| Cotton(Gossypium hirsutum)is an important cash crop.Cotton fiber is the most important natural textile raw material in the textile industry.Cotton fiber deposits a large amount of cellulose during the thickening period of the secondary cell wall,so that the cellulose content in mature cotton fibers accounts for more than 90%,becoming a unique secondary cell wall model.This unique secondary cell wall model is the best material for studying secondary wall synthesis.R2R3-MYB transcription factors are the largest type of transcription factors in plants.They play an important role in regulating plant cell differentiation,secondary metabolism,and hormone response.In recent years,due to the demand for biomass energy,plant cell walls have received extensive attention.The formation of secondary walls is controlled by a transcriptional regulatory network consisting of multiple transcription factor genes.In this network,NAC transcription factors act as a first-level switch to activate downstream MYB transcription factors,and MYB transcription factors located at the second and third levels of the regulatory network regulate the expression of secondary wall synthesis genes to execute specific functions.MYB transcription factors play an important role in regulating secondary cell wall synthesis.We previously identified 36 R2R3-MYB transcription factors that may be involved in the regulation of secondary wall synthesis in cotton fiber.We selected two of them and named them GhFSM2,GhFSM6 respectively.In this paper,the functions of these two genes were analyzed,and the following experimental results were obtained:1.GhFSM2 expression in Arabidopsis results in late flowering,more rosette leaves,and increased trichomesIn order to study the function of the GhFSM2 gene,we constructed an overexpression vector and transformed Arabidopsis,and obtained overexpressing transgenic plants.Phenotypic observations revealed that compared with wild-type Arabidopsis,the number of rosette leaves in transgenic Arabidopsis increased,and the shape of rosette leaves became slender,in addition,the flowering time was delayed,which was delayed for about two weeks,that is,the late flowering phenomenon appeared.The Arabidopsis seedlings were observed under a stereomicroscope,and the number of epidermal hairs of the overexpression transgenic Arabidopsis was more than that of the WT plants.When grown to six weeks,the number of branches and seeds of transgenic Arabidopsis increased.2.GhFSM2 affects the formation of vascular bundlesTransgenic Arabidopsis appears stunted,and the inflorescence stems become thinner,which may be related to changes in the secondary wall components.Therefore,we performed free-hand sectioning,It is seen that the interfascicular fibers are reduced,the vascular bundles are incompletely developed and the cells appear to be broken.HD-ZIPⅢ family genes also play an important role in vascular development and secondary wall development.We speculate that overexpression of GhFSM2 might affect several HD-ZIPⅢ family genes.The RT-PCR results showed that the expression of these genes was changed in the over-expressed transgenic Arabidopsis stem,suggesting that GhFSM2 may affect the development of vascular bundles by regulating REV,AtHB8 and AtHB 15.3.GhFSM6 regulates secondary wall biosynthesisTo investigate the function of GhFSM6 in the secondary wall,we constructed a 35S-driven overexpression vector and transformed Arabidopsis.Throughout the growth cycle of transgenic Arabidopsis unlike WT,GhFSM6 transgenic Arabidopsis has up-curlycotyledons when cultivated for 8-10 days.Three weeks later,the rosette leaves of transgenic Arabidopsis thaliana rose up,curled,and the siliques of transgenic Arabidopsis became smaller and partially sterile,and the plant height also became dwarfed.Furthermore,we found that the sepals of transgenic Arabidopsis flowers showed enhanced lignification.In order to study whether GhFSM6 is actually involved in secondary wall regulation,we performed freehand sectioning of the six-week old transgenic Arabidopsis inflorescence stem compared with the wild type,darker staining in overexpressing transgenic Arabidopsis stems,suggesting higher cellulose and lignin content.Further semi-quantitative analysis of the secondary wall synthesis gene analysis showed that genes involved in the synthesis of secondary wall cellulose,lignin,and hemicellulose were up-regulated in overexpressing plants.In addition,we demonstrated that GhFSM6 can specifically activate the cotton fiber SCW cellulose synthase promoter bytransactivation assay experiments.The above experimental results indicate that GhFSM6 is regulating secondary wall synthesis.In addition,compared with the wild type,the anatomical structure of the flowers of the transgenic plants did not change significantly,but the filaments of the transgenic Arabidopsis thaliana were significantly shorter than those of the wild type plants,and more pollen was attached to the pistil style of the wild type plants.The pistil style is relatively smooth without pollen attachment.Wild type anther pollen sheds much,while genetically modified anther pollen rarely sheds.Further observation and analysis of the anthers under the microscope showed that wild-type anthers had cracks visible to the naked eye,while transgenic anthers had no obvious cracks and no pollen grains were seen.This may be the main cause of male sterility in transgenic Arabidopsis.4.GhFSM6 negatively regulates proanthocyanidin synthesisThe seed coat color of GhFSM6 overexpression transgenic Arabidopsis seeds is lighter than that of WT plants.The color of the seed coat is caused by the accumulation of proanthocyanidins.Therefore,we used the proanthocyanidin-specific dye DMACA to stain the seed coat.It was found that the seed coat color of wild type seeds was black and brown,while the overexpressed transgenic Arabidopsis seed coat was still yellow,suggesting that GhFSM6 may be involved in regulating the deposition of proanthocyanidins.The proanthocyanidin biosynthetic pathway was regulated by a ternary complex composed of R2R3-MYB(TT2),bHLH(TT8)and WD-Repeat(TTG1)proteins.We isolated GhTT2-3A and GhbHLH130D from cotton genome and cotransformed them into tobacco,it was found that proanthocyanidin(PA)was deposited in the leaves via DMACA staining,but when GhFSM6 was added,the proanthocyanidin deposition was much less.Transactivation assay showed that GhFSM6 was able to activate the promoter activity of PA-specific LAR(leucoanthocyanidin reductase)gene。5.Regulatory relationship between R2R3-MYBs related to secondary wall biosynthesisWe previously isolated 36 R2R3-MYB transcription factors that may be involved in secondary wall biosynthesis.We isolated seven of these transcription factor genes from the cotton cDNA library,and examined whether these transcription factors activate cotton fiber SCW CesA4,CesA7,and CesA8 promoters using the dual-luciferase reporter assay system.At the same time,We checked whether some MYB TFs were transcriptional repressors through another set of pGreen-SKⅡ system.The results show that the three genes(Gh_D07G1305,Gh_D08G1356,Gh_D11G11108)may act as transcription activators to regulate the secondary wall synthesis of cotton fibers.Gh_A12G0349,Gh_A12G2460,Gh_D12G2588,and Gh_D11G1653 act as transcription repressors to regulate thesynthesis of secondary walls.Moreover,we found that GhFSM6 can regulate the above three transcriptional activators,but whether GhFSM6 can regulate transcriptional repressors remains to be further studied.6.RNAi vector construction of three lignin synthesis genes and cotton transformationThrough co-expression analysis,we found that GhFSM6 can be co-expressed with several lignin synthesis related genes We speculate that the lignin synthesis gene may be its downstream target gene.First we investigated whether lignin is involved in cotton fiber development.Through in vitro ovule culture experiments,different concentrations of lignin monomer synthesis inhibitor PA was added into the medium,the fiber elongation was suppressed,we conclude that lignin is necessary for cotton fiber elongation.Then we isolated three genes that may be involved in lignin synthesis,Gh_A13G2072:a key enzyme gene belonging to the conversion of C3H coumarate hydroxylase H-type lignin to G/S type lignin,which has advantages during the development of secondary walls Expression is also co-expressed with some transcription factors.Gh_D1G1805:A key enzyme gene that belongs to F5H ferulic acid hydroxylase G-type lignin to S-type lignin.It is predominantly expressed during the secondary wall thickening stage and also co-expressed with some transcription factors.Gh_A11G2936:belongs to the key enzyme genes for the polymerization of Laccase laccase lignin monomers into lignin.They are highly expressed during the cotton fiber secondary wall thickening period.we isolated the F5H promoter,and GUS staining in transgenic Arabidopsis showed that its promoter activity was mainly expressed in the trichomes of rosette leaves,and stem leaves,then the RNAi vectors of these three genes were constructed and transformed cotton... |
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