Cloning And Analysis Of Resistance-Related Genes To Phytophthora Capsici

Phytophthora capsici is a worldwide fungal disease of pepper. It has occurred for more than 60 years. Recently,it occurred in many provinces and cities,and tend to be much heavier year by year. There are many transmission routes for Phytophthora capsici. Breeding for disease resistant varieties is the most effective method of preventing or decreasing the harm of the Phytophthora capsici.So the main pepper production countries all take it as one of the most important target during the breeding work.This study used different resistant pepper varieties which were inoculated with Phytophthora capsici at six-leaf stage.Leaves were collected at 0 hour, 12 hours, and 24 hours after inoculation respectively. The different genes expression after inoculation were analyzed by DDRT-PCR.Finally, we gained the full length sequence of cDNA by RACE.The main results were as follows:1. The DDRT-PCR system that can be applied to show the mRNA differences of Phytophthora capsici in pepper was established according to gradient experiment.The first strand cDNA was prepared from 2.0μg of total RNA.cDNA products were amplified using combinations of anchored and arbitrary 10-mer primer of volume 25μL in total: cDNA 1.0μL , anchor primer 2.5μmol/L,random primer 2.5μmol/L,dNTP 250μmol/L,MgCl22.0mmol/L,10×Buffer 2.5μL,Taq DNA polymerase,1.0U.The PCR parameters were as follows:94℃for 3min;40 cycles of 94℃for 45s, 45℃for 2 min, 72℃for 1 min, and a final extension step 72℃for 8 min. The PCR products was separated on a 6% polyacrylamide gel and displayed by silver staining, cDNA bands on gels could be clearly observed.2. We studied the differential display of genes resistant to Phytophthora Capsici using DDRT-PCR, and finally seven differential gene fragments were obtained: T12C/B0312-251, T12C/B0312-248, T12G/B0312-339, T12C/B0307-170, T12G/B0317-374, T12A/B0320-235, T12A/B0320-195.3. The BLAST results show that T12C/B0312-251(GenBank accession No.EU280142) is homologous to Nicotiana glauca partial mRNA for putative ML domain protein (ml gene) at 90%.T12C/B0312-248(GenBank accession No.EU280143) is homologous to Capsicum annuum ERD15 mRNA,complete cds at 99%. T12G/B0312-339(GenBank accession No.EU280144) is homologous to Lycopersicon esculentum clone 113946R mRNA sequence at 92%.T12C/B0307-170, T12G/B0317-374, T12A/B0320-235, T12A/B0320-195 is lowly homologous to known genes.They are proposed to be pepper-specific cDNA sequences.4. Two gene-specific primers were designed based on the differential fragment T12C/B0312-251. Two terminal sequences were obtained by 5'RACE and 3'RACE.According to the 57bp overlap sequence we cloned, a new gene of 799bp was assembled. This gene is homologous to ml gene analyzed by bioinformatic tools.