Cd Tolerance Mechanism Of Trichoderma REMI Mutant And Its Role In The Improvement Of Oilseed Rape For Cd Uptake

Numerous studies demonstrate that accumulation of heavy metals in farming soil canthreaten crop production by its toxicity to plants. Also, through the food chain, heavymetals can be further accumulated in human body and eventually cause serious healthproblems. Oilseed rape （Brassica napus）, widely grown in Yangtze Delta region, wasexploited in the study for remediating Cd-contaminated soil in combination ofTrichoderma koningii. To improve oilseed rape phytoextracting efficiency by Trichoderma,restriction enzymes mediation integration （REMI） was used to construct Trichodermamutants with higher Cd resistance. Of200mutants,10mutants were shown with higherCd tolerance and ability of removing Cd from growing medium.Cd stress have been already ensured to result in more active oxygens released toinjury cell, even make its death. Based on our result, the insertional mutagenesis ofTrichoderma could lead to higher yield of ROS scaveging enzymes in mycelia than wildtype of Trichoderma, for instance, P6, a REMI mutant showed the higher activity of SOD、CAT and the higher amounts of GSH as compared with wild type of Trichoderma, whichthereby improve significantly its capability to alleviate damage to mycelia caused byover-accumulation of active oxygens in the presence of Cd stress. TEM photos showedthat Cd was mainly deposited in cell wall and vacuole of the mycelia of P6. the curves ofFTIR showed clearly that-OH,-COOH and protein in cell wall were responsible mainlyfor chelating Cd.The kinectic curve for P6mycelia uptaking Cd conform to quadratic equation,R²=0.9938. The maximum content of absorption was33.2mg Cd per gram mycelia. The pH of solution exerted the significant influence on the absorption of Cd by mutant P6, inshort, P6myceila could improve its power in absorption of Cd from environment as theincrease of environmental pH. The energy inhibitor NaN3was not able completely toinhibit the absorption of Cd.A tkpah gene encoding a protein homologous to platelet activating factoracetylhydrolase isoform II [PAF-AH （II）], a member of phospholiapase A2family, wascloned by plasmid rescue from a REMI mutant strain P6of Trichoderam koningii.Thegene contained a short intron of65bp and an open reading frame of1,719bp. Thededuced amino acid sequence comprises the potential conserved GHSFG motif andcatalytic triad （Ser-272, Asp-323, His-414）. Tkpah expressed in E. coli showed PAF-AHactivity. tkpah gene expression was weaker in spores than in mycelia, and wasconstitutively expressed during growth in PD media. The expressions were induced bynitrogen or carbon supplementation to minimal media and suppressed after12h treatmentwith Cd. The conidia of deletion mutants of tkpah displayed an enhanced resistance toH2O2and Cd stress, thus we inferred that the gene regulate mycelial tolerance to Cd innegative way.By2DE analysis, we found that numbers of protein spots of P6mycelia remarkablydecreased from330to255after4h Cd treatment. Among the differential spots,26spotswere identified as unique ones, and16spots were up-regulated. MALDI-TOF-TOF MSsuccessfully identified9spots which comprised14-3-3-like protein, phosphoglycero-mutase, HSP70etc.In pot experiment of Cd-contaminated soil, mutant P6could significantly alleviatethe negative impacts of Cd on oilseed rape （Hu17and Hu20） growth, and then improvethe Cd uptake of oilseed rape plant. At Cd-contaminated soil of20mg kg^-1the removal rateof Cd of Hu17combinated with P6were increased by58%and35%respectively,compared to wild type Trichoderma treatment and non-inoculated treatment（CK）.Similarly the figures were56%and31%respectively for Hu20-P6combination. At Cd-contaminated soil of50mg kg^-1, the removal rate of Cd was increased by110%and68%by Hu17-P6combination respectively, and by30%and23%respectively by Hu20-P6combination. The results suggested the Trichoderma mutant-oilseed rape symbiosissystem can be used in remediation of soil contaminated with heavy metal Cd.