Studies On The Resistance And Its Mechanisms Of Pepper To Phytophthora Capsici

Pepper (Capsicum annuum L) which was widely planted around the world is an important economic vegetable crop in our country, while the pepper blight caused by Phytophthora capsici has been epidemic in many provinces and areas and has become the major limiting factor of pepper production. In this research, the evaluation technique of pepper resistance to Phytophthora capsici was development, the resistant mechanisms in physiology and biochemistry was investigated, and the difference in gene expression profile of Capsicum annuum infected by different physiological races of Phytophthora capsici was analyzed by cDNA-AFLP. After its full length was cloned via RACE technology, the sequence and structure characters of positive cDNA and corresponding protein were analyzed using bioinformatics, including the secondary structure , transmembrane zones, signal peptide and evolution pattern. The expression and regulation of these were analyzed by semi-quantitative RT-PCR and real-time quantitative techniques. The aim of this study is to reveal the resistant mechanism of pepper to P. capsici and establish basis for breeding of new pepper cultivar with resistance. The main results of our research are showed as follows:1. First, in vitro evaluation technique with lateral shoots of pepper resistance to Phytophthora capsici using CM334 with high resistance, N3 with moderate resistance and EC with sensitiveness. When the lateral shoots from the first branch after the second branch shot were inoculated with P. capsici, the difference of disease symptom between resistant and sensitive cultivars showed mostly significant under zoospore concentration with 1×104mL-1, temperature with 28℃, light intensity with 4000lx and photoperiod with 12h/d. Compared to in vitro leaf-inoculation, stem-inoculation and root-irrigating method, the evaluation results of 34 pepper materials using this technique showed marked positive correlation with these three common methods, and the correlation coefficient were0.9150** , 0.8730** and 0.8384**. These results suggested that the new technique could effectively evaluate the disease resistance of pepper to P. capsici. 2. The changes of protective enzymes'activities were studied. The results showed that the activities of peroxidase (POD) were significantly higher in resistant cultivars than those in susceptible ones, though activities increased both in resistant and susceptible cultivars after inoculation. Results analysis showed that there was a positive correlation between the activities of phenylalanine ammonia lyase (PAL),polyphenol oxidase (PPO) ,β-1,3-Glucanase and chitinase and the pepper resistance to Phytophthora capsici.3. 80 ESTs of different expression gene were got from pepper tread with Phytophthora capsici. 22 ESTs were submitted to GenBank, whose accessed number are as follows: GO496263,GO496264,GO496265,GO496266,GO496267,GO496268, GO496269,GO496270,GO496271,GO496272,GO496273,GO496274,GO496275,GO496276,GO496277,GO496278,GO496279,GO496280,GO496281,GO496282,GO496283,GO496284.4. Six TDFs were selected and got full-length cDNA, they are CanPOD(FJ5961 78), CanZf(FJ596179), CanTF(FJ617518), CanNADPH(FJ617519),CanBPM4(FJ617 520), CanOBP(FJ617521). Bioinformatics analysis showed that these six genes all have a complete ORF.The deduced amino acid sequence of CanPOD and CanOBP were have typical trasmembrance protein and CanPOD protein has a typical singal peptide.Phylogenetic analysis showed taht CanNADPH had close relationship with Ricinus communisi NADPH:quinone oxidoreductase(EEF49550.1), CanZf had close relationship with oryza sativa Japonica zinc finger protein(Os03g0788800), the sequence of CanOBP is conservetive, it was conjectured CanOBP gene related to the interaction between pepper and Phytophthora capsici.5. CanPOD mRNA expression were analyzed by semi-quantitative RT-PCR. The results show that intensive expression of CanPOD gene was induced by Phytophthora capsici. Different dynamic changes in the expression of CanPOD gene of incompatible intera -ctions and compatible interactions, which of incompatible interactions the CanPOD was expressed early and quickly ,which of compatible interactions, the up-regulated time of this gene is long, from 8h to72h6. The expression of CanZf and CanOBP were detected by real-time quantitative PCR. After treat with different physiological races of Phytophthora capsici, the CanOBP gene is up-regulated expression in A3's root of incompatible interactions and compatible interactions. There are two expression peaks of incompatible interactions which were at 2h and 36h; but there is only one expression peak of compatible interactions, which was at 8h. The expression is also different between root and leaf after inoculation with ph3. The expression maximal peak (at 12h) in leaves is later than that in root, but it particularly higher than that in root. Furthermore, low temperature(at4℃), drought(400mmol/Lmannitol), high salt concentration(400mmol/LNaCl) could lead to up-regulated expression of CanOBP, and CanOBP is inhibited by SA(5 mmol/L), MeJA(50mmol/L) and H202(10mmol/L).The expression of CanZf is also up-regulated after treat with different physiological races of Phytophthora capsici. In incompatible interactions, the expression is 63 times at 36h contrast with CK, and in compatible interactions, the expression is 81 times at 8h contrast with CK. In leaves, the expression of CanZf is similar to CanOBP. Low temperature (at 4℃) and drought(400mmol/Lmannitol) could induced to up-regulated expression of CanZf, and H202.(10mmol/L) could suppressed expression of CanZf. After the expression of CanZf inhibition early which treated with SA (5mmol/L), MeJA (50mmol/L) and high salt concentration (400mmol/LNaCl), it is up-regulated expression later.The results indicated that CanOBP and CanZf play important roles in pepper resistant to Phytophthora capsici, and it can responded to many stress.7. Establishment of high efficient pepper haploid cultivation system. Genotype is a limit factor for pepper haploid cultivation. In this article, anther culture of 22 genotypes were tested, the embryoes could induced from 13 genotypes.The best culture medium is 1 mg·L-1 6-BA for P51 and that is 4 mg·L-1 NAA +1 mg·L-1 6-BA for P53. Changing temperature is best for embryos induced, first 4℃,3d,and then 35℃,4d. We created a method of embryoid obtainment from induced microspore of pepper, and have access to national patent.