The Auxin Responsible Elements Controlled GUS Transformation Of Cotton And Its GUS Detection

Cotton fiber is the main raw material in textile industry. The fiber is unicellular that developed from the epidermic cell of ovule. The fiber quality includes its length, the strength and the percentage of elongation. The phytohormone is actually the main factor that can influence the development process of cotton fiber cell and its quality.The auxin is an important phytohormone involves in regulation of the mutiply processes of development and growth and the morphogenesis of the plant. The ovule in vitro culture combine with ELISA test of the auxin illustrated that auxin can promote the development and differentiation of cotton fiber cell. But the effects of auxin on the development of cotton fiber cell have not been precisely pinpointed in vivo. In this research the auxin responsible elements DR5and GUS reporter gene are recombined and transformed into cotton to probe the auxin distribution on the development and differentiation of cotton fiber cell in vivo.The reporter gene DR5::GUS consist repeat sequences of the responsible elements AuxREs and the35S mini promoter and the β-glucosidase. The recombinant was transformed into cotton leave cells to perform transient expression at first. The GUS detection demonstrated that the reporter gene functional in cotton.The DR5::GUS was transformed into a cotton variety "Xiangza21" by ovary-injection of the Agrobacterium. After screening of Kanamycin resistance on seedling leaf and verify by PCR that the three transgenic seedlings were identified. The transgenics are in homozygous after several generations’ cleistogamy and it’s verified by PCR.When transgenic GUS staining test was performed with the ovule in different stage and the result was observed on microscope. Two days before bloom, the stained blue on the surface of cotton ovules was deep and there were no difference among cells, it showed that high-level auxin was distributed on the epiderm of cotton ovules. From two days before to the day of bloom, the blue stain gradually weak with no difference in cells. This implies the concentration of auxin in the epiderm cells gradually reduced. From the day and two days after the bloom that the blue stain gradually deepened shows that the concentration of auxin on the epiderm of the ovule risen gradually. In The two days after bloom the stain heavier on elongated fiber cells shows the fiber cell accumulate higher level auxin than other cells.From two days before bloom to two days after bloom, the concentration of auxin on the epiderm of ovule changed in a "V" shape and that may has some connection with the development of fiber cell and lump. From two days before bloom to one day before bloom, high concentration of auxin could promote differentiation and early development of fiber cell. On the bloom day, low concentration on the surface of cotton ovules can stimulate the lump of long cotton fiber cells. One day after bloom, the concentration of auxin on the surface of cotton ovules gradually increased and accumulated in the fiber cells, and that also promoted the elongation of the fiber cells.The flower primordium is also subjected into GUS detection and Observation. We detected the concentration of auxin is higher on the bottom border of receptacle and carpel, lower in stamen and filament, higher in pistil according to GUS stain. This typical auxin distribution must direct the development process of flower primordium and carpel and ovule identity. The distribution of auxin is also involved in the early development process of ovary.After the fiber quality test of the transgenic cotton that the DR5::GUS gene transformed into cotton had no obvious quality influence.