Transcriptome Analysis And Expression Identification Of Drought Induced Root-specific Gene In Common Wild Rice(Oryza Rufipogon Griff.)

The common wild rice (O. rufipogon Griff.) is considered to be the ancestor of Asian cultivated rice species, which contains many useful genes such as drought resistance, and indicates abundant genetic diversity. Drought is one of the most common abiotic stresses that influence plant growth, biomass production and crop yield negatively. When the plant was subject to drought stress, many genes that regulate metabolism at the physiological and biochemical level are highly expressed to enhance drought resistance.The roots of the plant is closely related to drought resistance. Discovering novel genes related to drought in common wild rice roots is one of the ways to rice genetic improvement. In this study, to study drought-related genes specifically expressed in roots, six cDNA libraries were generated from the seedling stage (shoot and root of control, root-treated) of common wild rice using Illumina deep sequencing. After removing adapters and filting the low-quality sequences,6.42million,5.74million, and4.59million clean100-bp reads were generated for the control shoot (CL1, CL2), control root (CR1, CR2), and drought-treated root (DR1, DR2) libraries respectively. We generated16.75million bases of high-quality sequence data for common wild rice and conducted de novo assembly and annotation of genes without prior genome information. These reads were assembled into119,332unigenes with an average length of715bp. A total of88,813distinct sequences (74.42%of unigenes) significantly matched known genes in the NCBI NT database; the majority of sequences (64.76%) were homologous to genes of Oryza sativa. A total of26,574potential simple sequence repeats (SSRs) were identified in20,586unigenes, and the Tri-nucleotide type was with the highest repeatability in the motif types.Differentially expressed gene (DEG) analysis showed that3617genes were up-regulated and4171genes were down-regulated in the CR library compared with the CL library. Among the DEGs,535genes were expressed in roots but not in shoots. A similar comparison between the DR and CR libraries showed that1393genes were up-regulated and315genes were down-regulated in the DR library compared with the CR library. To confirm the gene expression data,18up or down-regulated unigenes were randomly chosen from the three libraries for qRT-PCR analysis. For most of the18genes, their expression patterns in the real-time PCR analysis were similar to those predicted. The results show that the high throughput sequencing data obtained in this study is reliable.The present study found that the differentially expressed TF genes of belonged to a diverse range of TF families. Numerous transcription factors showed differential expression between the CR and CL libraries, including40MYB genes,31AP2/ERF genes,5NAC genes, and21WRKY genes, etc. These transcription factors may be related to tissue specificity. The comparison between the DR and CR libraries showed that4MYB genes,1NAC genes,7bZIP genes and2DREB genes were up-regulated in roots after drought stress. These transcription factors may be related to drought stress.After performing DEG analysis,37possible drought-related and tissue-specific genes were identified. We tested the expression patterns of four of the37differentially expression genes to testing the expression pattern in response to drought stress (15%PEG-6000) in root tissues. The transcription sequencing results showed that two of these four genes, comp83816_c0and comp60508_c0, gene were up regulated by drought, and the other two genes, comp78054_c0and comp88336_c0, were down-regulated by drought. In the qRT-PCR experiment, the transcription levels of the comp83816_c0and comp60508_c0genes gradually increased in response to drought stress at1to2days and then decreased at3days. The transcript level of the comp88336_c0gene gradually decreased in response to drought stress on days1to3, and the level of comp88336_c0quickly decreased in response to drought stress.In14of the535tissue-specific genes were analysed by RT-PCR. The results show that with the significantly specificity expression in root of seedling stage, comp84144_c0genes were not expressed in shoots of tillering stage and vegetative period.This study provides a transcriptome sequence resource for common wild rice plants and establishes a digital gene expression profile of wild rice plants under drought conditions using the assembled transcriptome data as a reference. These annotations provide valuable information for the investigation of specific processes, functions and pathways in the common wild rice. Several tissue-specific and drought-stress-related candidate genes were identified, representing a fully characterized transcriptome and providing a valuable resource for genetic and genomic studies in plants.