Functional Analysis Of ZmC2-15 In Maize Under Heat Stress

The objective of this study is to identify the members of maize ZmC2s gene family,to analyze the association between their genetic variations and heat tolerance,and to lay a foundation for clarifying its function and molecular mechanism in maize heat tolerance.Using the C2 protein domain PF00168,hmmsearch was applied to search for the members of the ZmC2s gene family from maize B73 genome.The protein isoelectric point,molecular weight,phylogenetic evolution,and gene family replication were analyzed.Using the method of candidate gene association analysis,the association between the natural variations of ZmC2s and the heat tolerance of maize seedlings was conducted,and the important heat-tolerant candidate genes of maize ZmC2s gene family were found.The relative gene expression level of the heat-tolerant candidate gene under stress was identified by Real time fluorescent quantitative PCR(RT-qPCR).The subcellular expression sites of heat-tolerant candidate gene were identified by transforming maize protoplasts.Obtain overexpressed Arabidopsis and zmc2-15 mutant maize and analyze their heat tolerance.A total of 95 maize ZmC2s genes were identified from the reference genome B73 in maize.According to the order of their physical coordinates,95 maize ZmC2s genes were named from ZmC2-1 to ZmC2-95,respectively.The length of the 95 proteins was 130–2141,the isoelectric point was 4.1–10.8,and the molecular weight was14.1–230.1.The evolution tree of C2 gene in maize,rice,and sorghum genomes was constructed.We found that C2 genes can be divided into three major cluster branches,and each cluster branch can be subdivided into two small cluster branches.Analyzing the whole genome collinearity data of maize,rice,and sorghum,59 ZmC2s genes were detected to have corresponding replication genes in rice and sorghum genomes.A candidate-gene based on the association analysis of ZmC2s showed that ZmC2-15/60/91 were important candidate genes for heat tolerance in maize(P ≤0.001,MLM),among which ZmC2-15 was the most significantly associated to heat tolerance at seedling stage(P ≤ 0.00001,MLM),and the relative expression level of ZmC2-15 was up-regulated under various stress treatments.Subcellular localization indicated that ZmC2-15 was localized in the cytoplasm,nuclear membrane,and endoplasmic reticulum.Transgenic Arabidopsis with overexpression of ZmC2-15 reduces ROS content,enhances photosystem recovery,and improves plant heat tolerance.The zmc2-15 mutant maize inserted μ transposon enhanced ROS content under high temperature stress,weakens the recovery function of the photosystem,and reduces plant heat tolerance.This reversely proves that ZmC2-15 is a key gene for improving plant heat tolerance.ZmC2-15 can be used as an important candidate gene for regulating heat tolerance in maize.