Mechanism Of Cadmium Stress On Mismatch Repair System And Cell Cycle In Arabidopsis Thaliana And Glycine Max

Cadmium（Cd）is a heavy metal element widely used in industry and exists ubiquitously in the water,air and soil,Cd is also one of the food contaminants and carcinogens,which is transferred to the food chain into the body causing harm to the body’s health and even inducing cancer.At present,most studies of Cd toxicology was focused on the way and mechanism of biological damage to Cd,and it is most focuses on Cd-induced DNA damage and cell cycle arrest.However,there are fewer studies on the respond mechanism of Cd-induced DNA damage repair and DNA mismatch repair.Under Cd stress,whether the MLH1,MSH2 and MSH6 of DNA MMR system were involved in G2 cell cycle aressed induced by DNA damage,there was unreported at home and abroad.Threrfore,Arabidopsis thaliana and Glycine max were used as tested plants in this study.Through the research changes of DNA damage,cell cycle arrest and related gene expression in Arabidopsis thaliana,we have cleared the respond mechanism of Cd stress in plant.we also have done Comparative study on the response mechanism of MMR system to Cd stress by using homologous genes in soybean,we found that respond mechanism of Cd stress has genotype differences between varieties,this difference provided new scientific theoretical basis for sensitive and resistant of Cd stress in soybean varieties breeding,and could also provided a new way for molecular marker assisted breeding,it can also provided the reference basis for the selection of safe food production varieties in middle and light polluted farmland.Arabidopsis seedings were grown hydroponically on 0.5×MS media containing cadmium（Cd）of 0-4.0 mg·L^-11 for 5 d.and the soybean seedings were grown on petri dish with double-layer filter paper,which was containing cadmium（Cd）of 0-2.5 mg·L^-11 for 4 d.Germination rate,fresh weight of seedings and root length of Arabidopsis thaliana and Soybean seeds were detected and analyzed.Under Cd stress,analyzing G2/M cell cycle retardation and changing trends of G2/M regulatory gene in Arabidopsis roots and the change trend of cell cycle retardation and DNA damage repair genes,regulatory gene in soybean roots by using flow cytometry（FCM）and real-time fluorescence quantitative polymerase chain reaction（PCR）,We analyzed the polymorphism of DNA damage by RAPD map.The results were as follows1.Effects of growth and development in Arabidopsis under Cd stress.There was no significant difference in the germination rate of WT,mlh1-,msh2-and msh6-deficient under Cd stress.the fresh weight and root length were increased at low concentration and decreased at high concentration in WT,the fresh weight and root length were decreased with Cd concentration in mlh1-,msh2-and msh6-deficient,the fresh weight was only about 60%of the control and the root length was only about 30%-50%of the control at 4.0 mg·L^-1Cd.2.The effects of expression G2/M cell cycle aress regulatory gene and DNA damage in WT,mlh1-,msh2-and msh6-deficient roots under Cd stressDNA damage could be induced by Cd stress at low concentrations of Cd,resulting in the instability of the genome.Polymorphism bands was similar after Cd(1.25-4.0mg·L^-1)treatment between WT roots and msh2-,msh6-deficient roots.However,we found that the extent of DNA damage was difference between mlh1-deficient roots and WT roots at2.5mg·L^-1Cd.Flow cytometry results indicated that Cd stress induced a G2/M cell cycle arrest both in mlh1-,msh2-,msh6-deficient and WT roots,The expression level of DNA mismatch repair genes MLH1,MSH2 and MSH6 were significant decreased;DNA damage response genes ATM,ATR and SOG1,DNA damage repair genes RAD51,BRCA1,KU70 and MRE11,The expression of these genes was inverted U-shape,expression level of G2/M phase marker genes CYCB1;1,CDKA;1,WEE1,CYCD4;1,MAD2 and CYCB1;2 were decrease or inverted U-shape.In mlh1-,msh2-and msh6-deficient Arabidopsis roots,expression level of MLH1,MSH2,MSH6,BRCA1,WEE1,CYCD4;1,MAD2,CDKA;1,CYCB1;1 gene was diminished compared with the WT control,whereas expression of ATM,ATR,SOG1,CYCB1;2 gene was sharply increase,Furthermore,Cd elicited endoreplication in msh2-and msh6-deficient roots.3.Select differences in tolerance to Cd of various soybean varietiesWe selected two soybean varieties with different tolerance to Cd,the Liaodou10 was sensitive to Cd,and Shennongdou 20 was insensitive.The sensitive type-liaodou 10 can be used as a variety for food safety production,while the insensitive type-shennongdou 20 can be used as a phytoremediation variety for soil Cd pollution.4.Effects of Cd stress on DNA damage,cell cycle aress and regulatory gene expression in Liaodou10 roots and Shennongdou 20 roots tipRAPD fingerprints indicated that DNA damage of Shennongdou20 seeding was more larger than Liaodou10 seeding,We suggested that there might be a cross-injury replication mechanism in Shennongdou 20 seeding,This mechanism was across the G1/S checkpoint,the DNA damage as a template for DNA replication,inducing DNA damagecascade reaction,as a result,The number of DNA polymorphic bands of Shennongdou20 more than Liaodou10.Cd-stress could induced G1/S cell cycle aress in Liaodou10 roots,however,Cd-stress induced G2/M cell cycle aress in Shennongdou20 roots.the expression of DNA mismatch repair genes MLH1,MSH2 and MSH6 was decreased significantly,We also observed the prominently inverted U-shaped dose-response effects of Cd stress on gene expression of RAD51,MRE11,E2Fa and CDK-A in Liaodou10 roots tip.The expression level of MLH1and MSH6 were decreased observely,the expression of MSH2,MRE11,WEE1 and CYCB1was significantly inverted U-shaped.the expression of PCNA1,E2Fa,RAD51 and MER11were increased as compared with the Liaodou 10 control.Taken together,we concluded that MSH2 and MSH6,components of the MMR system were involved in the G2 phase arrest induced by Cd stress in Arabidopsis roots and soybean,it might act as direct sensors of Cd-mediated DNA damage.MSH2 and MSH6 protein is a signal indicator of indicating relationships beween exposure-effect of Cd,We could quickly identify different soybean varieties with sensitivity or tolerance to Cd by measure MSH2/MSH6 protein expression,and it could also be used in preliminary identification and screening of soybean germplasm resources,and assisted molecular breeding,providing a theoretical basis for new soybean varieties breeding.On selecting varities reasonably,it could provide reference for safe food production which were grown in areas with moderate and mild cadmium pollution.At the same time,it also provided a reliable theoretical basis for sensitive-low accumulation variety breeding and apparent physiological research in the future.