Functions And Mechanisms Of MsDOG1 In Seed Dormancy Of Magnolia Sieboldii K. Koch

Magnolia sieboldii K.Koch is a famous ornamental and medicinal plant with important economic value.Seeds belong to the morphophysiological dormancy type,and its natural reproduction ability is very weak.In this study,the seeds of M.sieboldii were used as the research object and the imbibition condition for breaking physiological dormancy of seeds was obtained through hormone treatments.On this basis,the MEdarkturquoise module closely related to seed physiological dormancy release and the hub gene Ms DOG1 in this module were excavated by Illumina high-throughput sequencing and gene co-expression network analysis.The molecular structure,expression characteristic and gene function of Ms DOG1 were studied through PCR,bioinformatic analysis,RT-q PCR and genetic transformation of Arabidopsis thaliana to reveal the function of Ms DOG1 and its action mechanism in seed physiological dormancy release.The main results are as follows:1.The M.sieboldii seeds were soaked in 1500μM GA₃ solution at 4℃and then cultivated to germination.It was found that the physiological dormancy was gradually relieved during imbibition,and the physiological dormancy was basically relieved at 24 h.The germination rate of seeds imbibed for 24 h,48 h,72 h and 96 h were 84.44%,80.00%,82.22%and 81.11%,respectively,all of which were beyond 80%.At all time points during imbibition,the contents of ABA and GA₃ were higher than those of the control,while the ABA/GA ratios were lower than those of the control.At the early stage of imbibition,glucose is the main direct energy source during 0-6 h,sucrose was hydrolyzed to produce glucose for energy supply during 6-12 h and starch was hydrolyzed to produce glucose for energy supply during 12-24 h.2.A total of 35145 genes were obtained by Illumina sequencing of 21 samples from M.sieboldii seeds imbibed for 0 h,6 h,12 h and 24 h in 1500μM GA₃ solution and distilled water at 4℃.GO and KEGG enrichment analyses of up-regulated genes in seeds between the two imbibition conditions showed that physiological dormancy release of seeds includes three key stages and processes.They were（1）signal transduction（GA signaling pathway activation）,（2）regulation of cell cycle（changes in the cell cycle of the dormant state）and（3）starch and sucrose metabolism（regulation of glucose content by starch and sucrose metabolism）.The only MEdarkturquoise module positively correlated with seeds in dormancy state and negatively correlated with seeds in physiological dormancy release state was obtained.The co-expression network of MEdarkturquoise module gene was constructed,and the hub gene Ms DOG1 in this module was obtained.3.The encoding sequence of Ms DOG1 gene was obtained.It is the length of 870 bp and encodes 289 amino acids.The molecular formula of Ms DOG1 protein is C₁₄₅₄H₂₃₀₃N₄₃₃O₄₅₀S₁₁,and the molecular weight of it is 33.402 k Da.The isoelectric point p I of Ms DOG1 protein is 6.02,and it is a hydrophilic protein.This protein belongs to the same clade as dicotyledonous DOG1s protein and has a conserved domain of DOG1 family.It was located in the nucleus through subcellular localization prediction.4.The CYC and UCE were screened to be used as internal reference genes for different organs and seeds with different development stages in M.sieboldii.The Cu/Zn-SOD and Mn-SOD were screened to be used as internal reference genes for imbibition process of seeds in 1500μM GA₃ solution at 4℃.The Cu/Zn-SOD and RPS13 were screened to be used as internal reference genes for stratification process of seeds at 4℃.The expression of Ms DOG1was mainly expressed in seeds and maintained a high transcriptional level during seed development and maturation.The expression of Ms DOG1 in seeds was inhibited by imbibition in 1500μM GA₃ solution at 4℃and stratification at 4℃.5.Seed germination tests of dog1-3 mutant lines,dog1-3 mutant recovery lines,wild-type lines and Ms DOG1 overexpressed lines showed that overexpression of Ms DOG1could enhance seed dormancy level and inhibit seed germination of Arabidopsis thaliana.Its regulation effect on seed dormancy was regulated by temperature.