| We have identified the AhPR-1 sequence which can express active AhPR-1 protein by screening A.flavus resistance gene chip in sequenced peanut strain GT-C20 in our previous work and AhPR-1 gene is one of the most important resistance genes that are first identified in peanut.This paper studied the anti-fungal activity of AhPR-1 protein in peanut on the basis of AhPR-1 gene,optimized there factors that play a role in affecting anti-fungal activity;in further experiment,we constructed the vector used in Agrobacterium mediated pCAMBIA1300-AhPRl vector,and explored the efficient regeneration system of tested peanut line FUHUA12.The first part is based on the inhibiting function of recombinant AhPR-1 protein in E.coli against microbe,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 4×106CFU/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 PR-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 PR-1 protein under certain environmental circumstances.The second part is about the construction of Agrobacterium mediated plasmid,aiming to construct a transformed plasmid with inserted AhPR-1 gene,construct a pCAMBIA1300-AhPRl plasmid based on pCAMBIA1300 plasmid,which is modified by Liaoning Academy of Agricultural Sciences.The third part is about exploring the conditions needed to establish the regeneration system of transgenetic AhPR-1 peanut line.The key to the success of breeding via genetic engineering is the establishment of effective regeneration system in peanut,so how to increase the regeneration rate of peanut tissue culture has always been the focus of our study,this experiment used FUHUA12 as tested peanut line,whose plumular axis is used as explant,established its effective regeneration system and identified the best culture medium combinations with high regeneration rate,which are:Seed germination medium MS+0.7%agar;Dedifferentiation medium MS+6-BA 3.0 mg/L+NAA 0.4 mg/L+0.7%agar;subculture medium MS+6-BA 3.0 mg/L+NAA 0.4 mg/L+ 0.7%agar;rooting medium 1/2 MS+NAA 1.0 mg/L+0.7%agar. |
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