The Molecular Mechanism Of COI1/MYC2 And Cold Response Gene ICE41 In Regulating Winter Wheat Cold Resistance

Chilling injury critically limits the distribution and yield of crops worldwide.Plant hormone,as a signal molecule,plays an important role in biotic and abiotic stress.Wheat is one of the main grain sources in China,and"Dongnongdongmai 1"?Dn1?is a strong cold-resistant winter wheat variety.In the early stage,the research group constructed the mi RNA?Micro RNA?Library of tillers of winter wheat Dn1 and the analysis of the target gene by KEGG?Kyoto Encyclopedia of Genes and Genomes?showed that under low temperature stress the expression levels of Ta COI1and Ta MYC2 genes in the JA signal transduction pathway of winter wheat changed.The proteomic study of Dn1 showed that under low temperature stress,the expression levels of Wcor14,Wcor15,Wcor18,and Wcor413 proteins encoded by four cold response genes changed significantly,suggesting that JA may be involved in the response of winter wheat to low temperature stress.As a positive regulator of the CBF pathway,ICE1 interacts with MYC2 and participates in response to low temperature stress in some plants.In this study,Dn1 was used as the material to investigate the effects of exogenous Me JA treatment on the variations of expression levels of Ta MYC2,cold response genes Ta ICE41,Wcor14,Wcor15,Wcor18,and Wcor413 in winter wheat leaves and tillers under natural cooling in the field;combined with bioinformatics analysis the Ta COI1,Ta MYC2,and Ta ICE41 overexpression vectors were constructed to transform Arabidopsis thaliana to obtain T₂-positive plants,and cold-resistant biological verification was performed in the T₃transgenic Arabidopsis thaliana;the yeast two-hybrid method was used to initially verify the existence of vitro interaction between Ta MYC2 and Ta ICE41 in winter wheat.The research aims to reveal the mechanism of JA's response to cold resistance in winter wheat,and provides a theoretical basis and production basis for the study of the molecular mechanism of hormone regulation of winter wheat's cold resistance.The results are as follows:?1?The results of bioinformatics analysis of Ta COI1,Ta MYC2,and Ta ICE41 showed that Ta COI1 was localized in the cytoplasm,while Ta MYC2 and Ta ICE41 were localized in the nucleus.Phylogenetic tree analysis showed that the Ta COI1 protein and the monocotyledonous Oryza sativa Os COI1 protein belong to the same cluster with the close relationship of 93%;the Ta MYC2protein and the monocotyledonous plant Zea mays Zm MYC2 protein belong to the same cluster with the close relationship of 83%;the Ta ICE41 protein and The Bd ICE1 protein of the monocotyledon plant Brachypodium distachyon belongs to the same cluster,with the close relationship of 97%.Motif analysis showed that:Ta COI1 protein has 10 identical motifs with Oryza sativa,Glycine max,Arabidopsis,Nicotiana tabacum,and Solanum lycopersicum;Ta MYC2protein has 10 same motifs with MYC2 protein of Solanum lycopersicum,Nicotiana tabacum,Arabidopsis,and Zea mays;Ta ICE41 protein has 8 identical motifs with the Bd ICE1 protein of Brachypodium distachyon has,which further supports the results of protein evolutionary tree analysis.?2?The expression levels of Ta MYC2,Ta ICE41,Wcor14,Wcor15,Wcor18,and Wcor413genes in winter wheat showed that the expression levels of Ta MYC2 and Ta ICE41 in the tillers section of the control group reached the peak at-10°C,and the expression levels of Wcor14,Wcor15,Wcor413 reached the peak at 0°C,The expression level of Wcor18 reached the peak at-25°C;Ta MYC2 and Wcor18 in the leaves reached their peaks at-25°C;Ta ICE41,Wcor14,Wcor15and Wcor413 all reached their peak at 0°C.Exogenous Me JA treatment increased the expression of the above genes to different degrees.The gene expression of the tiller reached the peak at-10°C,and the difference was significant;the change of gene expression in the leaves was consistent with the control group,the expression levels of Ta ICE41,Wcor14 and Wcor413 were significantly different from the control group.?3?Functional verification of Ta COI1,Ta MYC2 and Ta ICE41 genes:construction of Ta COI1,Ta MYC2 and Ta ICE41 Arabidopsis expression vectors,and genetic transformation of wild-type Arabidopsis thaliana were performed,and positive plants were screened and identified,and T₃generation plants were obtained.Using WT Arabidopsis as a control,two-week-old Arabidopsis thaliana was cold acclimated at 4°C for 3 d,and treated at-10°C for 4 h,and cultured for 4 d after photoperiod recovery.Phenotype observation,survival rate statistics,Cold resistance physiological detection and downstream cold response gene expression detection.The results showed that the survival rate of ox-Ta COI1,ox-Ta MYC2 and ox-Ta ICE41 plants was significantly higher than that of WT;the relative conductivity and the content of Malondialdehyde?MDA?of ox-Ta COI1,ox-Ta MYC2 and ox-Ta ICE41 plants were lower than WT,and the content of Proline?Pro?was higher than WT;The expression levels of cold response genes At KIN1 and At RD29A in ox-Ta COI1,ox-Ta MYC2 and ox-Ta ICE41 plants were significantly higher than those in WT,and the expression levels of At COR15A and At COR47 in ox-Ta COI1 and ox-Ta MYC2 plants also increased significantly compared with WT.?4?In vitro yeast two-hybrid verification of the interaction between Ta MYC2 and Ta ICE41:p GBKT7-Ta MYC2 and p GBKT7-Ta ICE41 were co-transformed with p GADT7-T into yeast competent cells,respectively,and grew well on DDO?SD/-Trp-His,second deficiency?medium PGBKT7-Ta ICE41 does not grow on TDO?SD/-Trp-His-Leu,three-deficient?medium,while p GBKT7-Ta MYC2 self-activates activity on QDO?SD/-Trp-His-Leu-Ade,four-deficient?medium was completely suppressed.p GBKT7-Ta MYC2 and p GADT7-Ta ICE41 or p GBKT7-Ta ICE41 and p GADT7-Ta MYC2 co-transformed on AH109 yeast competent cells on QDO medium,all can grow normally.The yeast two-hybrid experiment showed that the Ta MYC2protein interacts with the Ta ICE41 protein in vitro.