Study On The Molecular Mechanism Of Regulation Of Dormancy And Germination Of Arabidopsis Seeds By Auxin Response Factors ARF10,16,17

Seed dormancy is a measure for higher plants actively adapting to harsh environments,and it is a feature of the higher plants that has evolved over a long period of time to gain advantage and avoid damage.Seed dormancy in agriculture can effectively prevent sprouting and reduce yield loss.Auxin is a second hormone found in recent years in addition to abscisic acid,that induces seed dormancy,And the auxin downstream response factor(ARF)ARF10/16/17 is an important regulatory factor involved in its process.However,the molecular mechanism regulating seed dormancy is not clear.This study screened and obtained the key gene DOG1 that regulates seed dormancy downstream,and further studied its molecular mechanism,obtained the following research results:1.Through bioinformatics search,the functional domains of ARF10,ARF16 and ARF17 were obtained and the downstream genes DOG1 that regulate seed dormancy of ARF10,ARF16 and ARF17 in Arabidopsis were screened by yeast one-hybrid method.It was verified that ARF7,ARF10,ARF16,ARF17,ARF19 and ABI3 could all interact specifically with the RY element in the promoter region of DOG1 gene,But the binding characteristics of ARF7 and ARF19 with the RY element in DOG1 promoter region were much lower than ARF10,ARF16,ARF17,ABI3.It is speculated that ARF7 and ARF19 may have corresponding differences in binding stability due to differences in amino acid composition.At the same time,it was speculated that DOG1 may play a central role in the process of auxin and ABA regulated seed dormancy and is a key node gene that regulates seed dormancy.2.The overexpression mutants of DOG1,ARF10 and ARF17 genes driven by the 35S promoter were constructed,and the dog1 mutant was constructed by using the CRISPR/Cas9 genome editing technology.Both of them can be used for the late double mutant function research.3.Realtime fluorescence quantitative detection of DOG1 gene expression in ARF7,ARF10,ARF16,ARF17,ARF19 mutants of fruit pods and immature seeds revealed that only ARF10,ARF16 and ARF17 mutants were found to be differentially expressed in preserved seeds and imbibed seeds.The difference of embryonic development was more significant than that of seed imbibition.No significant difference was detected between ARF7 and ARF19 in seeds of fruiting and sucking.It was presumed that this may not be involved in mediating DOG1 regulation of seed dormancy.4.Semi-quantitative RT-PCR detected the expression pattern of ABI and NCED family genes in ARF10,ARF16 and ARF17 overexpression mutants.The results showed that the mutants could promote the expression of ABI3,4,5 and NCED9,and improve the mutant seed dormancy and ABA sensitivity.The difference was significantly higher than that of the wild type.It was speculated that the mutant may mediate the ABA biosynthetic pathway by promoting the expression of NCED9 during seed imbibition and mediate the ABA signal transduction pathway by promoting the expression of ABI3,ABI4 and ABI5.5.The DOG1 promoter and RY element mutations of DOG1 promoter to drive DOG1 and GUS mutants were Constructed and were genetically identified respectively.Under the background of ARF10,ARF16 and ARF17 overexpression,they activated and upregulated DOG1 expression.After RY element mutation,ARF10,ARF16,ARF17 no longer have a regulatory effect on DOG1.Based on the above research results,Arabidopsis thaliana ARF10,ARF16,and ARF17 were regulated by direct binding to the RY element in the DOG1 promoter region and were involved in auxin-mediated regulation of seed dormancy and germination.that process which may be related to abscisic acid metabolic pathways.This result provides strong evidence for further revealing the molecular mechanism of Auxin-regulated Arabidopsis seed dormancy and germination.