Studies On Molecular Mechanism Of Transcription Factor GhNAC140 In Regulating Cotton Fiber Development

Cotton is one of the most important commercial crops in the world.Cotton fiber is a raw material for textiles and fine chemicals,and an important strategic material.Secondary cell wall(SCW)deposition directly affects the fiber yield and quality.The SCW biosynthetic process is regulated by multilayer transcriptional regulatory network,among which transcription factor(TF)plays an important role.NAC transcription factor is one of the largest plant specific transcription factor families.GhNAC140 is a NAC transcription factor that is specificly expressed in cotton fiber.The molecular mechanism of GhNAC140 in regulating fiber development in this paper was analyzed,and the results were summarized as follows:(1)Expression analysis of NAC140 gene.Quantitative analysis showed that NAC140 gene is expressed exclusively in fiber tissues among roots,stems,leaves,fibers and ovule tissues of different periods in upland cotton TM-1.In TM-1,the expression pattern of GhNAC140-At and GhNAC140-Dt is same,but the expression level of GhNAC140-Dt is significantly higher than that of GhNAC140-At.However,the expression level of GhNAC140 in upland cotton was significantly higher than that of sea-island cotton.(2)Analysis of GhNAC140 downstream regulatory genes.Co-expression analysis identified 33 genes that might be the targets of GhNAC140 transcription factor.This includes the secondary wall specific expression gene GhCOBL9,which has been reported in our laboratory.Analysis on the promoter of GhCOBL9 revealed that there were 13 NAC transcription factor.The dual luciferase system was used to determine the Luc/Ren value,which further demonstrated that the GhNAC140 transcription factor enhanced the activity of GhCOBL9 gene promoter.(3)Overexpression and RNAi transgenic cotton generation of GhNAC140 gene.Cloning the ORF sequences of GhNAC140-At and GhNAC140-Dt in upland cotton TM-1,and inserting the Flag tag to the 3’-end of GhNAC140-At/Dt gene in pCAMBIA2301 to construct the five Flag-tagged or untagged overexpression vector.453 bp ORF sequence of GhNAC140 was ligated into the pART27 vector with forward and reverse complementary sequences,respectively,to construct the GhNAC140 RNAi expression vector.Together with control vector,the constructed six vecotrs were transformed into Agrobacterium LBA4404,respectively.Transgenic cotton plants were generated using Agrobacterium mediated method.Different transgenic cotton TO plants were obtained and PCR-based genotyped.(4)Structural analysis of NAC140 promoter in different cotton species.By analyzing the promoter sequence of GhNAC140,it was found that the promoter of GhNAC140-Dt contains a 5107 bp Copia-LTR type retrotransposon insertion in TM-1.By PCR detecting on diploid cotton,wild,simi-wild and cultivated cotton varities,the retrotransposon were not presented in diploid,wild and sea-island cotton genomes,but presented in some semi-wild cotton and some upland cotton varieties.The above studies indicate that the retrotransposon was inserted into semi-wild cotton during domestication of upland cotton,and it was further retained in the selection of upland cotton varieties exists in some upland cotton varieties.(5)Correlation analysis between retrotransposon presence and yield traits in upland cotton.By using two sets of natural populations of upland cotton from different sources,combined with the phenotypic data from nine environmental conditions,the association analysis was conducted between retrotransposon presence and yield traits.In the natural population composed of 277 upland cotton core germplasm materials,167 materials contained the retrotransposon sequence in the GhNAC140-Dt promoter,and 110 materials did not contain the retrotransposon.Phenotypic data from nine environmental conditions were used for correlation analysis.The results showed that compared with the materials without the retrotransposon,materials with the retrotransposon had significantly higher lint percentage and boll number,while boll weight and seed index is significantly reduced.Using phenotypic data from a population consisting 331 upland cotton varieties,it was found that the lint percentage and lint index were significantly higher in materials containing retrotransposon than that in materials without retrotransposon.These results indicated that the retrotransposon in the promoter region of GhNAC140 was closely related to the cotton yield traits,especially to the lint percentage.(6)Retrotransposon insertion enhanced the promoter activity of GhNAC140.Sequences of-7100 bp promoter region of GhNAC140-Dt in TM-1 with retrotransposon and-2300 bp promoter region of GhNAC140-Dt in W0 without retrotransposon were cloned and inserted upstream of the luciferase gene to construct the dual luciferase system report system.It was found that the activity of GhNAC140-Dt promoter from TM-1 with retrotransposon was higher than that of GhNAC140-Dt promoter from WO without retrotransposon.(7)Establishing of a method for gene transient expression in cotton fibers.Using cotton fibers at different developmental stages as receptor,the transient infection conditions were investigated via comparative analysis on Agrobacterium strains,different infection times,and different co-culturation times.GFP and GUS were used as reporters.Specific conditions for Agrobacterium mediated transient expression in cotton fibers were determined.Using the established method,transient expression of fiber expressed genes GhFSN1,GhMYB212,GhCFE,GhSusc4 were conducted.The promoter activity of the GhFSN1,GhMYB212 was also investigated via transient expression in the fibers.The establied method demonstrated that the activity of GhNAC140-Dt promoter with retrotransposon was higher than that of GhNAC140-At promoter without retrotransposon.