Functional Analysis For APX1Gene And TMS1Gene Response To Oxalic Acid Stress In Arabidopsis Thaliana

Oxalic acid (OA) was a key pathogenicity factor of plant pathogen Sclerotiniaclerotiorum. The resistance of crops to both OA and Sclerotinia clerotiorum keptconsistent, so OA was applied to distinguish the resistance to Sclerotinia clerotiorumin this study. In previous study, we found that APX1and TSM1genes wereup-regulated in the differentially expressed gene chip under the stress of OA.Furthermore, it was confirmed by RT-PCR analysis.Ascorbate peroxidase (APX) coded by APX1was located in chloroplast andcytoplasm. APX used ASA as its main electron donor to scavenge H2O2in the plantcells and protect cells from damage. Research showed that over-expression mutants ofAPX gene enhanced the resistance to stress.TSM1gene coded DNA J heat shock protein which was a member of J domainprotein family. Most of J domain proteins possessed their own molecular chaperone. Jdomain protein played an important role in growing development and resistantresponse in plants.We constructed over-expression vector of APX1and TMS1which were noted asAPX1-OE and TMS1-OE respectively. APX1-OE and TMS1-OE were transformed intoArabidopsis mediated by Agrobacterium, screened with basta and confirmedsubsequently the homozygous candidates by three primers mehod.We also attained the homozygote of T-DNA insertion lost of function mutants(apx1and tms1) and confirmed that the expression level of OE mutants was folderenhanced. Meanwhile, the lost of function mutants were real null allele mutants.Using the Arabidopsis-Sclerotinia sclerotiorum as host-microbial interaction model,we inoculated two sorts of homozygous mutants with detached leaves and entireplanst.Histological H2O2production in WT and mutants upon infection with Sclerotiniasclerotiorum were examined via the3,3-diaminobenzidine (DAB) and trypan bluestaining. Two OE mutants exhibited remarkable resistance to Sclerotinia clerotiorum,which indicated that APX1-OE mutant broke out the hypersensitivity to reduce thenumber of dead cells. We also quantified the length of root of mutants with OA stress, and found thatthe OE mutants significantly resistant to OA when compared to WT. It proved theconsistency of the resistance between OA and Sclerotinia sclerotiorum.