Gene Function Study For OsCPK12 And Screening For Interacting Proteins

There are a lot of signal transductions of the plants itself and accommodative responses for external stimuli along the growth and development process.As an important second messenger,Ca²⁺plays a key role in plant response to in-vitro and in-vivo stimuli,signaling and regulation of various physiological and biochemical reactions in the body.Calcium ion-dependent protein kinase?CPK?is a kind of calcium ion-binding protein widely existing in plants.As important part of plant calcium ion-signaling cascade reaction,CPKs are involved in the regulation process of plant growth,development,biological and abiotic stress.In this study,OsCPK12 gene was researched in stress regulation and hormone signal regulation.The knockout and overexpression mutants of OsCPK12 were constructed in the way of reverse genetics.Under the 150mmol/L NaCl treatment,the plant height,root length and leaf phenotype of the over-expressed lines was much better than that of the wild type,whereas compare to the wild type,the plant height of knockout mutants was shorter,the root development were affected,and the leaves were withered and yellow.So,it was confirmed that OsCPK12had positive regulation effect on salt tolerance in rice.The expression level of hormone signal transduction pathway genes in different tissues of mutant lines was analyzed by real-time PCR.Expression of multiple hormone receptor coding genes and downstream regulatory genes in the overexpression lines were significantly up-regulated compared with the wild-type.This result indicated that OsCPK12 was involved in signal transduction of hormones,which affected the stress response in rice.In order to further characterize the gene function of OsCPK12 and explain the mechanism,subcellular localization of OsCPK12 was done by fusion expression of target protein with fluorescent protein.This experiment proved OsCPK12 was mostly located in the plasma membrane which also laid the foundation for further research.Two water channel proteins,OsPIP1-1 and OsPIP2-7 were screened from Zhonghua 11's membrane protein library and further confirmed its interaction with OsCPK12 by Yeast Two-Hybrid test.We speculate that OsCPK12 may interact with OsPIP1-1 and OsPIP2-7 and regulates the entry and exit of water molecules which improve the salt tolerance of rice.