Functional Research Of Receptor-like Kinase With RNA Interference In Rice

As the receptor of extracellular signaling, receptor-like protein kinases play an important role in plant growth and development. The technique of RNA interference is also an useful tool in functional genomics research. It is reported that the CR4 gene encoded a predicted receptor kinase and is required for the normal differentiation of leaf epidermis in maize, the same research result was also reported in Arabidopsis; and the PRK2 gene encoded a LRR receptor like kinase and might function in compatible pollinations, it is specifically expressed in mature pollen and germinating pollen tubes. In order to confirm the functions of receptor-like kinases (OsCR4 and OsPRK2), the RNAi method was used in this study.The plasmid pTCK303 was as the back bone for constructing RNAi vectors. RNAi fragment was selected from extracellular receptor domain of the two target genes and named 0sCR4i , OsPRK2i, which was cloned and sequenced successfully. Then two-step cloning method was used for RNAi vector construction and at last formed an inverted repeat separated by the rice intron. Agrobacterium tumefaciens EHA105 harboring the binary vector pTCK303-CR4i or pTCK303-PRK2i was used for rice transformation.A rapid and high-efficient protocol has been developed for Agrobacterium-mediated transformation of rice in this study. NB medium supplemented with proper amino acids was used for mature embryo-derived callus induction, in which the combinations of 2,4-D(2.0mg/L) and 6-BA(0.5mg/L) was probaly the best offered. After 3 weeks in the dark at 28℃, embryogenic calli initiated on the scutellar surface of mature seed embryos were selected and subcultured at 3-week intervals on the same medium. 3-5 day was the proper time of co-culture, when the OD600 of bacterium was 0.8-1.0. The Agrobacterium could be efficiently blocked after washing with sterile distilled water. The resistant calli were transferred to regeneration medium to regenerate into plantlet. Regenerated plantlets were transplanted into soil in greenhouse. The result showed that a rice transformation protocol for highly efficient and reproducible production of large...