| Maize is a thermophilic crop,which requires.high temperature.However,due to the influence of geographical environment,the cold damage in early-spring has become one of the main natural disasters in Northeast China.Germination stage and bud stage are the key periods of maize growth and development.At this time,if the temperature is low,the germination rate and emergence rate will be reduced,and the yield of maize will be reduced by 22.5%-34.3%,which will threaten the stable yield of maize.Therefore,it was of great significance to strengthen the research on cold tolerance of maize in germination and bud stage,especially to clarify the mechanism of resistance to low temperature stress.Transcriptome can reflect gene expression and regulation in cells on the whole level.Therefore,the research on maize cold tolerance by the digital gene expression profile is of great significance to explore genes tolerance to cold and understand the function of related genes.Combined with the evaluation on chilling tolerance of ninety-five inbred lines at germination stage and at bud stage,two inbred lines were used to transcriptome sequencing,which were tolerant and sensitive to cold stress at both stages,respectively.The temperature for the treatment was 10℃ at the germination stage and 2℃ at the bud stage.The samples of germination embryos and young shoots were taken at 0 h,6 h,12 h,24 h,36 h.and the samples were used to explore the changes in gene differences by the digital gene expression profile.The main results are as follows:(1)In this study,the germination rate,germination index,average germination time and relative values of 95 inbred lines were measured under 10℃ low temperature stress,integrate statistical methods such as principal component analysis and cluster analysis to evaluate the chilling tolerance of inbred lines during germination and screen chilling tolerance germplasm.The results showed that six single indicators at germination stage were synthesized into two independent comprehensive indicators by principal component analysis.ninety-five inbred lines were divided into 5 groups by systematic clustering,in which the materials with excellent cold resistance,strong cold resistance,medium cold resistance,weak cold resistance and very weak cold resistance were 11,25,26,15&18,respectively.Based on the aboved results and the identification of the bud stage,Zhongxi 091/O2 tolerant to cold stress and chang7-2 sensitive to cold stress at two periods were used to the further research of expression profile sequencing.(2)The results of the expression profile sequencing showed that with 0 h as the control,when treated at 10℃ for 6 h,12 h,24 h and 36 h at low temperature,there were 286,2920,4008 and 7170 differentially expressed genes(DGEs)for the cold-resistant individuals(MA),and 112,701,2485 and 4003 DGEs for the cold-sensitive individuals(MB)at germination stage.When treated at 2℃ for 6 h,12 h,24 h and 36 h,there were 505,1240,3370 and 5180 DGEs for the cold-resistant individuals(YA),and 876,2396,3846 and 3681 DGEs for the cold-sensitive individuals(YB)at bud stage.(3)The analysis of GO enrichment for DGEs found that MA,MB,YA,YB had 11859,5937,8078,8244 DEGs for 6 h,12 h,24 h and 36 h,respectively.52 functions were annotated by the aboved different materials.Common enriched GOterms had biological processes such as response to cold,transcriptional regulation,abscisic acid-activated signaling pathway;cell components such as membrane components,ubiquitin ligase complex;binding function,catalytic activity,transporter activity and other molecular functions.(4)The analysis of pathway enrichment analysis for DGEs found that the significantly enriched pathway of two materials at two periods was plant hormone signal transduction pathway,and two significant enrichment pathways related to cold tolerance were specifically analyzed.The DGEs and the expression level for the materials tolerant to cold stress were more than those sensitive to cold stress.The genes such as coding ARF,PYR/PYL,etc were specifically expressed by the materials tolerant to cold stress.(5)WGCNA analysis of DGEs for MA,MB,YA and YB was carried out by using dynamic shear algorithm.2,2,2 and 3 tissue-specific modules were selected respectively,and 5 key genes closely related to the modules were selected from each module,which may play a role in the response to low temperature.(6)In this study,25 genes related to cold tolerance were screened,including 8 DEGs for cold responsive(GO:0009409)in GO term,12 DEGs specifically expressed for the materials tolerant to cold stress in pathway enrichment analysis,5 DEGs specifically expressed in WGCNA.Ten genes were selected for the verification of qRT-PCR,which included Zm00001d002533,Zm00001d003601,Zm00001d010863,Zm00001d022125,Zm00001d028401,Zm00001d030310,Zm00001d034099,Zm00001d039718,Zm00001d045557,Zm00001d047705.The results showed that Zm00001d003601,Zm00001d034099 and Zm00001d028401 were specifically expressed for the materials tolerant to cold stress,and had significant difference between different time points.These genes could play an important role in the process of low temperature stress. |
