| Low temperature stress is one of the important abiotic stress factors that harm the growth and development of maize.The cultivation of low temperature tolerant germplasm is an important way to solve the low temperature damage.Local germplasm of maize in Tibet is a special germplasm formed by natural and artificial selection in alpine environment.It is of great significance to study its low temperature adaptation mechanism for the improvement of maize low temperature tolerance.In this study,cold resistant variety L56 and cold sensitive variety L78,which were obtained from the low temperature tolerance screening of Tibet maize inbred lines in the previous stage,were used as experimental materials for low temperature stress experiment.Through transcriptomic and proteomic sequencing,the expression changes of differential genes and differential proteins under low temperature stress of Tibet maize were analyzed,and important cold resistant genes and proteins related to response to low temperature stress were discovered.The function of maize in response to low temperature stress was analyzed to provide reference for further revealing the low temperature adaptation mechanism and cold resistance breeding of maize in Tibet.The main conclusions are as follows:(1)Transcriptome analysis showed that 12425 differentially expressed genes were identified under low temperature stress,including4074 up-regulated genes and 8351 down-regulated genes.A total of11,241 differentially expressed genes were identified in the L78 variety,among which 4,368 and 6,873 genes were down-regulated,respectively.Functional annotation analysis showed that these differentially expressed genes were widely enriched in biological processes such as cell composition,metabolic regulation,stress response,signal transduction and transcriptional regulation.Weighted gene co-expression network analysis(WGCNA)was used to cluster differential genes with similar expression patterns,and key genes related to low temperature response in related modules were analyzed.24,219 genes with different expression levels in 12 transcriptome samples were clustered into 16 different gene modules.The expression levels of differential genes in black module were up-regulated in the two varieties under low temperature stress.The expression of differential genes in turquoise module was downregulated.In the black up-regulation module,SNAP33,sweet protein-like gene TLP1,NAL1,phospho-inducible gene PHI-1,transmembrane protein TET8 and ethylene insensitive protein EIL1A were screened.From the down-regulated turquoise module,plasma membrane aquaporin gene PIP2-1,endoplasmic network channel protein SEC61A,polyamine oxidase PAO,stress-related protein kinase SAPK2,transcription regulator WRKY24,cold acclimation negative regulator ESK1 and heat shock protein Dna J were identified.These results indicated that Tibetan corn may adapt to low-temperature environments through the relevant differential genes screened above.(2)Proteome analysis showed that 1616 differentially expressed proteins were identified under low temperature stress,803 differentially expressed proteins were up-regulated and 813 differentially expressed proteins were down-regulated.A total of 1206 differential proteins were significantly expressed in variety 78,among which 813 were up-regulated and 393 were down-regulated.Further cross-comparison analysis of the differential proteins of the two test varieties showed that 341differentially expressed proteins were co-expressed in the two varieties,462 differentially expressed proteins were specifically expressed in the L56 variety,and 472 differentially expressed proteins were specifically expressed in the L78 variety.Among the down-regulated proteins,a total of 163 differentially expressed proteins were co-expressed in the 2varieties,650 differentially expressed proteins were specifically expressed in the L56 variety,and 230 differentially expressed proteins were specifically expressed in the L78 variety.The differential proteins in response to photosynthesis were screened,including the thylakoid membrane protein ELIP1,the early photoinducible protein FTSZ1,the chloroplast coding genes psb A and psb D.Different proteins in response to substance metabolism and energy metabolism:proline synthesis rate-limiting enzyme P5CS1,key enzyme genes for trehalose synthesis TPS1,TPS7,sucrose metabolism pathway key enzyme SPS;Stress response related differential proteins:heat shock protein HSP70,latex protein MLP423,glutathione transferase GSTU8,caffeic acid oxymethyltransferase COMT,calcium receptor protein CML16,Na~+homo-transporter BASS5,etc.Maize in Tibet may respond to the adaptation mechanism of low temperature stress through the above cold-tolerance related differential expression proteins.(3)Based on the transcriptome sequencing data and proteome sequencing data of the two tested varieties that have been obtained,the association analysis between the two was conducted for genes or proteins involved in the same biological process that have the same or similar change laws.The results showed that a total of 4074 upregulated differential genes and 8351 downregulated differential genes were detected in the cold resistant variety L56;Upregulated 803 differentially expressed proteins and downregulated 813 differentially expressed proteins.In the low temperature sensitive variety L78,a total of 4368upregulated differential genes and 6873 downregulated differential genes were detected;Up regulated 813 differentially expressed proteins and down regulated 393 differentially expressed proteins.In cold resistant variety L56,the expression of cold resistant transcription factor SBP1,phosphorus transport related transport protein PHO1,S-adenosylmethionine synthetase SAMS1,etc.,which were significantly up-regulated at both the level of the transcriptome and the middle level of the protein group,were screened;Among the genes whose expression levels were significantly down regulated at the same time,the expression levels of photosynthetic antenna proteins LHCB5 and,LHCB7,genes involved in glucose metabolism HXK5,and galactoside synthase gene GOLS2 were significantly down regulated.The cold resistant related gene glutathione peroxidase GPX6,key factor involved in MAPK cascade signaling pathway MKK5,natriuretic peptide Gh PNP1 gene,raffinose synthase RFS,etc.,whose expression levels were significantly up-regulated at both the level of the transcriptome and the level of the protein group,were screened from the cold sensitive variety L78;The PPO gene of polyphenol oxidase,the LHCA2 gene composed of chloroplast light harvesting complex,and Calreticulin CRT were found to be significantly down regulated among the genes whose expression levels were significantly down regulated at the same time.Functional enrichment analysis was conducted on differentially expressed genes or proteins,and differentially expressed genes with the same expression trend were mainly annotated in biological processes such as metabolic processes,cellular physiological processes,and single organism processes under the association analysis of the two experimental varieties;Differential genes are mainly enriched in binding pathways and catalytic activities in molecular functions;In terms of cell composition,differential genes are mainly annotated to each organelle.The enrichment analysis of metabolic pathways indicates that the main annotated metabolic pathways include:aminoacyl t RNA biosynthesis,starch and sucrose metabolism,photosynthesisα-Metabolic pathways such as linolenic acid metabolism suggest that Tibetan maize may regulate low-temperature stress response mechanisms through these biological processes.The above results preliminarily revealed the molecular regulatory mechanism of maize response to low temperature stress in Xizang Province,and screened out some cold-tolerance related genes and cold-tolerance proteins,which laid a foundation for the future breeding of cold-resistant maize germplasm resources and the study of low-temperature adaptation mechanism. |
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