Cloning And Functional Identification Of Auxin Transport Protein PIN-like Gene In Rice

As one of the most important phytohormones, Auxin participates in the modulation of many developmental and physiological processes in plant, such as the extending growth of root, tropic response, adventitious rooting, differentiation of vascular tissue, vegetable tropism, apical dominance, cambium cell division, cell elongation. Auxin transport in plant is an active transport process which needs energy, and its polar transport is very complicated. It has different types in different organs, it is transported basipetally in coleoptile and stem while acropetal transport occurs in root. Up to now, auxin has been the only phytohormone that polar transported. And this is determined by the asymmetric localization of auxin efflux protein in plasma membrane of auxin transport cell. It can be concluded from many studies that the auxin transport protein plays a crucial role in post-embryonic development of plant, plant photomorphogenesis and histological differentiation.Rice (Oryza sativa L.) is not only one of the most important food crops in the world, but also a model plant for the study of molecular developmental biology in cereals. In this research, a promoter and cDNA of a putitive PIN gene which may encode a auxin efflux transporter in rice were isolated based on the previous study. In order to confirm the function of the putative PIN gene, Two binary expression vectors named p1300-promoter::GUS and p1300-cDNA in our rearch were constructed on purpose. One was used for the gene expression and localization in tissues, the other one was used for the identification of gene function. Agrobacterium tumeffaciens EHA105 harboring the two binary vectors was used to rice transformation.A rapid and high-efficient rice transformation protocol was developed in this study. 3-5days was the proper time of co-culture, when the OD₆₀₀ of bacterial concentration was 0.1-0.15 and the transformation efficency was quite high. Sufficient rinsing with sterile distilled water and drying treatment to callus after co-culture could efficiently restrained the growth of Agrobacterium. Low temperature and appropriate drying treatment before inoculation could promote the differentiation of rice callus. TO transgenic plants, which generated from the transformation of the vector p1300-promoter::GUS, were GUS dyed, and the expression patterns of the putative PIN gene in different tissues were initially identified. The putative PIN gene was found expressed in plumule, stem, shoot, coleoptile, the leaf trace vascular bundle and old leaves. And the gene was expressed differently in different tissues. High expression was detected in plumule and stem where auxin was synthesized while the gene was only faint expressed in other tissues. Thus we conclude that the expression patterns of the putative PIN gene may relate to its function. The wild rice and mutant rice were transformed with p1300-cDNA to further validate the gene function.