Study On Preparation Of Prototype S-RNase Microarray And Molecule Hybridization Condition

Pear is a typical kind of gametophytic self-incompatibility plant, which is controlled by single S-RNase multiple alleles. Because of the low fruiting during the natural pollination, it is necessary to arrange varieties for pollination to increase the yield. So the identification of S-genotype of pear can provide the scientific foundation for its cultivating and breeding. Now in the world PCR-RFLP,DNA sequence, cDNA cloning and sequence analysis combined with crossing pollination testing, which are with large operations, high cost and low automatization, are used more often for S-genotyping. It's very important to establish a new method for detecting of S-genotype efficiently, directly and simply. The lake of reports on identification of S-genotype of pear by genechip impels us to research it by designing some oligonucleitide probes according to the structure characteristic of S-RNase, preparing the prototype oligonucleitide microarray, exploring the molecule hybridization condition and examining the genechip's characteristics. The main studies are following as:1. Primer "FTQQYQ"(P1) and "anti-IIWPNV"(P2) labeled by fluorescence Cy3 were used to amplify S-alleles fragments from the pear's genomic DNA by PCR. Cy3-labeled target sequence, which would be used to hybridize with the oligonucleitide microarray, were gained and purified.2. According to the Genebank information,relevant papers and the designing rules of oligonucleitide probes, seven 32mer oligonucleitide probes were designed and synthesized, every probe was modified by-NH2 at the 5-end.3.0.1M pH=9 carbonate buffer was used as the spotting solution and the oligonucleitide probes,whose optimal concentrations were adjusted in carbonate buffer to 100 ng/μL, were printed on a glutaraldehyde modified glass slide. So the oligonucleitide genechip for detecting S-genotype of pear was prepared. These genechips were hybridized with the amplified S-RNase fragment of pear and the effective hybridization fluorescence signals were obtained.4. The molecule hybridization conditions were optimized by exploration of hybridization temperature,hybridization time and hybridization concentration of PCR products. The results showed that the optimal hybridization temperature should be 47℃, optimal hybridization time should be 4h and the optimal concentration of PCR products should be 100 ng/μL.5. detecting known S -genotype pear varieties such as Huanghua,Guiguan,Huangjin,Xianhuang,and Dahebai by oligo microarray showed that the microarray results of S-genotype were similar with the RFLP and sequenced results. repeated hybridization showed the same results. It was estimated that the specificity and accuracy of the oligonucleitide microarray was good.In conclusion, the oligonucleotide microarray is an effective method in detecting S-genotype of pear. With the future improvement, it could widely used in the study of self-incompatibility of pear and other fruiter.