Role&Mechanmism Of MdCIPKs In The Regulation Of Apple Fruit Developement

The study of Fruit development and ripening is one of the most important aspects in fruit researches. Fruit are generally classified into two physiological groups, climacteric and nonclimacteric, according to the presence or absence of ethylene biosynthesis peaks and respiratory bursts during ripening. Over the years, a lot of studies focused on the changes of fruit development, ripening process and biochemical metabolism, however, it is little to know the molecular mechanism of the fruit development and ripening. With the rapid development of molecular biology, people gradually realized that the nature of the development of the plant is affected by a complex series of signal transduction process. Here we use Golden Delicious apple as the material to deeper insight of the mechanisms of the apple fruit development and ripening.In the the apple genome, we identified eighteen MdCIPK numbers through the bioinformatics methods, all of them are the serine/threonine protein kinase, which contains kinase domain, regulatory domain and connective domain, the kinase domain contains ATP binding site, serine/threonine protein kinase active site; the regulatory domain has NAL/FISL motif and PPI site. MdCIPKs temporal and spatial expression analysis showed that the MdCIPK12expressive characteristic was relative to the fruit development and ripening, which indicate MdCIPK12may be related with the apple fruit development and ripening regulation.Pharmacological experiments showed that the MdCIPK12expression was strongly induced by ethylene former ACC. meanwhile, it was notably inhibited by the ethylene receptor inhibitors MCP. The MdEIN2is the key role of ethylene signal transduction. In apple fruit calluses, MdEIN2-RNAi can significantly inhibit the expression of MdCIPK12, and the culture medium with ACC did not induce MdCIPK12expression. These results suggest MdCIPK12plays an important role in the ethylene signal transduction, and also imply MdCIPK12may play an important role in the growth and ripening of the fruit.Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of MdCIPK12homologous gene SICIPK12can prolong the tomato fruit development process and ripening. To further confirm the importance of MdCIPK12in regulation of fruit development and ripening in tomato, we get four stable transgenic lines of antisense interference SICIPK12, their fruit development and ripening need much longer time than control’s. Transgenic lines aslo reduced series ethylene signaling pathway genes (SIETR3, SIETR4, SIETR6, SIEIN2, SICTR1), and fruit ripening related genes expression (such as pigment metabolism pathway genes SIGGPS1, SIGGPS2, SIPSY1, S1PSY2, SIZDS, SIPDS, SIBcyc genes and glucose transport-related SlSUT1). These results support that MdCIPK12plays an important role fruit development and ripening process.To further reveals the mechanism of regulation of fruit development and maturation of MdCIPK12, bimolecular fluorescence (BiFC) analysis showed an interaction between MdCIPK12and MdNDPKl, and they both located in plasma membrance regions and endoplasmic reticulum. Co-immunoprecipitation aslo validates interaction relationship between them. We aslo found during the apple fruit development and ripening, MdNDPKl and MdCIPK12have similar expression and response characteristics, for example, the expression of MdNDPK1and MdCIPK12were induced by ACC treatment and inhibited by MCP treatment, both of them respond to ABA, sucrose, salt stress and drought stimulation. These results indicated MdCIPK12-MdNDPKl signal pathway plays an important role in the development and ripening of fruit.In conclusion, this study use a variety of modem molecular biology technologies, reveals that MdCIPK12plays an important role in fruit development and ripening. Moreover, MdCIPK12interact with MdNDPKl. This study deepens our understanding the molecular regulation mechanism on fruit development and ripening. It has important theoretical value for the molecular basis of the further analysis of apple fruit development and maturation of the regulation. And it is essential to use gene engineering to control the fruit development and quality formation in the future.