Cloning And Functional Analysis Of PagC3H3 Gene In Populus Alba ×P.glandulosa

As the main component of wood,lignin is usually polymerized from three monomers.During its biosynthesis,a total of 10 enzyme families are involved in the conversion of phenylalanino to monmeric lignin,of which C3H is the key control enzymes in the hydroxylation process of p-coumaroyl CoA to caffeoyl CoA and in the formation of G/S monomer.Exploring the function of PagC3H3 Pgene is of great significance for tree selection and material improvement.In this paper,the PagC3H3 gene was cloned from 84K poplar,and the gene-specific expression analysis and promoter function were explored to verify the expression pattern of the gene.Preliminary study on the biological function of PagC3H3 gene by overexpression and RNAi inhibition of expression and genetic transformation of Populus alba×glandulosa,the results are as follows:(1)The full-length CDS of the PagC3H3 gene of Populus alba×P.glandulosa is 1527 bp,encoding a protein sequence consisting of 508 amino acid residues;Tissue-specific expression analysis showed that the expression of this gene was different in different tissues of Populus alba× P.glandulosa,and the expression level was higher in tissues with high degree of lignification such as root,middle stem segment and basal stem segment,and the expression level was lower in other parts;The 2035 bp long PagC3H3 promoter sequence was cloned and transiently transformed into Arabidopsis thaliana and genetically transformed Populus alba x P.glandulosa.The GUS staining results showed that the staining was deeper in the part with high degree of lignification.It is speculated that the PagC3H3 gene regulated by the Populus alba × P.glandulosa PagC3H3 promoter is strongly expressed in tissues with high degree of lignification.(2)The subcellular localization of PagC3H3 protein was verified.The results showed that the green fluorescent signal of PagC3H3-GFP was only displayed in the cytoplasm,suggesting that PagC3H3 is a functional protein localized in the cytoplasm.(3)The overexpressing line(OE-PagC3H3)has obvious growth advantage,the plant height is significantly higher than wild type,while the RNAi inhibitory expression line(RNAi-PagC3H3)grows slowly,the plant height is lower than wild type;The leaves of the overexpressing lines were larger than the wild type leaves at the same position,and the leaves of the RNAi-inhibited expression lines were smaller than the wild type leaves,and the seventh leaves of the same line were larger than the sixth leaves.(4)The xylem layer of the overexpressing line is more than the wild type,while the xylem layer of the RNAi inhibitory expression line is less than the wild type;the xylem of the eighth,ninth and tenth stem segments of the same plant from top to bottom,the number of layers is gradually increasing.(5)Determination of lignin,cellulose and hemicellulose content in different transgenic lines showed that overexpression of PagC3H3 gene significantly increased lignin content.inhibited expression significantly reduced lignin content while the difference in the content of cellulose and hemicellulose was not significant and it was speculated that the PagC3H3 gene played a role in lignin synthesis.(6)The insect resistance test of transgenic lines showed that the 3rd instar larvae of Gypsy moths tend to choose less lignin content when feeding,and the leaves of the expression-inhibiting lines with lower degree of lignitication,but the leaves of the overexpressing lines were eaten less,which further confirmed that the PagC3H3 gene plays an important role in the regulation of lignin synthesis in plants.