| Secondary wall deposition in plants are regulated by a series of transcription factors and genes.Lignin,as the main component of secondary wall,accounts for 10%?30%of the dry weight of plants.It has important functions such as maintaining the shape and strength of cell wall,promoting water and nutrient transport,and resisting biological and abiotic stress.Laccase,as a polycopper oxidase,can oxidize and polymerize lignin monomer to form lignin.In addition,laccase also is involved in a variety of other biological processes,such as wound repair,iron metabolism,and maintaining the structure and integrity of cell walls.The function and the mechanism of cell wall biosynthesis of laccase genes PtrLAC16,PtrLAClike and PtrLAC17 were studied.The main research results are as follows:1.Laccase genes PtrLAC16,PtrLAClike and PtrLAC17 were specifically expressed in vascular tissue.The prediction results of cis-acting elements showed at least three AC elements and one SMRE element(the MYB response element in secondary wall synthesis)in the three laccase genes.The three promoter sequences of laccase gene were fused with GUS gene to construct the fusion expression vector,respectively.The results of GUS histochemical staining showed that all three laccase genes were specifically expressed in vascular tissues of tobacco roots,stems and leaves,but there were localization differences among the three genes in vascular tissues.Root localization:PtrLAC16 had high expression in the middle column and endodermis.PtrLAClike was mainly located in secondary vascular tissues and endodermis of the root.PtrLAC17 was located in the secondary vascular tissue of the root.Stem localization:PtrLAC16 was mainly located in the primary xylem of the stem.PtrLAClike existed in primary xylem and cambium cells of the young stem.PtrLAC17 expressed in the cambium and secondary xylem of the stem.Leaf localization:PtrLAC16 was located in the vascular bundles sheath of main leaf vein.The location of PtrLAClike was mainly concentrated in vascular bundles of leaf veins,including catheter,sieve tube and thick-walled cells.PtrLAC17 existed in the middle column and endodermis of the leaf veins.2.The location of vascular-specific elements were identified in PtrLAC16 promoter.Using the 5'-end deletion analysis of promoter sequence,the two AC and SMRE elements,which located in-275?-408 region in PtrLAC16 promoter,determine the vascular-specific expression.The loss of AC elements in the-90?-275 region results in the loss of root-specific expression.And the SMRE element at-77 region was important for leaf-specific expression.3.Subcellular localization differences of PtrLAC16,PtrLAClike and PtrLAC17 indicated that functional differentiation was possible.The results of immunocolloidal gold hybridization indicated that PtrLAC16 was mainly located in cell walls of fiber cell and tracheary elements,and PtrLAC17was mainly located in the cytoplasm of cambium cells and the cell wall of wood fiber of the stem,and PtrLAClike existed in the cambium cell wall and the wood fiber cell wall near the cambium.4.The active laccase PtrLAC17 was obtained and the enzyme activity was analyzed.A eukaryotic expression system of laccase was established based on tobacco leafs.Laccase proteins were expressed in tobacco leaves using co-expressed p BIPtrLACS-His and p19(as a cofactor),respectively.PtrLACs-overexpressing cell lines were also obtained by using the available transgenic materials in the laboratory.The active laccase proteins were purified by ammonium sulfate and antibody column.The michaelis constant values were obtained by enzyme activity analysis:Km=0.33mmol/L,Vmax=0.53 mol·L-1·min-15.Histochemical analysis of transgenic poplar plants.Gene and protein expression levels of overexpression or antisense expression transgenic plants were analyzed by q RT-PCR and Elisa.The results showed that the three laccase genes had different effects on the thickness of the cell wall and the morphology of the vessels.Raman examinations also indicated that three laccase genes were involved in lignin synthesis and affected the distribution and content of lignin in the stem.he most significant changes of lignin in PtrLAClike transgenic lines indicated that it plays an important role in xylem development.Our study showed that there were differential expression among the three laccase genes.It indicated that there were functional differentiation of the three genes during xylem development.The three laccase genes may have different mechanisms involved in xylem development.The active laccase which was successfully obtained by eukaryotic expression system,was used for the study of the enzymatic characteristics of laccase.This study provides a new thought and theoretical basis for the further realization of lignin synthesis regulation in poplar. |
PDF Full Text Request