The Construction Of Transformation System Of Puccinia Striiformis F.sp Tritici

Wheat strip rust is an air-borne disease caused by Puccinia striiformis West. f.sp tritici, which is one of the most important wheat diseases in our country and all over the world. The studies and practices in China and the other countries showed that growing rust-resistant cultivars was the most economical, effective and feasible environment-friendly measure to manage wheat strip rust integrally. However, the virulence of wheat strip rust varied frequently and produced new virulent races which led to the breakdown of resistance genes and the periodical prevalence of wheat strip rust. Therefore, formulating the reasons and rules on virulence variation of wheat strip rust and putting forward effective countermeasures to prolong service life of resistant cultivars, which is the key to control wheat strip rust and wheat breeding. Mutation and heterokaryosis are the two main ways of Puccinia striiformis virulence variation, the construction of pathogens mutation library is an important means to molecular genetic analysis as well as a short cut to clone pathogenicity-related genes. This study explored the ways to produce P. striiformis mutation and establish a set of effective genetic transformation system in P. striiformis1. Compared the effects of electroporation transformation, EMS chemical mutation and particle bombardment to produce P. striiformis virulence mutation showed that electroporation transformation cannot produce virulence mutation effectively while EMS chemical mutation and particle bombardment can produce virulence variation effectively and established the transformation system.2. Constructed the best virulent mutation system of wheat rust by chemical inducement. EMS has extensive biological effects on the viability, pathogencity and reproduction of stripe rust. Germination rate of radiated spores was remarkably reduced, its disposal time and concentration curve have shown that urediospores of stripe rust were sensitive to lower disposal concentration and to longer time. The dose-germination curve were followed MMF and Harris mode in CurveExpert (1.3), they were inverse S shape. Implied its has a optimized parameter among in the space. On primary leaf of susceptible cultivars inoculated mutated urediospres, the latent period of rust infection was extended, rust incidence and severity were reduced, reproduction of urediospores of stripe rust decreased, and the host-pathogen interaction changed into incompatible with low-infection type. All of responses strengthened with the increase of disposal dose. An optimized mutagenic system of wheat stripe rust urediospore was designed,The results indicated that when the buffer PH value was7.0 in the normal temperature conditions, the uredospore of tripe rust disposed 7-8 minutes with 0.03mol/L EMS solution the death rate has reached 80% to 90%. This number value adapted to the selection criterion of the optimum mutagenic dose of the microbe chemical mutagenesis experience value.3. Establishing and optimizing the transformation system of P. striiformis by gene gun: there are many factors to affect the transformation rate including the speed and gun shot of microprojectiles, the dosage and the diameter of gold powder, the concentration and hydrate time of urediospores of P. striiformis, He pressure in bombardment, the membrane-cracking pressure,and so on. The study showed that when the diameter of gold powder is 0.6μm ,the transformation rate is higher than 1.5μm. Under the parameters of the distance between the membrane-cracking and membrane-bearing was 2.5cm, the distance between membrane -bearing and blocked net was 0.8cm, the distance between blocked net and target cell was 6cm, the He pressure was 2.7×104Pa and the diameter of golden powder was 0.6μm, when the membrane-cracking pressure was 650,900Psi, the germination rate was comparatively high but the transformation rate was relatively low. However, when the membrane-cracking pressure was 1100,1350 and 1550Psi, the germination rate was comparatively low but the transformation rate was relatively high. Moreover, our experiment also showed that when the dosage of rust fungi in each gun was 0.2mg, the dosage of golden powder in each gun was 500μg, plasmid was 1μg, setting time was 2h, the transformation rate was higher. These results were the main parameters of transform Puccinia striiformis gene gun.4. Transient expression of reporter gene in P. striiformis transformants using biolistics: urediospores of P. striiformis is transformed with the plasmid pGUS6L20 carrying the GUS reporter gene. The GUS in transformed urediospores are able to express temporarily under the delivery pressure of 1100, 1350, 1550psi. The transformation efficiency is high up to 0.2% and 0.34% under the delivery pressure of 1350psi and 1550psi respectively. However, the germination rates of these urediospores are as low as 25%. After stained with x-gluc, some of the spores are deep blue and some are light blue which shows that there are difference in the copies and expression quantity of GUS gene within the transformed urediospores. When transformed with the pKLHyg14 which contains the hygromycin B resistance gene hpt, under the delivery pressure of 650psi, 900psi, 1100psi, the urediospores have higher germination rates on media containing 50μg/ml hygromycin B compared to the control. This indicates that hpt gene has expressed in those urediospores. Therefore, reporter gene GUS and antibiotic resistance gene hpt can both be used as selection marker for P. striiformis transformants.5. Screening the EMS induced mutants of CY17 and CY31: Urediospores of parents culture, predominant race CY17-5 and CY31-3 were treated by EMS, induce mutant were screened on the Chinese differential cultivars and the near-isogenic lines (NILs) of rust. 11 and 8 mutant isolations were obtained with estimated mutation rates 10-5～10-6. 4 mutant groups isolate form CY17-5, code 17M1 to 17M4, they have 86% similarity with its patents culture. And 3 mutant groups form CY31-3, code 31M1 to 31M3, using 92% similarity cut-off point. The virulence of the mutants to a set of differentials used in Chinese race survey was different from all of the known races of stripe rust. Most mutant isolates kept stable virulence such as 17M2 to Mentana, 17M3 to Mentana,Suwon11 and 17M4 to Abbonanza,Suwon11. 31M1 were virulent on Jubilejina 2 and Trigo Eureka. 31M2 and 31M3 were detected to avirlent on several differential cultivars. Most of the CY17 mutants were avirulent mutants while CY31 mutants were virulent mutants, indicating that most of feeble virulent strains produced avirulent mutation, and high virulent strains produced virulent variation agter the inducement of EMS.6. Generation of virulence mutants of P. striiformis using biolistics transformation and their biological characteristics: Single spores of albino strain-5 are used as the recipients transformed by biolistics with vectors carrying GUS gene and hygromycin resistance gene hpt. When P. striiformis is transformed with the vector pGUS6L20 carrying GUS gene under the delivery pressure of 1350psi and 1550psi, the GUS gene expression in progenies is detected. When P. striiformis is transformed with the vector pKLHyg14 carrying the hpt gene under delivery pressure of 1100psi, two stable virulence mutants M1100-4-2 and M1100-4-3 are generated. Mutant M1100-4-2 shows reduced virulence on Mentana with reaction type changing from 4 (wild-type) to 2. M1100-4-3's reaction type on Mentana decreased from 4 (wild-type) to 2 and 3 which shows the virulence differentiation. Infection assay of mutants on Chinese differential host shows that virulence of all the mutants changes significantly in contrast with wild-type isolates. Their biological traits also show considerate variation by losing the virulence on most screening cultivars and expanding the latent periods. PCR amplification in the two mutants yields the hpt gene fragment and this indicates that the mutation in the two mutants results from the insertion of hpt gene.This study used chemical mutagenesis to generate the virulence mutant strains of P. striiformis. Using particle bombardment, foreign gene markers are introduced and pathogenicity mutants of P. striiformis are generated. Therefore, a novel technical system for studying inheritance of P. striiformis pathogenicity is established, which lays a strong foundation for cloning virulence genes in P. striiformis and elucidating the mechanism of virulence variation in P. striiformis.