Establishment Of Transformation Systems For Gibberella Fujiuroi And Effect Of P450-3 Gene Deletion On Gibberellins Biosynthesis

Gibberellins?GAs?act as efficient plant growth regulator,promoting stem elongation,break dormancy,flower induction,the elongation of fruit cells and so on.The rice pathogen Gibberella fujiuroi produces a large amounts of gibberellins with highly activity,but the fermentation level of GAs is low in industry.Study on the gene of GAs biosynthesis pathway can help us to build high-yield strains.This study construct an efficient gene knockout system of protoplasts rely on SOE-PCR?Splicing by Overlap Extension?and use this system to knockout the p450-3 gene of G.fujiuroi.In this study,several fungal degrading enzymes were tested to prepare G.fujiuroi protoplasts.An efficient method for preparing protoplasts of G.fujiuroi was developed:keeping the mix of driselase and yaterlase at the ratios of 3:7,the concentration of enzymes were maintained at 15mg/mL,hydrolyzing the mycelium 3h under 37?.Generally,5.13×10⁷protoplasts/mL were obtained with this method.An optimization of SOE-PCR was carried out.The upstream?800 bp?and downstream?800 bp?homologous fragment of p450-3 gene and internal hygromycinB resistance sequence?1860 bp?was connected to a long gene fragment?3460 bp?.The thermo-cycling conditions are 94?for2 min,35 cycles of?94?for 30 s,58?for 10 min,72?for 5 min?and72?for 10min.The G.fujiuroi protoplasts were transformed with knockout fragment and 108 transformants were achieved.A totally of 93transformants with stable resistance were screened and the transformation rate was 56 transformants/?g DNA.Seven transformants?T3?T5?T9?T10?T23?T27?T34?randomly selected form 27 transformants which had been identificated by PCR were analysed by RT-PCR and Southern blot.The result showed that five p450-3 gene deletion mutants?T5?T10?T23?T27?T34?were confirmed.Finally,wild-type and p450-3 deletion strains were studied in phenotype and GAs producting capacity.When compared with WT,the p450-3 deletion mutant showed no significant difference on growth rate on PDA medium.The significant difference was found on mycelial morphology.More cracked mycelia were found and more irregularity on thickness of mycelia in p450-3 deletion mutants.This result indicated that the deletion of p450-3 may affect the development of mycelium.Detecting GAs producting capacity by HLPC,the result showed that p450-3 deletion mutants lost the capacity of producting GAs?GA₃?GA₄?GA₇?.This result showed that p450-3 not only in charge of catalyticing GA₇ to GA₃.We deduced that p450-3 gene may be a multifunctional gene which involves more complex catalytic steps in GAs biosynthesis pathway.The GAs producting capacity of twenty transformants that have been identified by PCR and five p450-3 deletion mutants were detected by HLPC.The result showed that eleven transformants can not produce any GAs?GA₃?GA₄?GA₇?and the GAs titer of fourteen transformants were similar with wild type.This study would provide the basis for establishing the gene knockout system of G.fujiuroi,constructing the high-yield strain of gibberellin and analyzing the fuction of the gene in gibberellin biosynthesis pathway.