Metabolomics Analysis Of Reaction Wood Formation From Betula Platyphylla And BpMYB4 Gene Expression And Functional Analysis

MYB transcription factor family refers to a class transcription factor containing MYB domains.They are widely involved in various regulation of development and metabolism,such as cell differentiation,cell cycle regulation,hormonal and environmental factors response,and regulation of morphogenesis and secondary metabolites.Through homologous sequence alignment to eambium tissue eDNA libraries,obtained AtMYB4 of homologous genes,named BpMYB4.In this paper,we did a series of studies of BpMYB4.RT-PCR results showed that BpMYB4 gene was expressed in different tissues,the highest expression in flower tissues,which is consistent with ithibition factor expression characteristics;The expression level of tension wood increased,initially identified BpMYB4 was lignin biosynthesis inhibitor.Obtaining BpMYB4 gene promoter by chromosome walking method.PLACE analysis found that the promoter containing the cyrokinin-related components,the anthers and pollen development related components,GA-responsive element,Dof protein binding sites and cold stress and shedding acid response elements.GUS staining revealed that the GUS gene specific expression in stems,petioles and shoots,which is consistent with the results of quantitative analysis,suggesting that BpMYB4 may be involved in the development of vascular tissue and xylem development.Constructing overexpression vector pROK ?-BpMYB4 and inhibit expression vector RNAi-BpMYB4,electric shock shift to Agrobacterium,and infection instantaneous,RT-PCR analysis showed that overexpression lines upregulation 10 times;inhibiting the expression lines down 28 times.Indicating that the expression vector had a function of overexpression and inhibit expression.Constructing fusion vector pROK ?-BpMYB4-GFP,onion epidermal transformation by gene gun bombardment,the results show,BpMYB4 gene expression in the nucleus.Using Agrobacterium-mediated method,we tansformed the pROK ?-BpMYB4-GFP into Arabidopsis thaliana.Found that the transgenic plants compared to the wild-type stem growth speed,increased branching of the stem.We presumably not inhibit the lignin synthesis,the cellulose content is also increased.Determination of lignin and cellulose showed that transgenic lines have significantly increased cellulose content,lignin content decreased.Using yeast one-hybrid technology,screened interaction components,namely ACGTATERD1,CGACGOSAMY3,DOFCOREZM,LTRECOREATCOR15,TAAAGSTKST1.These elements have dehydration response,participate in carbon metabolism and stem tissue-specific expression,cold induced,stomatal control and so on.Suggesting that BpMYB4 may be involved in carbon metabolism in response to dehydration,stem tissue by specifically identify these components.The mainly cis-acting elements in cellulose synthase genes and seven Arabidopsis MYB transcription factor interaction analysis,and found that the MYBST1 and AtMYB61 gene interaction.Further analysis component where cellulose synthase gene promoter fragment AtCEV1 and AtMYB61 gene interaction.Indicate that AtMYB61 regulates cellulose synthesis through.AtCEV1 gene interaction.AtMYB61 and BpMYB4 61%homology,and are associated with cell wall formation genes,suggesting that BpMYB4 may regulate cellulose anabolic.Metabolomics analyzed metabolites in the xylem of birch wood stress-related formed layers,found that there are 142 kinds of changes in metabolites,such as glucose,a phosphoric acid,glucuronic acid,xylitol,ribitol,shikimic acid,coniferyl alcohol and so on.The study found,tension wood promoting the synthesis of cellulose,inhibit lignin synthesis by changing lignocellulosic metabolite.