Functional Characterization Of Poplar PtrGARP1

Wood is important renewable energy source in nature,which derives from secondary growth in stems and roots of plants.Plant secondary growth is a process that vascular cambium cells continually differentiate and develop with the participation of many genes.There are many ways to identify the molecular mechanism of plant wood formation.In this study we explore the function of a unknown Populus trichocarpa gene PtrGARP1;1,which may be involved in plant secondary growth.The main research results are as follows:The Locus Name of PtrGARP1;1 is Potri.002G223100.The full CDS consists of 510 nucleotides that encode 169 amino acids.It is the most characteristic that the amino acid sequence contains abundance of glutamates.Homology search showed that the homologous genes of the PtrGARP1;1 exist in various plants such as Populus euphratica,Theobroma cacao,Gossypium hirsutum and other species.Sequence alignment revealed these genes also have conserved glutamate sites,suggesting that glutamate sites may be required for the gene performing function.RT-PCR analysis indicate that the PtrGARP1;1 gene was highly expressed in the xylem of Populus and the increase of transcriptional expression level was in accordance with the aggravation of lignification of young stems.The expression of PtrGARP1;1 was further explored in situ hybridization,indicating that the PtrGARP1;1 gene was highly expressed in the xylem and phloem of Populus trichocarpa.The result was consistent with RT-PCR analysis.PtrGARP1;1::GFP plant expression vector was constructed by fusing reporter gene GFP using Gateway technology,then the construct was transformed into Arabidopsis.We monitor GFP fluorescence of the roots of transgenic Arabidopsis and found that PtrGARP1;1 may be localized to the cytoplasm.The overexpression of PtrGARP1;1 material was obtained by genetic transformation of Arabidopsis thaliana.Cell morphology observed by tissue anatomy and chemical staining did not change in the stem cells of overexpressing PtrGARP1;1 compared with the wild type.We created PtrGARP1;1 mutant based on CRISPR/Cas9 technology.Phenotypic observation exhibited that compared to wild type,the mutant plants grow more slowly and the leaves were more narrow and smaller;the lignification of tissue developed more slowly and the cell wall of the xylem tissue were thinner in the stems of high lignification.We presume that the gene may associate with the formation of secondary cell wall and participation in the development and formation of wood.