Analyses Of Cotton Glycosyltransferase Genes(GT43A1 And GT47B)and Their Promoters

Cotton?Gossypium hirsutum L.?is an important fiber crop.Cotton fiber cells are differentiated from ovule epidermal cells.Their development process can be divided into four stages:fiber initiation,primary wall formation,secondary wall thickening,dehydration and maturation.In dicotyledonous plants and most monocotyledonous plants,xylan is the predominant hemicellulose polysaccharide in the secondary wall.It may be involved in determining the quality of cotton fiber.However,the specific role of xylan in the thickening of cotton fiber secondary wall is unclear.In our previous study,two putative xylan synthesis related genes:one gene from the GT43 glycosyltransferase family,GhGT43A1 and one from the GT47 family,GhGT47B were isolated,Also three MYB transcription factor genes,GhMYB1G,4G and 13G,which may be involved in the regulation of secondary wall synthesis were isolated.In this study,we analyzed the phenotype of GhGT43A1-RNAi silenced transgenic cotton.We also examined the activities of GhGT43A1 and GhGT47B promoters,the main results are as below:1.Phenotypic analyses of GhGT43A1-RNAi transgenic cottonGhGT43A1-RNAi vector was constructed and transformed into cotton,positive transgenic lines were obtained.Analysis of T2,T3 generation transgenic lines showed that GhGT43A1 expression was markedly reduced in 15DPA?day post anthesis?and 20 DPA fibers in transgenic plants compared to wild type control.The mature fibers from transgenic lines were shorter,also the bolls and the seeds were smaller as compared with the wild type.2.Activity analyses of GhGT43A1and GhGT47B promotersWe constructed the GhGT43A1/GhGT47B Promoter::GUS vectors and transformed into Arabidopsis thaliana.GUS staining was performed in the transgenic Arabidopsis plants.The staining results revealed that GhGT43A1 and GhGT47B can drive GUS expression in vascular bundles of flowers,rosette leaves and stems in the transgenic plants,suggesting that these genes may be involved in the secondary wall synthesis.Furthermore,Mannitol and NaCl treatments can repress the promoter activity,while MeJA and GA treatments can induce the expression of GUS.The results implied that drought and salt stresses can inhibit GhGT43A1 and GhGT47B promoter activity,whereas MeJA and GA is able to activate the expression of GhGT43A1 and GhGT47B.3.GhMYB4G may regulate secondary wall biosynthesispBI121-GhMYB4G overexpression vector was constructed and introduced into Arabidopsis thaliana to obtain transgenic plants.Phenotypic analysis showed that the 7-day-old roots of the transgenic seedlings exhibited ectopic patchy lignin deposition.In seven-week-old stem sections,no ectopic secondary wall deposition was detected,the expression of genes related to the secondary wall synthesis was down-regulated,and the expression of AtMYB46 was suppressed as well.It seemed that GhMYB4G might play a negative regulatory role in stems of transgenic Arabidopsis but act positively in roots.Additionally,GhMYB4G promoter::GUS vector was constructed and transferred to Arabidopsis thaliana.GUS staining results showed that GhMYB4G promoter could induce the expression of GUS in flowers and rosette leaves,the GUS staining was strong in the flower.4.Transactivation assay of GhGT43Al and GhGT47B promotersOur previous studies revealed that GhMYB1G and GhMYB13G may regulate secondary wall biosynthesis.To investigate whether this two MYB genes can regulate expression of GhGT43Al and GhGT47B promoters.We crossed the homozygous transgenic Arabidopsis harboring GhGT43A1/47B Promoter::GUS vector and transgenic Arabidopsis carrying pBI121-GhMYB1G/GhMYB13G vectors.GUS staining results displayed that GhMYB13G can activate the expression of GhGT43A1 and GhGT47B.5.Yeast two-hybrid assayWe isolated one phylogenetically related GhGT43A1 genes,namely CotAD₅2122,and one phylogenetically related GhGT43C1 genes,namely CotAD₃3550 from cotton genome.We used yeast two-hybrid assay to examine if these GT43 family proteins can interact.We constructed AD and BD vectors,that is:AD-GhGT43A1,AD-GhGT43C1,AD-52122,AD-33550,BD-GhGT43A1,BD-GhGT43C1,BD-52122,BD-33550.The experiments are still ongoing.