using bioinformatics method, we identify firstly R. glutinosa mlncRNAs. Furthermore, by DGE and qRT-PCR we analyze differences of mlncRNAs expression in continuous cropping obstacles. We suggest that mlncRNAs play an important role in forming continuous cropping obstacles. The ncRNAs accounted for most of the transcriptome, several times higher than the number of messenger RNA (mRNA), but about its function is little known. This is a preliminary study to cognize them and reveal their function. |
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