Functions And Mechanisms Of MsARF5 In Embryo Development Of Magnolia Sieboldii K.Koch During The Seed After-ripening

The Magnolia sieboldii K.Koch is a kind of national protected wild plant under third class state with high ornamental and medicinal value,which has broad prospect of development and application.To study the role of MsARF5 in the seed of M.sieboldii during after-ripening,the molecular structure,spatio-temporal expression,gene function,promoter activity,and regulation mechanism were tested and analyzed by using Iso-Seq sequencing,SMARTer RACE sequence amplification,chromosome walking,Agrobacterium-mediated tobacco transient expression,Arabidopsis thaliana stable expression,and enzyme-linked immunoassay technique.The main results are described as followings:1.The m RNA-mi RNA sequencing of M.sieboldii in 0 d,45 d,75 d and 90 d,4 significant turning points of maturation and germination in the process of seed stratification and germination,were sequenced.In total,158,083 high-quality single genes,14,499 differential genes and 571 m RNA-mi RNA relationships were obtained.The results reveal that the seed germination regulatory network consists of 4 critical parts:(I)degrading lipids and macromolecular carbohydrates;(II)reducing the seed germination inhibitors;(III)involved in stress response;(IV)regulating the hormone related to the seed germination and dormancy.2.The spatiotemporal expression pattern of MsARF5 shows high expressions after imbibitions,and the lowest expression appearing at mature period.Also,2 peaks of expression appear at 30 and 75 days during the seed stratification.These changes are consistent with auxin content.Auxin signal transduction genes PIN1,IAA6,IAA16,etc.are positively correlated with the expression of MsARF5,while IAA14 and ARX3 are negatively correlated.3.The full-length c DNAs of MsARF5 was acquired.It harbours a 2,958 bp open reading frame(ORF)encoding a predicted protein of 985 amino acid residues with a calculated mass of 109.277 k Da and a theoretical isoelectric point of 5.33.The analysis of MsARF5 shows that it has strong hydrophilicity,no transmembrane structur,and sharing 84% identity with MsARF5 of Chinese lotus(Nelumbo nucifera).The MsARF5 promoter fragment which is 2,568 bp was achieved by PCR strategy.The cis-acting elements on the promoter sequence were predicted by bioinformatics software.The putative cis-regulatory elements of MsARF5 promoter was analyzed and 20 types of potential cis-acting elements about hormone response and regulation were found.The response to 0.1 m M IAA and IBA indicate that-2544 to-1310 bp and-859 to-400 bp are the major regions regulating promoter activity.4.The overexperssion vector of MsARF5 was transformed into A.thaliana by floral dipping method.After screeningand PCR identification,transgenic plants were obtained.The hormone treatments on transgenic A.thaliana shows that the expression of MsARF5 in A.thaliana can promote the development of plant vascular tissue,advance the seed germination and bolting time,increase the seedling height and leaf width,and restore the characters of A.thaliana arf5 mutants that are not fruiting and late-germinating.All these results prove that MsARF5 has the functions of promoting the formation of plant vascular tissue,regulating seed development and germination.