| Sugarcane smut is a worldwide disease that seriously affects the yield and quality of sugarcane.The Sporisorium scitamineum is a dimorphic fungus,namely,haploid yeast cell and diploid mycelium in life history.Sporisorium scitamineum is dimorphic fungus that nonpathogenic with monophyletic cell.The sexual mating of two haploid cells from compatible mating-typ can fuse a dikaryon with white hyphae to infect the host.Therefore,sexual mating is a foundation for the pathogen to infect the host.This study investigated and analyzed the functions of several key genes in cAMP/PKA signaling pathway that affect the sexual mating in Sporisorium scitamineum.In order to get mutants the PEG-mediated protoplast transformation system was established for the first time.To further analysis these key genes of c AMP/PKA signal pathway,we constructed the mutants by homologous disruption,the main results are as follows:1.To establish an efficient PEG-mediated protoplast transformation system by exploring the effection of preparation of protoplasts,regenerating and screening to transformation efficiency.This part main study at these aspects: cell age,treatment temperature of enzyme,treatment time of enzyme,enzyme dosage that may influence the preparation of protoplast,the optimum treating condition of protoplast preparation in S.scitamineum(preparation rate of protoplast is more than 60% and regeneration rate is more than 40%): a single colony was inoculated in 5ml liquid Ye PS at 28? for 24 h,transfer it to 60 ml liquid Ye PS to OD600 0.1,continuing inoculating to OD600 0.7,add SCS to wash the precitation,add 2ml 10mg/ml Lysing solution to incubate at 28? for30 min.The antibiotics' types and concentrations were selected by the gradient concentration from two different antibiotics:the optimum condition is the hygromycin that final concentration is 200?g/ml.2.Successfully knockout genes Ss UAC1,Ss ADR1,Ss PDE1,Ss PDE2 and Ss PRF1 by using PEG-mediated protoplast transformation system,obtained the homologous disruption mutants ?Ssuac1,?Ssadr1,?Sspde1,?Sspde2 and ?Ssprf1.3.We found that the mutants ?Ssuac1,?Ssadr1 and ?Ssprf1 are all defective in mating means ?Ssuac1,?Ssadr1 and ?Ssprf1 respectively mating with WT18,the hypha is less than WT17 mating with WT18 and no hypha is found when ?Ssprf1 mating with WT18.Also there are no difference about hypha between ?Sspde1,?Sspde2 and wild type when mating.4.Add exogenous 5m M c AMP in Ye PS soild medium,the recovery of hyphal growth of?Ssuac1 can reach the same degree like wild type,?Ssadr1 has weaker recoverability,however,the hypha was inhibited in ?Sspde1,?Sspde2,?Ssprf1 and wild type.The results show that Ss UAC1 positively regulate c AMP signal transduction and sexual mating in S.scitamineum.5.Add exogenous 0.7m M H2O2 in Ye PS soild medium,the haploid of mutants' growth were all inhibited compared to wild type,but with 0.7m M H2O2 and 7.5m M c AMP in Ye PS,the growth returned to normal.We guess that there is a relationship between c AMP/PKA signal pathway and oxidative stress.6.Add exogenous mental ions 7.5m M Zn2+ in Ye PS soild medium,the mating was significantly inhibited in mutans ?Sspde1,?Sspde2 and wild type.The growth of haploid ?Ssuac1,?Ssadr1 and ?Sspde2 are more sensitive to Zn2+ than wild type;also,their hypha recover in c AMP Ye PS soild medium.It is suggested that these genes may be independent of c AMP pathway to resist high concentration of Zn toxicity or participate in the physiological function of Zn in cells.This study successful established the PEG-mediated protoplast transformation system for the first time,constructed the mutants ?Ssuac1,?Ssadr1,?Sspde1,?Sspde2 and?Ssprf1.We found that Ss UAC1 positively regulate c AMP signal transduction;and there is a certain connection between c AMP/PKA signal pathway and oxidative stress,resistance of Zn2+.These are all provide a theoretical basis for the prevention and control of sugarcane smut. |
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