Functional Analysis Of Cotton(Gossypium Hirsutum)HOX4 Gene In Fiber Development

Cotton is one of the most significant economic crops around the world.Cotton fibers are classified as long,unicellular seed trichomes originated from cotton ovule epidermal cells.As the structures of cotton fibers are similar to Arabidopsis leaf trichomes,it is suggested that the cotton fiber may have the similar mechanism to Arabidopsis trichomes.Currently,the molecular mechanism of cotton fiber initiation and elongation still remains unclear.Therefore,it is of great significance to address the regulation of cotton fiber development to improve the quality and productivity of cotton.In Arabidopsis,a HD-ZIP IV protein,GL2,is required to the trichome development.Here,we isolated a GhHOX4 gene from cotton(Gossypium hirsutum)which is homologous to Arabidopsis GL2.Some works that mainly focused on the function of GhHOX4 gene were carried on in this thesis.The results are shown as follows:1.Isolation and characterization of GhHOX4 and phylogenetic analysisGhHOX4 gene was isolated from a cotton fiber cDNA library.The sequence showed that the open reading frame(ORF)of GhHOX4 was 2367 bp in length and it encoded a protein with 788 amino acids.Phylogenetic relationships among GhHOX4 and several HD-ZIP IV proteins demonstrated that GhHOX4 is much closer to AtANL2 and AtHDG1.2.Expression pattern of GhHOX4 gene in cotton tissuesQuantitative RT PCR was employed to detect the expression of GhHOX4 gene in different cotton tissues.The results showed that this gene was preferentially expressed in fibers.And it began to express in ODPA ovule,but it showed high levels in 6 DPA to 12 DPA fibers.However,there was weak expression or no expression in roots,hypocotyl,stem,cotyledons,leaves,petals.3.The subcellular localization of GhHOX4 proteinThe GhHOX4:eGFP fusion construct was constructed and delivered into the onion epidermal cells via microprojectile bombardment of particle gun.The GFP fluorescence signals were detected in the cell nucleus of the onion by confocal microscopy.Moreover,pGL2:EYFP:GhHOX4 was constructed and introduced into the wild type Arabidopsis.We isolated the leaf trichomes of pGL2:EYFP:GhHOX4 transgenic plants and observed that the fluorescence was also localized in the nuclei and cytoplasm of trichome cells.These results indicated that GhHOX4 protein is mainly located in the in nuclei of cells.4.The transcription activity of GhHOX4 proteinTo investigate whether GhHOX4 has the transcriptional activation activity,the fusion vector pGBKT7-GhHOX4 was constructed and transferred into yeast(Saccharomyces cerevisiae)strain AH109 and Y187,respectively.The positive transformants of AH109 strain were cultured on SD/-Trp/-Ade and SD/-Trp/-His medium,respectively,while the positive transformants of Y187 strain were detected by X-gal assay.The results showed that GhHOX4 has no transactivation activity.5.The interaction among four cotton GhHOX proteins and interation between GhHOX4 and other proteinThe vectors pGADT7-GhHOX1/2/3/4 and pGBKT7-GhHOX1/2/3/4 were constructed to investigate the interaction among these proteins via the yeast two-hybrid system.Intriguingly,the results showed that only GhHOX1 can interact with itself so that can form a homodimer.Also,another protein,GhGL21ike was found to interact with GhHOX4.6.The morphological analysis of GhHOX4 overexpression yeast(S.pombe)The pREN5N-GhHOX4 construct was introduced into yeast(S.pombe)and the positive transformants were cultured on induced and non-induced medium,respectively.The morphology of yeast cells showed that the length of GhHOX4 overexpression yeast cells became dramatically longer than control.7.The phenotypic analysis of GhHOX4 overexpression transgenic Arabidopsis35S;GhHOX4 and pGL2:EYFP:GhHOX4 constructs were constructed and transformed into Arabidopsis WT and the transgenic Arabidopsis plants were obtained.We counted the number of trichomes on the 9th leaf of 27-day-old plants of WT,gl2 mutant and GhHOX4 overexpression plants.But no significant differences were found between the overexpression plants and WT.Then the leaf trichomes of 35S:GhHOX4,pGL2:EYFP;GhHOX4 transgenic plants and WT were isolated.The average lengths of tricomes were measured and the branches of trichomes were also photographed.The results showed that the trichome lengths of 35S:GhHOX4 and pGL2:EYFP:GhHOX4 transgenic plants were much longer than WT.Also,the trichome branches of pGL2;EYFP:GhHOX4 transgenic plants changed.The percentage of 4 branches of trichomes raised and a few trichomes with 5 branches existed in transgenic plants.These results showed ectopically expression GhHOX4 could affect the trichome development and pattern formation.Additionally,to investigate the relationship between GhHOX4 protein and GL2 protein,we transferred 35S:GhHOX4 and pGL2:EYFP:GhHOX4 into gl2 mutant,respectively.The transgenic plants showed the similar morphology to gl2 mutants as the the leaves still remained glabrous.This indicated that GhHOX4 may function differently from GL2.8.PA interacts with GhHOX4 proteinWe constructed the vector pET28a-GhHOX4,and then induced the protein to express in Rosetta(DE3).It was found that only a little protein was soluble.The PA-binding result showed that PA could bind to GhHOX4 protein.9.GhHOX4 transgenic cotton35S:GhHOX4 and GhHOX4 RNAi constructs were constructed and the DNA was transferred into cotton hypocotyls via agrobacterium LBA4404 to perform cotton transformation,respectively.Currently,we have got a host of embryogenic calluses after a year of subculture.Particularly,a part of embryogenic calluses have differentiated into transgenic seedlings and several transgenic cotton plants were obtained.However,futher studies are currently underway in order to address the function of GhHOX4 gene in cotton fiber development.