| Aflatoxins,produced mainly by Aspergillus flavus(A.flavus)and Aspergillus parasiticus(A.parasiticus,)are the most toxic fungal secondary metaboilites and the most potent carcinogens that contaminate agricultural commodities such as peanuts,cotton and corn.Understanding the underlying mechanisms of crop resistance to fungal infection is an important first step toward for safe production of human food and animal feed.First and foremost in the first three chapters,in order to understand the mechanism of peanut resistance to A.flavus infection or aflatoxin production,one aflatoxin resistant peanut line GT-C20(R)and one aflatoxin susceptible line Tifrunner(S),were inoculated with A.flavus NRRL3357 from 1 to 7 day to determine the differences in the expression of aflatoxin biosynthetic pathway genes,the expression of peanut resistance-related genes,and the content of peanut nutrient compositions.Based on the data obtained from these studies from 1 to 7 day,mechanisms of fungal defence were infered and a time-course model of of resistance in peanut against A.flavus,was proposed.On one hand for A.flavus on different peanut lines,the mycelial growth of A.flavus NRRL3357 on the resistant peanut line GT-C20(R)was much slower than that on the susceptible(S)peanut line,Tifrunner.Besides reduced fungal growth,the R line inhibited aflatoxin production completely,whereas the susceptible line Tifrunner produced 85452 ppb aflatoxin.Real-time RT-PCR analyses of fungal gene expression in the mycelia recovered from A.flavus inoculated R and S lines revealed that the expression of five aflatoxin biosynthetic pathway genes,the regulatory gene aflR and the aflD,aflM aflP,and aflQ structural genes,was delayed significantly in the mycelia grown on the R line.The results suggested that resistance factors of the R line acted negatively on A.flavus growth and also altered fungal development.The dysfunction in development changed the timing and the pattern of aflatoxin gene expression,which in part rendered A.flavus unable to produce aflatoxins.On the other hand for infected different peanut lines by A.flavus,in order to understand the resistance mechanism employed by the peanut line GT-20C,14 putative resistance related genes identified in a previous microarray study were selected and their expressions in mycelia grown R and S lines were compared to determine whether they are really involved in peanut resistance to A.flavus infection.The results revealed diverse time-changing patterns of gene expression during the germination process after A.flavus inoculation.Based on the expression levels and the relative-expression patterns over a 7-day period,the 14 peanut genes could be classified into 6 different groups belonging to three main biochemical or gentical processes of counter-attacks.A network of gene expressions was activated in proper sequential order in response to A.flavus invasion in both resistant and susceptible peanut lines.In addition to examining the differences of peanut lines in the contents of nutrients,such as carbohydrate,fat and protein,which can affect the growth of A.flavus and production of aflatoxin,were also compared between the R and S line.The reducing sugar detected by HPLC,free fatty acid determined by Soxhlet extraction method and certain amino acid such as glutamic acid measured by Amino Acid Analyzer contributed to suppression of fungal growth as well as aflatoxin production on peanut.In the second place,one of constitutively up-regulated peanut gene,AhPR-1(pathogenesis-related)used as a marker of the capacity of defense in PRs,was selected for further investigation.A clear difference in the expression of AhPR-1 gene between R and S peanut lines was detected by PCR and Real-time PCR.Because the biological functions of PR-1 proteins have not been well studied,AhPR-1 cDNA was cloned into pET28a plasmid and overexpressed in E.coli Rosetta2(DE3)pLysS.In the same time,Agrobacterium mediated pCAMBIA 1300-AhPR1 plasmid was constructed aiming to construct a transformed plasmid with inserted AhPR-1 gene.Antibacterial and antibacterial activity of purified recombinant AhPR-1 protein expressed by E.coli Rosetta2(DE3)pLysS were determined against A.Flavus,E.coli and S.aureus in culture medium,followed confirmed by LC-MS/MS and WestBlot.In the third place,based on the inhibiting function of recombinant AhPR-1 protein in E.coli against microbes,environmental factors of protein inhibiting A.flavus growth on peanut were optimized by using surface response method.The inhibiting effects of AhPR-1 on peanut,including protein concentration,temperature and water activity,were estimated using apparent evaluation method on A.flavus growth level and HPLC on aflatoxin production.Results show that,when inoculated with 4×106 CFU/mL spore suspension of A.flavus NRRL 3357 on peanut,the resistance effect of PR-1 increased with the concentration protein,while there was no significant difference of inhibition effect between 0.32ng/?L and 0.40ng/?L of AhPR-1 protein,respectively;in the same time the inhibition effect of PR-1 protein reached its peak under 0.3-0.4 water activity at 28?.Thus,growth A.flavus and aflatoxin synthesis on peanut could be suppressed to some level by AhPR-1 protein under certain environmental circumstances.In the forth place,applicationof different detection techniques including fungal growth score and aflatoxin measured by HPLC in the model pathway one,can not only idenfiy applicability of methord in model,but also screen the potential resistant peanut lines.Eighteen The 18 peanut lines,chosen from Institute of Windy and Sandy Soil Improvement and Utilization of Liaoning Province and Huludao City,the peanut lines were inoculated with A.flavus NRRL3357 and cultured for 7 days.The resistance level capacity of these peanut lines against A.flavus were evaluated according to the level of infection index deduced from the traditional classification standard.The suppression of aflatoxin;the capability of blocking the biosynthesis of Aflatoxin was determined using estimated on the basis of the value measured from TLC and HPLC.The levels of infection index of different peanut lines correlated well were almost accordant with the aflatoxin data from values of TLC and HPLC.Two resistant peanut linesgenotypes,Baisha1016 and Fuhuall,were identified as highly resistancte against A.flavus with low levels of aflatoxin.Last but by no means least,AhPR-1 proteins have potential capability of inhibiting fungi,so recombinant AhPR-1 protein is potential fungal inhibitors.Recombinant AhPR-1 protein and other fungal inhibitors were conducted to surface of peanut followed inoculation with A.flavus.After estimated growth scores and detected aflatoxin by HPLC on peanut,relative quantification expression level of fungal biosynthetic genes nor1,ver19 omtA and aflR via Real-time RT-PCR were investigated in different treatment groups after incubating 5 d.Scores as well as aflatoxin B1,in recombinant AhPR-1 protein treatment group,were far lower than in negative group's and a little higher than in positive inhibitor group's.Inhibition function of recombinant AhPR-1 protein was observed and detected markedly,and it may bring promising contribution in the respective of peanut storage. |
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