Chracterization Of Cotton MADS And BZR Genes And Phenotypic Analysis Of Transgenic Cotton Plants

Cotton (Gossypium hirsutum) is a widely grown textile crop which is cultivated mainly for its fibres. Cotton fibres, which are seed hairs, are single-cell trichomes that arise from the epidermal layer of the seed coat. To improve the fiber yield and quality is always the most important object of cotton breeding in the China which is always treated as a important cotton production and consumption country.MADS-box genes which form a large family for transcription factors are defined by the highly concerved motif known as the MADS box.Through forming homodimer or heterodimer complex,MADS-box protein are involved in various aspects of plant development,including floral genes,fruit formation,seed pigmentation,control of root structure and so on. Up to now,homeotic abnormalities and MADS-box genes have rarely been reported in cotton.This reasearch has fouced on the chracterization of two cotton fibre MADS-box genes,GhMADS9and GhMADS10.As a group of plant steroid hormones,BRs (Brassinosteroid) play essential important roles in plant development,metabolic pathway and biosynthetic pathway.Genetic and molecular studies in Arabidopsis and rice has greatly advanced the understanding of the BR signaling pathway.It has been reported that the BZR1and BZR2are two major transcription factor in this passway. Previous study revealed that the accumulation of unphophorylated BES1and BZR1in the nucleus resulted by interaction between BZR1and14-3-3protein enable the two factor could interact with additional factors to regulate genes for various BR responses. Although the study in Arabidopsis and rice has justified that the the14-3-3proteins participating in various signalling pathways, little is known so far about how the cotton14-3-3proteins regulate fibre development through interacting with BZR1.Our previous study has reported that isolation and characterization of six cotton14-3-3s and their possible proteins during fibre development and a BZR1protein involved in the response to phytohormone signalling were identified as GM4-3-3interactors.This research has fouced on the chracterization of GhBZRl and phenotypic analysis of14-3-3transgenic cotton plants.1. The GhMADS9/10genes were not active transcriptional activators in yeast cells.Transactivation activity assay indicated that GhMADS9/10could not activate the expression of the two reporter genes and were not active transcriptional activators in yeast cells. It is known that the MADS-box proteins physically interact and form dimers as higher-order complexes to regulate the down-stream target proteins. Thus, we hypothesized that GhMADS9/10are a transcriptional factors that may interacts with other MADS proteins as heterodimers to perform its function on regulating the expression of downstream target genes.2. In situ hybridizationIn situ hybridization analysis further demonstrated that the GhMADS9gene is pollen-specific during anther development. At very early stages of cotton anther development, no or very weak signals of GhMADS9expression were observed in archesporial cells and primary sporogenous cells (PSCs) of about5-day-old anthers. As anther developed, weak to moderate activity of the gene expression was found in pollen mother cells (PMCs) as well as the surrounding cells of about10-day-old anthers. Very strong GhMADS9signals were detected in single-nucleic pollens in15-day-old anthers and maturing pollens in25-day-old anther, but little or very weak signals were observed in other anther tissues. On the contrary, no signals were found in any anther tissues using sense chain transcript probes of the GhMADS9gene as controls in in situ hybridization. These results suggested that GhMADS9gene is mainly expressed in pollens and may function in pollen development of cotton.3.the functions of GhBZRl in regulating cotton fiberThe DNA sequence of GhBZRl included1536bp,encoded a protein that included243amino acids.Through the PCR methods,we isolated the AtBIN2sequence from the cDNA library and constructed on the vector. The yeast two-hybrid assays results justified that a AtBIN2-GhBZRl could interaction and it is a prerequisite for the cotton GSK3kinase to phosphorylate its substrate. To investigate the subcelluar localization of GhMADS9, green fluorescent protein fused to the GhBZRl was expressed in cotton calli under the control of cauliflower mosaic virus35S promoter. The results Showed that GFP fluorescent signals in the transformed cells with GhBZRl: eGFP expression were predominantly localized in cell nuclei, suggesting that GhMADS9protein may be a transcription factor.4the analyse of14-3-3transgenic cottonThe over-expression transgentic plants showed phenotype in cotton fibre development.The sections of ODPA ovules showed that at the very early stage of cotton fibre development,the processes of fibre cells in RNAi transgenic cotton plants are less than the processes in wild type,while the processes in Overexpression transgenic cotton plants are more than the processes in wild type. This results indicated that the expression levels of the genes related to fiber development were declined, resulting in slower initiation and elongation of fiber cells, compared with the wild type.