Analyses Of Cotton HAP5Gene Expression And The Identification Of Its Target Proteins

Cotton (Gossypium hirsutum), is originated from Subtropical zone, belongs to Malvaceae, Gossypium. Cotton is one of the most important crops in the world. There are higher cotton yields in China, the United State, India and so on. Recently there is little report about the study of molecular mechanisms of the cotton growth and the development as well as the yield and quality of fiber.The CCAAT box is a widespread regulatory sequence found in promoters and enhancers of several genes, whose functional importance has been well established in different systems. The HAP complex was independently identified by different groups and is also known as CCAAT box factor (CBF) or nuclear factor Y (NF-Y). The HAP complex consists of the three subunits:HAP2(NF-Y; CBF-B), HAP3(NF-YB; CBF-A), and HAP5(NF-YC; CBF-C). In this study, a cDNA, GhHAP5, encording a typical HAP protein with259amino acids was isolated from cotton fiber cDNA library. This study research the DNA and protein sequence analysis, the expression of GhHAP5and screen and identify GhHAP5target proteins.1. DNA and protein sequence analysisa777bp genomic DNA fragment of GhHAP5gene was isolated in cotton by PCR for characterizing GhHAP5gene structure. Compared with its cDNA sequence, the genomic DNA is same with cDNA, which means that the genomic DNA has no intron. Sequence analysis revealed that GhHAP5protein contains the conserved structure of81-204amino acid, which is belongs to H2A subfamily. The GhHAP5protein also has a nuclear localization sequence (RRTLQKN).2. GhHAP5gene is expressed at higher lever in fiber and is developmentally regulated in cotton fiberThe GhHAP5was predominantly expressed in10DPA fiber, but strong expression was also detected in leaves and6-day-old hypocotyls. In other tissues, GhHAP5was expressed at very low levels.The experiment result showed that the expression of GhHAP5was different in every stages of developing fibers. GhHAP5transcript was accumulated at relatively high level in early fiber development stage (3-5DPA), and its expression reached a peak in10DPA fibers, and then gradually declined to very low level with further fiber development.3. Subcellular localization of GhHAP5proteinGhHAP5:eGFP (enhanced green fluorescent protein) fusion genes under control of CaMV35S promoter were introduced into Arabidopsis thaliana. Roots of the transgenic Arabidopsis plants expressing GhHAP5:eGFP fusion protein were examined wih a leica confocal laser scanning microscope. Very strong fluorescent signals were observed in cell nuclei, suggesting that GhHAP5protein may be a transcription factor.4. Transactivation activity assay of GhHAP5proteinIn order to determine whether GhHAP5protein has transcriptional activation activity, yeast one-hybrid system was employed. we transformed Y187yeast strain with the fusion plasmid pGBKT7-GhHAP5. The scribing and color reaction indicated that GhHAP5protein functions as a transcriptional repressor.5. Screening and identification of GhHAP5target proteinsWe screened and identified the target proteins of GhHAP5by yeast two-hybrid technology. Result demonstrated that there were five kinds proteins which can interact with GhHAP5:transcriptional factor NF-YB, transcriptional factor MYB109, glycosyltransferase, hydrolase, WD-repeat protein. We confirmed the interaction between them by yeast mating.6. The expression of GhBCP in cotton fiber when treated with heavy metal ionsIn order to syudy the effect of heavy metal ions on the expression of GhBCP, we used different concentrations of heavy metal ion to process10DPA fiber with3h, then tested the expression level of four GhBCP. When we treated with different concentration of CuSO4and ZnSO4, the expression of GhBCP showed increased tendency. But when we treated with Al2(SO4)3,these gene expression level is declined.7. Overexpression of GhBCP gene enhances the fission yeast (S.pombe) tolerance for heavy metal ionsWe transformed the fission yeast with GhBCPl/4and treated them with CuSO4and ZnSO4. We found that the growth rate of transformed yeast was faster than control group. The results indicated that the GhBCP protein can improve tolerance to heavy metal ions on yeast.