Establishment Of The R-PCR Method And Its Application

Removing PCR(R-PCR), based on the Polymerase Chain Reaction(PCR) and restriction enzyme, was developed for fishing out target genes from gene pool by eliminating undesired genes. Comparing with the existed methods which were developed for isolating specifically expressed genes, for example, subtractive suppression hybridization (SSH), microarray, and high-throughput next-generation sequencing technologies, R-PCR was easily manipulated, lowly cost but with less false positive, better repeatability, and it could be used more widely.Maize banded leaf and sheath blight(BLSB), mainly caused by Rhizoctonia solani K uhn, is a destructive disease that widely spread in the world. Isolation of the specifically expressed genes in maize, which was induced by Rhizoctonia solani Kuhn, is an efficient way to understanding the resistance mechanism of maize at the gene level and provide fundamental knowledge for breeding new varieties with efficient, broad-spectrum, stable resistance to the maize BLSB.In our research, we put forward the concept of R-PCR and try to provide a new scheme for screening target genes from gene pool by preliminarily build up the R-PCR system both at single gene level and gene population level. At the same time, we applied the R-PCR successfully into maize up-regulated genes isolation. All these results lay the foundation of constructing gene expression profiling of disease resistance in maize as well as studying the resistance mechanism against maize BLSB at global transcriptional level. We use maize inbreed lines Ye-478and high pathogenic fungus anastomosis groups AG-1-IA of Rhizoctonia solani Kuhn as the material. The up-regulated genes compared with the genes in healthy maize, in the mix of leaves which were obtained at6hrs,12hrs,24hrs,36hrs,48hrs,60hrs,72hrs of the post-inoculation, were fished out by R-PCR. Also the real-time PCR was used to verify the pathogen induced genes cloned from the R-PCR. The obtained main results were as follows: 1. The single gene level R-PCR system was established preliminarily by using the fragment of Triticum aestivum small GTP-binding protein gene as starting material, and it conformed that R-PCR did work as its principle.2. On the base of single gene level R-PCR system, we also built up the multiple gene level R-PCR system whose starting material was the genome DNA of Ye-478after further optimizing. Excellent elimination effect was obtained and it illustrated the feasibility and high efficiency of R-PCR.3. We used inoculated Ye-478cDNA and healthy Ye-478cDNA as the starting material, following by dealing with R-PCR procedure, and got the R-PCR product of up-regulated genes. These R-PCR products were cloned into pGEM-T Easy Vector and transformed into E. coli DH5a. In total,122clones were selected to sequence and51uni-EST sequences were obtained.4. All of the sequences of inserted fragments were analyzed by using bioinformatical methods. About41EST sequences have significant homology with known genes and the remaining12EST were unknown about its functions. According to the results of the others’, we found8genes were relative to resistance and defence. The rest need further research.5. In total,23EST were randomly chosen to design primers of real-time PCR following by relative quantifying these genes’ expression level between inoculated maize and healthy maize. The results indicated that14genes were evidently up-regulated and the other9genes were up-regulated less than2times.