Cloning And Function Analysis Of Expansin-Like Genes In Cotton (Gossypium Hirsutum)

As the significant distinctive component in cells between palnt and animal, cell wall was obtained increasing concern by scientists since it was discovered by Hooker from cork slice in 1665. Plant cell wall plays a crucial role in cell growth differentiation、division and death. In addition, many pathways involved in plant development such as mechanical construction、material transportion, defective raction, cellular recognition, was contolled and regulated by cell wall proteins.Expansins are identified as the proteins encoding by multiple genes family. As members of cell wall protein, they can be divided into four subfamilies such as a-expansin (EXPA),β-expansin(EXPB), expansin-like A (EXLA)and expansin-like B (EXLB). Recently, EXPA and EXPB subfamilies were studied in Arabidopsis, tomato, zeatin and rice. The research indicated that Expansin proteins participated in cell elongation, leaf primordium generation, polle tube permeation, Fruit ripening, cell wall degradation, seed germination and growing. Moreover, expansin proteins were affected by treatments as flooding, lighting, salt and hormone.Based on the SSH library derived from the cell wall regeneration from cotton protoplasts, RACE and homologous sequence cloning, we obtained four expansin genes from Gossypium hirsutum L. Sequence alignment analysis suggested four genes have high similarity with expansin-like genes in GenBank. These four genes can be predicted to be the members of expansin-like subfamily and denominated by GhEXLAl, GhEXLA2, GhEXLBl and GhEXLB2. Four expansin-like genes expression were analyzed by real-time PCR. The result revealed that these genes preferentially and highly expressed during cell wall regeneration from cotton protoplasts. GhEXLBl expressed at the lowest level in root, stem and leaf compared with the other genes. GhEXLB2 was shown to expressed higher in globular embryo than the other three genes.In order to study the functions of expansin-like genes in cell wall establishment, gene overexpression and RNAi expression vectors (pCAMBIA2300S and pHELLSGATE4) were constructed and transformed to the hypocotyl of YZ1 and callus of C201. The statistical analysis with all of transgenie plants and monoclonal lines indicated that the transformation of exogenous expansin-like genes or fragments negatively influenced the regeneration of plants and embryogenic callus, especially GhEXLA1 and GhEXLB1. Transgenic plants identified from the DNA detection, Southern blot or RNA expression were used for further analysis. The main results are as follows:(1) We have acquired 15 transgenic plants. Among them, there are 4 plants transformed with GhEXLA2 RNAi expression vectors,4 plants of overexpression with GhEXLB2 gene and 7 plants with RNAi of GhEXLB2 gene. All the young leaves and stems from transgenic plants were studied in cytological observation with paraffin sections. The observation demostrated that the premature leaves and stems of transgenic plants appeared normal compared to wild plants. The result indicated that expansin-like genes have invisible function to development and cell size determining of leaves and stems.(2) One monoclonal line were chosen from transgenic monoclonal lines of each expression vector except GhEXLBl gene overexpression vector for the analysis of cell wall regeneration. In this experiments, we isolated protoplasts from transgenic monoclonal lines and then transferred them into cell wall regeneration culture medium. The cultures were observed after Oh,12h,24h,48h and 72h staining with Calcofluor White M2R and the Ratio of Wall Regeneration were counted. The data discovered that the regenration speed of somatic wall would be reduced when transformed with whatever expression vectors. In which, the regeneration speed of cell wall from transgenic monoclonal line with GhEXLB2 exhibited the most of all. The speed of transgenic monoclonal line with GhEXLA2 appearred less than GhEXLB2. The protoplasts from cell wall of GhEXLAl suggested the lowest effecience of regeneration.In conclusion, Variety of expansin-like genes expressing influenced the regeneration of plants from explants and embryogenic callus and the reestablishment of cell wall in Gossypium hirsutum; however expansin-like genes hadn’t the same functions as expansin genes in this study.