Effects Of DNA Damage Repair Genes On Agrobacterium-mediated Genetic Transformation In Rice

Agrobacterium-mediated genetic transformation has been widely used as a powerful tool for genome analysis and the predominant technology for production of transgenic plants.The gene transfer mediated by Agrobacterium is a very complex biological process involving in the synergetic effect of genetic factors of both the bacterium and the host plant cell.T-DNA integration is the most critical step of the transformation process.More and more evidences suggest that plant DNA damage repair machinery plays an important role in Agrobacterium T-DNA integration.However,much more attention was paid to the genes related to DNA break repair and the genes in other DNA damage repair pathway awaits further investigation.The main results are as follows:(1)The response of DNA damage repair related genes to Agrobacterium infection.Expression of ku70 and lig4 in NHEJ repair pathway,rad51 and reca in HR repair pathway,msh2 and msh6 in MMR repair pathway,fgp and maglp in BER repair pathway was detected by RT-qPCR at different time after Agrobacterium infection.The expression level of ku70,lig4 and rad51,msh2 and msh6,ku70,lig4 and rec,fgp and maglp was significantly increased at 12h,24h,36h and 48h in callus,respectively.The results suggest that these DNA damage related genes may be involved in Agrobacterium mediated transformation process.(2)Effects of gene mutation in mismatch repair(MMR)and base excision repair(BER)pathway on transient expression of transgene gus.Transient expression frequency of gus gene in the callus after co-cultivation was analyzed by GUS histochemical staining method.There was no significant difference in the transient expression frequency between the MMR gene insertional mutant(NF9010 and NF7784)and the BER gene insertional mutant(NC2747 and ND1052)with the control Nipponbare,indicating that the early stage of Agrobacterium-mediated transformation is not affected by the mutations of MMR gene(Os09g0407600)and BER genes(Os04g0673400 and Os09g0420300).(3)The effect of MMR and BER gene mutation on T-DNA integration.The T-DNA integration was analyzed by screening and differentiation test of calli with hygromycin resistantce and PCR characterization of regenerated plants.The MMR gene mutant NF7784 and NF9010 showed significant decrease in screening rate of the resistant callus and differentiation rate of regenerated plants,the BER gene(Os04g0673400)mutant NC2747 also showed significant decrease in differentiation rate,but no differences in both screening rate and differentiation were detected between the BER gene Os09g0420300 mutant ND1052 and the control Nipponbare.Results of PCR characterization for hpt gene and gus gene showed that the stable transformation frequency for MMR mutants decreased by 49.33%?64.53%,while it decreased by 11.2%?36.53%for two BER gene mutants.These results suggest that the integration efficiency of T-DNA was affected by the mutations of MMR and BER genes tested in this research.