The Research On The Function Of The Key Gene DiCCoAOMT1 In The Endocarp Lignification Of Davidia Involucrata Baill.

Dove tree(Davidia involucrata Baill.)is a deciduous tree of the family Davidia involucrata in the family Davidia involucrata.The species is listed as a national key protected wild plants.The endocarp of Davidia involucrata rapidly lignifies to form a thick,hard,dense,and tight suture structure,which affects the material exchange between the seed and the external environment,leading to long-term dormancy of the seed.Based on the data of Davidia involucrata seed transcriptome,the research team selected 14 members of the CCoAOMT gene family whose expression level continued to increase as the accumulation of lignin in the seeds deepened,and selected the most significant differential expression transcript Cluster-149.63013 for cloning.And the gene was named DiCCoAOMT1(KY243330).The gene is specifically expressed in the endocarp,and in vitro activity verification shows that the product encoded by the gene has a specific and strong O-methyltransferase activity.CCoAOMT catalyzes the formation of ferulyl-CoA during the synthesis of lignin monomers.Based on the research results obtained,we believe that this gene plays an important role in the lignin synthesis of Davidia involucrata endocarp.In order to further analyze the function of DiCCoAOMT1 gene,this research constructed a plant expression vector of DiCCoAOMT1 gene,through Agrobacterium-mediated genetic transformation of Arabidopsis thaliana and poplar,and observed the overexpression of the target gene on the growth and lignin accumulation of recipient plants to verify the function of this gene.It will lay the foundation for the further exploration of the molecular regulation mechanism of the physiological processes of Davidia involucrata rind development,lignin accumulation and seed dormancy.1.Overexpression of DiCCoAOMT1 gene and phenotype in Arabidopsis thaliana The recombinant vector containing the gene of interest was genetically transformed into Arabidopsis thaliana by inflorescence infusion.After resistance and PCR screening,a total of 18 transgenic Arabidopsis strains were obtained.After homozygous screening,5 homozygous transgenic Arabidopsis strains were finally obtained.The length of transgenic Arabidopsis pods length increased by 12.7%and the plant height increased by 9.87%compared with wild type.The determination of lignin content showed that the lignin content of transgenic Arabidopsis thaliana increased by about 45%.The results of paraffin-stained sections showed that the lignin content of transgenic Arabidopsis thaliana increased and the secondary xylem thickened.2.Overexpression of DiCCoAOMT1 gene and phenotype in poplarThree positive transgenic poplar seedlings were obtained by genetic transformation of poplar.Through observation,transgenic poplars had significantly increased stem nodes and petioles compared to wild-type poplars,with growth rates of 27.69%and 27.02%,respectively.The determination of lignin content showed that the lignin content of transgenic poplars increased by an average of 20%.Paraffin-stained sections showed that the lignin content of transgenic poplars increased and secondary xylem thickened.Gas chromatography was used to determine the lignin monomer composition.The results showed that the G and S monomers of transgenic poplars increased significantly,the H monomers did not change significantly,and S/G increased.In this research,heterologous overexpression of Davidia involucrata DiCCoAOMT1 gene proved that this gene can promote lignin biosynthesis and provide more evidence for enriching the function of the CCoAOMT gene family.