Molecular Cloning And Expression Analysis Of Fiber Development Ralated GhAQP And GhNAC Gene In Cotton

Cotton is one of the most important economic crops in the word and is important raw textile material in textile industry, and plays an important role in the agriculture and national products. However, with the development of textile industry, new textile technology requires high cotton fiber quality, so it is the one of main work for cotton breeders to breed varieties with longer, stronger, and thinner fiber. Therefore, it is effective to improve the quality and yield of cotton fiber, and enhance the international market competitiveness of cotton fiber industry and textile industry through isolating and identifying some genes involed in cotton fiber development, and analyzing their expression profile and regulation mechanism on the molecular level.AQP is a kind of membrane channel proteins that allow plants to rapidly and efficiently transport water molecules and other small molecules by altering their membrane permeability, and had the consensus sequence HINPAVTFG of MIP family and two highly conserved peptides Asn-Pro-Ala (NPA), which play a different role in the process of the plant important life and physiological activities. NAC transcription factors are unique to higher land plants, which were discovered in the last decade. They contain a conserved NAC domain which is composed of150amino acids in N-terminal ends and a highly variable transcription regulatory domain in C-terminal ends. The current research about NAC transcription factors mainly focus in the model plant Arabidopsis and rice, less for the other plants. Gene isolation and functional identification of cotton NAC is little reported; the role of the NAC protein in cotton fiber cell growth, cell wall morphogenesis currently has been not reported.The excellent recombinant inbred line (69307) with high fiber strength was selected as material, which developed from an F6.g population of upland cotton(Gossypium hirsutum L.) cross of sGK9708X0-153. sGK9708is the commercial transgenic cultivar resistant to budworm. and0-153with high fiber strength. Based on bioinformatic database, including cotton EST, Tigr and the Plantgdb and SSH-cDNA library constructed in our lab, we carried out cluster analysis and expression profile analysis by real-time quantitative RT-PCR technology. Three AQP genes and four genes that code NAC transcription factors were identified, which associated with cotton fiber development, and preliminary function study was conducted by cloning, expression pattern analysis, phylogenetic analysis and constructing expression vectors into Arabidopsis. The study is expected to provide an important functional gene through genetic engineering techniques in order to improve the cotton fiber quality.This not only laid a solid foundation for in-depth research function of GhAQP, and GhNAC genes in the cotton fiber development, but also provides the useful gene elementis on fiber quality molecular breeding. Therefore, this study not only has important theoretical significance, but also has a strong application value. The main results are as following:1. One gene which contains the complete ORF was isolated from cotton SSH library which encoded aquaporin, named GhAQP2. We obtained the full-length cDNA sequences of GhAQP3and GhAQP4by homology cloning, and the full-length genomic DNA sequences of GhAQP2, GhAQP3and GhAQP4with cotton genomic DNA as a template. GhAQP2and GhAQP3are composed of four exons and threeintrons, GhAQP4has three exons and two introns in comparison of their sequences of genomic DNA and cDNA. Tese genes encode308,291,278amino acids, respectively.2. We obtained53ESTs about NAC transcription facors in Gossypium hirsutum L by using the programs tBLASTn withArabidopsis NAC protein as a template in the NCBI database. By quantitative RT-PCR to build their expression profiles, and screen four predominantly expressed genes in fiber cells:GhNAC1, GhNAC12, GhNAC17, and GhNAC28. We obtained full-length cDNA sequences and full-length genomic DNA sequences of GhNAC12and GhNAC28by gene cloning. For these genes, the full length of ORF is1038,1236bp; code345,411amino acid residues. Full-length genomic DNA sequences of GhNAC12and GhNAC28is1431,1554bp; all contain three exons and two introns.3. Quantitative Real-time PCR assay was performed to analyze the expression patterns of seven genes in cotton different organs and fibers in different days post anthesis. GhAQP2is preferentially expressed in the late stage of fiber elongation; GhAQP3is strongly expressed in cotyledons and hypocotyls in cotton; GhAQP4has an expression advantage in the early stage of fiber elongation. So we speculated that the three genes play a role in the different tissues. Especially, GhAQP2predominantly expressed in20DPA fiber cells, which provides an importantinformation for the further research on the gene-expression regulation during developmental stages from fiber cell elongation to the secondary cell wall synthesis. GhNACl and GhNAC17are preferentially expressed in fiber elongation to the turning of the secondary cell wall thickening period; GhNAC12is ubiquitously expressed in fiber cells and non-fiber cells; GhNAC28is expressed specifically during developmental stages from fiber cell elongation to secondary cell wall synthesis, and not expressed in non-fiber cells.4. In order to investigate the function of GhNAC12, the gene was cloned into the PCRI121plant vector to obtain overexpression vector, then the combined plasmid was transformed into Arabidopsis. Compared with the wild Arabidopsis, we detected and determined the transgenic lines. This laid the foundation for the study of its function in the cotton.