Differentially Expressed MicroRNAs In Sugarcane Challenged By Sporisorium Scitamineum And Their Target Gene Function Identification

Sugarcane (Saccharum officinarum) is the most important sugar crop and energy crop in China. It accounts for 92% of the total sugar production of China. Sugarcane smut caused by Sporisorium scitamineum(S. scitamineum), may cause serious loss to the sugarcane production which reduces sugar yield and sucrose content and creates a big enormous economic loss to the Chinese sugarcane industry. Cultivating smut resistant sugarcane varieties has been proved to be the most economic and effective measure to control this disease. The molecular mechanisms of plant and pathogen interaction are complex, including the transcription level, the post-transcriptional level, the translational level, the post-translational level, and even some other regulatory mechanisms. Along with the development of biotechnology, it has been a great progress on the research of resistant mechanism to sugarcane smut. However, there is not any report on the analysis of differentially expressed microRNAs (miRNAs) in sugarcane challenged by S. scitamineum using high-throughput sequencing technology and their target gene prediction and function identification. In the present study, we conducted comprehensive analysis and comparision on the regulatory mechanism of miRNAs between the compatible interactions and incompatible interactions of sugarcane-smut fungus at the post-transcriptional level. The research is from three aspects, which involves high-throughput sequencing of small RNA and miRNA annotation, differenent miRNAs screening and expression and expression pattern analysis by qRT-PCR, functional analysis of differenent expressed miRNAs and their target gene prediction and expression analysis. we hope that this study could provide evidence for sugarcane smut resistance mechanism at miRNA level, enrich and deepen the research on the correspondingmolecular mechanism, and also aim to set up the miRNA-level theory basis for the cultivation of smut-resistant sugarcane variety.miRNAs, which have important biological functions, play an important role in the regulation process of plants under biotic and abiotic stresses. Gene regulation at post-transcriptional level mediated by miRNAs is an important small RNA regulatory pathway in vivo. In this experiment, two sugarcane varieties, Yacheng 05-179 which is resistant to sugarcane smut and ROC22 which is susceptible to sugarcane smut, were used as the plant materials. Sugarcane buds 48 h after inoculation of S. Scitamineum was taken as treatment group and those treated by water as the control group. Fristly, we used high-throughput HiSeq sequencing technology to analyse the differentially expressed microRNAs in sugarcane challenged by S. Scitamineum at 48 h, then small RNA sequences were annotated and the differentially expressed miRNAs were screened. Secondly, using bioinformatics technology and the database, we predicted the target genes of these miRNAs and conducted the GO and KEGG enrichment function of these target genes. Finally, the expression of parts of miRNAs and their targeted genes were validated by qRT-PCR and the expression patterns of several target genes was further analyzed at different time points of 0 h,12 h,48 h,96 h. The aim of this study was to find the key miRNAs and their target genes in sugarcane challenged by S. Scitamineum, and provide miRNA and gene resources for smut resistance molecular breeding. The main results are as follows:1. The quality of four sugarcane samples was good. The result showed that the clean sequences of RCK, RT, YACK and YAT were36396588,27812972,27464468 and28290231, respectively. The rate of impurity sequence was smaller than 1%, indicating the quality of small RNA obtained by high throughput sequencing was very high. Combined with multiple databases, clean sRNAs sequences were screened and annotated. The results showned that the number of known miRNAs in RCK, RT, YACK and YAT was each 264,263,260 and 262, respectively, while the number of novel miRNAs in RCK, RT, YACK and YAT was 137,140,111 and 119 respectively. In addition, the statistical analysis of base distribution of novel miRNAs showed that most of the first point bases of 21 nt and 22 nt in novel miRNA sequences that had more contents in plants were U, and eleven loci bases of candidate miRNAs sequence tended to be A. According to the miRNA sequence characteristics, the prediction results for novel miRNAs sequences was assured to be feasible.2. In ROC22, we identified 231 expressed known miRNA. Of them,35 known miRNA were significantly differentially expressed (|log2-ratio|> 1, P< 0.05) in between the treatment group treated by S. scitamineum and the control group treated by water. Among these 35 miRNAs,10 were expression up-regulated and 25 were expression down-regulated, and the highest difference ratio of expression quantity was up to 4.6 between control group and treatment group. In YA05-179, we identified 208 expressed known miRNA. Of them,11 known miRNA were significantly differentially expressed (|log2-ratio|>1, P<0.05) in between treatment and control group. Among these 11 miRNAs,2 were expression up-regulated and 9 were expression down-regulated, and the highest difference ratio of expression quantity was up to 2.1 between control group and treatment group. Besides, most of significantly differentially expressed know miRNAs in ROC22 and YA05-179 were the same with lower expression and the expressed trend of these miRNAs in the two varieties were also the same. Thermore, we identified 16 significant differentially expressed (|log2-ratio|>1, P<0.05) novel miRNAs in ROC22 samples, six in YA05-179 samples. Most of differentially expressed novel miRNAs were unique in a single sugarcane variety, and there was no novel miRNAs which was significantly differentially expressed in both varieties. In addition, qRT-PCR validation was conducted on the 16 differentially expressed miRNAs and their corresponding 27 expression values in high-throughput sequencing, and the results showed that the consistent rate reached 85.2%, indicating that the reliability of the sequencing results of this study was higher.3. Sugarcane is a highly heterozygous allopolyploid and aneuploid crop, and its whole genome sequencing has not been completed. Therefore, the annotation results of microRNAs showed that the majority of small RNA was unknown sequences. The date from further analysis of target genes of miRNAs was not completely in this case, and the annotation and analysis of unknown small RNA sequence need to be furthered. The predicted number of target genes of known miRNAs in YA05-179 was less than that in ROC22, but the number of differentially expressed novel miRNAs was higher in YA05-179 than in RO22. Analysising biological function of the target gene, GO analysis could be divided into biological process, cellular components and molecular function. In both varieties, the classification statistics results of the target genes of differentially expressed know miRNAs and differentially expressed novel miRNAs were the same. Target genes were mainly distributed in cell process and metabolic process in the biological process, and mainly distributed in the cell, cell components and organelles in cellular components, and mainly distributed in binding and catalytic activity in molecular function. KEGG pathway enrichment analysis showed that target genes regulated by differentialy expressed miRNAs involved in a series of physiological biochemical pathway or disease resistance related biochemical metabolism and signal transduction pathways, indicating that sugarcane-smut interaction mechanism was a complex network system. We found that, the target genes of miR5077, miR5671, miR5044, miR5261, miR5783, miR5221, miR6478 and miR948 play an important role in the MAPK signaling pathways, plant hormone signal transduction and plant pathogen interactions. Besides, we constructed the related protein network diagram.4. In this study, the miRNA target genes which had a sequence of biological annotation in sugarcane EST library were screened, and qRT-PCR expression analysis was conducted for parts of these target genes of known miRNAs and novel miRNAs. The results showed that the regulatory mode between most of the miRNAs and their target genes was negative, which means that when the expression of miRNAs was up-regulated, the expression of their target genes was down-regulated. Besides, the expression of 7 out of 8 target genes of these miRNAs was basically consistent after 12 h and had the highest matching degree at 48 h, which from the other side demonstrated that the regulation effect of these target gene of miRNAs reached the highest at 48 h after sugarcane smut challenging. In addition, this study revealed that the expression abundance prediction and function analysis of known miRNA and their corresponding target genes were highly reliable. However, for the novel candidate miRNA, the coincidence rate of the negative regulatory trend between target genes and novel miRNAs was not that high, and we still need to further excavate and improve the measures to forecast and analyse the novel miRNA.