| Rice(Oryza sativa) is one of the important food crops in the world. However, stable and high rice yields were restricted severely by rice diseases such as rice blast. Breeding new varieties of rice with blast resistance especially durable resistance has become one of the main ways to control the devastating disease. The durable resistant pi21allele, which is found in some strains of japonica rice, could improve blast resistance of rice worldwide. Pi21-RNAi transgenic rice lines and Nipponbare were employed as materials in this study. Using high-throughput RNA-Seq technology, the interaction between rice and Magnaporthe grisea was surveyed after materials were inoculated by M. grisea isolates. The main results were obtained as follows:1. In this study, Pi21-RNAi transgenic rice line241(resistance) and Nipponbare (susceptible and as controls) were inoculated by two M. grisea isolates (TMC-1and GUY11), respectively. Rice leaves were sampled from transgenic line and control at five time points (0h,12h,24h,48h,72h) post-inoculation, and thus20samples were totally obtained. Semi-quantity RT-PCR analysis of pathogen-related genes (such as WRKY TF genes, ABC transporter genes, protein Kinase genes, MYB TF genes and pathogen-related genes) in isolate-induced materials showed that gene expression differences occurred between resistance and susceptible materials, and that similar changes of gene expression level were seen in the materials infected by TMC-1and GUY11, respectively. Those results above indicated that good performances were achieved in the inoculation experiment and the samples created here could represent the true interaction relationship between pi21and M. grisea.2.20infected samples were sequenced by high-throughput RNA-Seq producing about217million Clean Reads, which accounted for97.41%of Raw Reads. The number of Clean Reads in each sample was over10M and the nucleotides in each sample over500Mb.Clean reads were mapped to reference sequences of the rice genome of Nipponbare using SOAPaligner/SOAP2. No more than2mismatches were allowed in the alignment. The results showed that clean reads mapped to genome reached more than86.97%in17samples, while the percentage was lower in the rest three samples, TMC-Nip-72h (75.85%), GUY-Nip-72h (68.54%) and TMC-241-72h (75.19%). Also, consistent mapping results were achieved when clean reads mapped to reference gene set by the same alignment algorithm. Analysis of sequencing saturation showed that when the number of clean reads reached more than10M, the growth curve of detected genes flattened, indicating that the number of detected genes tended to saturation. In addition, the distribution of reads on the reference genes from5’ to3’ were approximately evenly, showing that mRNAs were broken into short segments well randomly. All sequencing quality assessments mentioned above gave a good score to the data obtained by RNA-Seq.3. The gene expression level of43222genes in each sample was calculated by using RPKM method, and the average RPKM of all genes in each sample ranged from15.2to16.9, indicating that there was no difference in the total mRNA transcript level between all20samples. Screening of differentially expressed genes (DEGs) was conducted by using "FDR≤0.001and the absolute value of log2Ratio≥1" as the threshold to judge the significance of gene expression difference, and the DEGs laid a solid foundation for genes related to pi21resistance to be screened next.4. The putative genes interacted with pi21were screened through the whole genome by setting a series of screening conditions for differentially expressed genes. By semi-quantity RT-PCR and qPCR assays, We preliminary made a conclusion that OsPIG1may play an important role in no-species specificity resistance mediated by pi21. Further research on OsPIG1may give us a clue that leads to understand the resistance mechanism of pi21. |
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