Cloning And Functional Characterization Of BnNAC19,BnNAC24 And BnMYB46 Genes In Ramie

Ramie(Boehmeria nivea L.Gaud)is one of the most important natural fiber crops.The biosynthesis of secondary cellular walls(SCWs)has fiber cells has an important influence on fiber yield.In Arabidopsis,the biosynthesis of SCWs was regulated by the regulatory network mainly consisted of NAC and MYB transcription factors.However,in ramie,it is still unknown for the function mechanism that the NAC and MYB transcription factors conferred to fiber development.In this study,Phylogenetic analysis revealed that the BnNAC19,BnNAC24,and BnMYB46 were highly homologous with Arabidopsis SND1,ST1/2 and AtMYB46 in protein,respectively.Two NAC genes(BnNAC19 and BnNAC24)and one MYB gene(BnMYB46)were cloned in ramie.The vectors of these three genes for overexpressed,subcellular cellular located and trans-activated experiments were constructed.Overexpression of these three genes in Arabidopsis and tobacco found to confer more fiber cells,along with a remarkable thickening observed in the secondary wall of these cells in transgenic plants of BnNAC24.A significant increase of crude fibers was also observed in transgenic tobacco of BnNAC19.Expression analysis in transgenic Arabidopsis showed that the expression of AtMYB46(which is a key gene involved in SCWs biosynthesis of fiber)was dramatically up-regulating by BnNAC19 and BnNAC24.We performed a subcellular localization experiment for three genes and found that their encoding protein expressed in the cell nucleus,indicating that the protein contains an authentic NLS.Transactivation assay in the yeast using the full-length or C-terminal region sequences of three genes fused to the DNA-binding domain of GAL4 produced the same transactivation activity,which suggested that the C-terminal region in three genes had an activation domain.Three genes(aspartic acid protease gene,fructosediphosphate aldolase gene,and serine hydroxymethyltransferase gene)that interacted with BnNAC19 were identified by yeast hybrid experiment,of which aspartic acid protease gene was mainly involved in programmed cell death.Expressed pattern analysis revealed that BnMYB46 highly expressed in stem bark with mature fibers.In conclusion,these results revealed that BnNAC19,BnNAC24,and BnMYB46 were three transcription factor-encoding genes.The BnNAC19 and BnNAC24 conferred the SCW biosynthesis by upregulating the expression of AtMYB46 in Arabidopsis.BnNAC19 are potentially involved in the programmed cell death in the process of fiber formation,and BnMYB46 probably has an important role in the stage of fiber mature.The identification and functional characterization of these three genes provide new insights into bast fiber formation in ramie.