Function Characterization Of PtLIM1 Gene In The Regulation Of Secondary Wall Synthesis In Populus Trichocarpa

As a kind of bioenergy with great economic value,wood is widely used in all aspects of human life and industrial production.The structure of wood is mainly composed of secondary xylem,Lignin and cellulose are the main components.Understanding and studying the mechanism of plant secondary wall biosynthesis not only has important scientific significance for the basic research of plant secondary development but also has laid a solid foundation for the selection and breeding of new tree varieties that meet the needs of human production and life.The plant LIM protein family generally contains two LIM domains(Lin-Isl-Mec domain)separated by 40～50 amino acid residues,with one or more zinc finger structures in the molecular structure.In different development periods and different development types of cells,LIM protein through the zinc finger protein structure influence the interaction between proteins.the structure of protein and protein kinase,transcription factors and other biological activities are changed,plant LIM proteins can be used as lignin biosynthesis and actin transcription factor binding protein.Therefore,the study of LIM protein is of great significance for the growth and development of cells.Based on phylogenetic tree analysis and tissue expression analysis of poplar PtLIM1,combined with previous research reports,we speculated that PtLIM1 gene may be involved in the formation of plant secondary wall.In addition,tissue sections,genetics,molecular biology and other research methods were used to further study the effects of PtLIM1 expression changes on wood property,in order to provide molecular theoretical and technical support for the improvement of wood property of fast-growing tree species.The main research results are as follows:(1)Cloning and Sequence Analysis of PtLIM1 GeneA 621 bp full-length CDS sequence encoding 206 amino acids was cloned from the xylem of Populus tomentosa.Through the prediction of the gene structure,the protein encoded by it contains three zinc finger protein domains.The phylogenetic analysis of this gene showed that the PtLIM1 gene was closely related to the At PLIM2 b gene of Arabidopsis thaliana,and was mainly expressed in mature pollen,followed by high expression in Apoplar,which played a role in the synthesis of secondary wall lignin and cellulose.(2)Expression specificity analysis of PtLIM1PtLIM1 is highly expressed in xylem,relatively high in phloem,low in leaf,and not expressed in root.It suggest that PtLIM1 may be involved in biosynthesis of secondary wall.The synthesis process of secondary wall is complex,which is coordinated by hormones,signal molecules and transcription molecules in time and space.The expression of PtLIM1 gene was up-regulated in different degrees when treated with a certain concentration of gibberellin,auxin,ethephon,abscisic acid and brassinolide,these results suggest that hormones may affect the synthesis of lignin and cellulose by affecting the expression of genes.(3)Functional analysis of PtLIM1 in transgenic poplarA 35S:PtLIM1 overexpression vector was constructed to transform the leaves of Poplar sinensis by Agrobacterium-mediated transformation.The transgenic seedlings screened for initial Kan resistance were used to extract RNA for quantitative PCR to identify the changes in gene expression,and the transgenic positive plants were obtained.The PtLIM1 overexpression of transgenic poplar thinned the stem tip and reduced the biomass.Stem tissue sections showed that the overexpression of PtLIM1 gene resulted in a narrower xylem region and a larger pith core.Secondly,the determination of lignin and cellulose content showed that lignin content decreased while cellulose content increased.It is concluded that PtLIM1 negatively regulates the formation of lignin and positively regulates the formation of cellulose in the secondary wall of poplar.Quantitative PCR detection of PtLIM1 overexpressed transgenic plants growing in greenhouse for 3 months showed that the expression levels of key enzyme genes in the PtLIM1 lignin pathway were decreased,the expression levels of key enzyme genes in the cellulose synthesis pathway were up-regulated.Thus,we can conclude that PtLIM1 affects the synthesis of substances by regulating the expression of key enzymes in lignin and cellulose pathways.