Response Of ZmHMA2-1 And ZmHMA2-2 Genes To Cd Stress In Maize A Preliminary Study Of Function

Lead and cadmium are the most serious heavy metals in soil in China.cadmium is an indispensable element in the process of biological growth and development.It is one of the most harmful heavy metals to animals,plants and human beings in nature.Corn is a global consumption of gramineous crops,which is widely planted in China.Southwest China is one of the main maize planting areas in China.However,the heavy metals content in soil in these areas is high,mainly lead,Cd,Cr and other pollution,which brings great risks to maize growth and food security.Previous studies have reported that Cd can be absorbed and transported by maize roots under Cd stress,and stored in root coat and cortex tissue.Maize tolerance to Cd can be enhanced by depositing Cd in root cell wall and forming apical stratum.However,the molecular mechanism of maize responding to Cd pollution at the molecular level is still poorly studied.The function of two genes of Zm HMA family（Zm HMA 2-1 and Zm HMA 2-2）was further analyzed in this experiment.The mechanism of Cd uptake and transport by maize HMA family genes was studied,and the molecular mechanism of Zm HMA gene responding to Cd stress was analyzed.It could improve the characteristics of Cd enrichment in existing maize varieties and provide some references for phytoremediation technology of Cd-contaminated soil.In this study,the functions of two Zm HMA family genes in maize response to Cd stress were studied by q T-PCR,subcellular localization,yeast heterologous expression and gene knockout techniques.1.Two Zm HMA family genes,Zm HMA2-1（gene number:GRMZM2G175576）and Zm HMA2-2（gene number:GRMZM2G099191）,were cloned from Cd-tolerant maize inbred line B73 with reference to the genomic sequence of maize B73.Bioinformatics analysis showed that these two genes of Zm HMA family were similar to Os MAH2 and 3genes of rice.The genetic relationship of At HMA2 and 3 genes in Arabidopsis thaliana is close,among which Zm HMA2-1 and Os HMA3 genes have the highest homology,Zm HMA2-2 and Os HMA2 genes have the highest homology,and protein alignment structures show that Zm HMA2-1,Zm HMA2-2 and Os HMA2,3,At HMA2 and 3 have the same conserved domains of heavy gold transport,such as TGE（phosphatase domain）,DKTGT（histidine kinase domain）.2.The results showed that Zm HMA2-1 and Zm HMA2-2 genes were expressed on the cell membrane of tobacco cells.Quantitative analysis of two genes in B73 and MO17under Cd stress showed that both genes had related response mechanism to Cd stress in maize,which could improve the defense function of maize against abiotic stress in this environment to a certain extent.3.Yeast heterologous expression vectors with two genes were successfully constructed and transfected into Cd-sensitive yeast mutant strains.The results showed that Zm HMA2-1gene could enhance the Cd sensitivity of yeast mutants under Cd Cl₂stress of 30 and 50micromol/L,and make them more sensitive,while Zm HMA2-2 and no-load mutant yeast strains could enhance the Cd sensitivity of yeast mutants.The difference is not obvious.Therefore,Zm HMA2-1 has more obvious function in response to Cd Cl₂.4.Phenotypic identification of the Arabidopsis homologous mutant athma2 showed that the mutant was very sensitive to Cd when compared with WT（wild type）at 25 and 40micromol/L.The overexpression vectors Zm HMA2-1-PRI101 and Zm HMA2-2-PRI101were constructed and transformed into Arabidopsis thaliana.At present,positive transformation events of Zm HMA2-2 were obtained.The Cd stress test showed that the phenotype of Zm HMA2-2 was not significantly different from that of WT（wild type）.5.A maize Zm HMA2-1 knockout vector was constructed.CRISPR/Cas9 was used as the carrier skeleton and maize B104 was used as the transformation receptor.At present,21maize plants survived and fruited.