| Endophytic fungi plays an important role in plant resistance against biotic and abiotic stress.At present,researches focus on the endophytic fungi species diversity,ecosystem distribution and biology function.However,the mechanism of symbiotic interaction between endophytic fungi and plants much leaves to be investigated on.Fusarium nematophilum NQ8GII4 is endophytic fungi isolated from the root of Lycium barbarum L..In this study,NQ8GII4 strain was used as the material.The protoplast preparation and regeneration conditions were optimized by single factor experiments and response surface methodology.The expression vector pDL2,pFL2,r-KNT,KNTG were transformed into NQ8GII4 protoplast by PEG-mediated transformation.The transformants were tested for their phenotype,fluorescence,antagonistic activity on Colletotrichum gloeosporioides and pathogenicity on leaves of L.barbarum L.,Using GFP-labeled NQG8II4 transformant fermentation broth to irrigation L.barbarum,the colonization of NQG8II4 strain in L.barbarum and the effect on the growth and resistence of L.barbarum were clarified.The results were as follows:1?NQG8II4 strain was used as the material.The effects of mycelial age(12?20 h),enzymolysis time(0.5?3 h),enzyme mass concentration(15?35 g/L Driselase+10 g/L Lysing enzyme)and osmotic pressure stabilizer concentration(0.6?1.2 mol/L NaCl)on the protoplast yield,regeneration rate,and available protoplast volume were investigated by single factor experiments.Protoplast preparation and regeneration conditions was optimized through response surface methodology,at a 3-factor,3-level experiment Box-Beheken design.Three equations were obtained to fit the empirical evidence between protoplast volume,regeneration rate,available protoplast volume and enzymolysis time,enzyme concentration,and osmotic pressure stabilizer concentration.The optimum conditions for formation and regeneration of protoplasts from NQG8II4 strain were obtained as follows mycelial age of 16 h,stabilization of the osmotic pressure using 0.713 mol/L NaCl,collecting 0.05 g hypha and hydrolyzing them in 1 mL enzyme solution which contained 30.30 g/L Driselase and 10 g/L Lysing enzyme for 2.5 h.The protoplast volume,regeneration rate and available protoplast volume reached 7.12×107/mL,5.56%and 39.59×105/mL,respectively,which well accorded with the model prediction value.2?Screening the expression vector for GFP-labeled endophytic fungus F.nematophilum NQ8GII4 from L.barbarum.It was transformed by PEG-mediated protoplast transformation.The expression vectors pFL2,pDL2,r-KNT and KNTG were transformed into the protoplast of NQ8GII4 respectively.The best expression vector was obtained by comparing the fluorescence observation,mitotic stability,antagonistic role against C.gloeosporioides and pathogenicity on leaves of L.barbarum L.of the four types of NQ8GII4 transformants.The sensitivity assessment of NQ8GII4 to hygromycin B showed that the lowest concentration was 20 mg/L.The sensitivity assessment of NQ8GII4 to G418 showed that the lowest concentration was 80 mg/L.The expression vectors pFL2,PDL2,r-KNT and KNTG were successfully transformed into the protoplast of NQ8GII4.272 pFL2 transformants,57 pDL2 transformants,73 r-KNT transformants and 226 KNTG transformants were obtained,the transformation frequency of NQ8GII4 was 13,6,2.85,3.65 and 11.3 transformants per microgram,respectively.Transformants observed under fluorescence microscopy,the pDL2 and KNTG transformants could produced stronger fluorescence than PFL2 and r-KNT transformants.From the transformants that were obtained and assessed for mitotic stability,89.47%pDL2 transformants were mitotically stable for resistence to hygromycin B at a concentration of 30 mg/L after five generations.67.28%pFL2 transformants,68.47%r-KNT transformants and 58.85%KNTG transformants were mitotically stable for resistence to G418 at a concentration of 100 mg/L after five generations.Four types of transformants with similar antagonistic activity and pathogenicity as wild type strain.The expression vector pDL2 was suitable for GFP-labeled of NQ8GII4 strain.3?The cell walls of young hypha of NQ8GII4 strain was lyzed with enzymes to generate protoplasts,which were used to insert exogenous DNA randomly under PEG8000 mediated transformation.Fifty seven transformants had been get and the effeciency reaches to 2.85 transformants per microgramme.The growth rate and sporulation of 12 transformants showed have no difference with wild strain.It was confirmed that the gfp gene had been successfully transferred into the NQ8GII4 strain by fluorescence observation and PCR analysis.After colony and microscope observation,the characterization of the colony,spore,hyphae and conidia fructification have no difference with wild strain.One transformant was screened by antagonistic activity on C.gloeosporioides.The transformant had no difference on pathogenicity and H2O2 sensitive with wild strain.4?To understand the colonization of NQ8GII4 in L.barbarum L.and effects of different concentrations on the growth and resistence of L.barbarum L.,the colonization of NQ8GII4 was observed by fluorescence microscope using GFP-labeled NQ8GII4(NQ-D-47).The results showed that the NQ-D-47 strain could colonization in root of L.barbarum.When using 10%fermentation broth for root irrigation,the colonization rate reached the maximum,93.95%.Using 5%?20%NQ-D-47 strain fermentation broth could promoted the growth of plants and root development.When using 5%fermentation broth,the height and above-ground weight reached the maximum,which increased 79.68%and 100%compared with the control.When using 10%fermentation broth,the root weight and dry weight reached the maximum,which increased 195.56%and 85.19%compared with the control.At the same time,the activity of defensive enzymes such as POD,PAL,PPO,SOD in L.barbarum L.were higher than that normal control groups,and colonization of NQ-D-47 in the L.barbarum L.root could reduced significantly the incidence of anthracnose. |
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