Cloning Of OsCIPK Gene Family And Analysis Of Tolerance Of Transgenic Plants With OsCBL8to Abiotic Stresses In Rice (Oryza Sativa L.)

Calcium as a second messenger in cells plays an important role in the stress-signaling pathways in plants. The spatial and temporal Ca2+signal in plant cells, induced by the different stresses and decoded by many Ca2+-sensors, will activate downstream cascades such as phosphorylation leading to leasing expression of stress-related genes. Calcinerurin B-like proteins (CBLs) are plant-specific calcium banding proteins. CBL-interacting protein kinases (CIPKs) are Ca2+-dependent serine/threonine kinases with a high conserved SNF kinase domain and a NAF motif. CBL/CIPK signaling system has an important function in the response of plants to stresses. Rice is a model monocot plant and also one of the best important crops. The researches on CBL/CIPK in rice will help us not only better understand molecular mechanisms of CBL/CIPK signal transduction under stresses in plants, but also genetically improve the resistance and productivity of rice or other crops.In this study,12full-length cDNA of CIPK genes were obtained by silico cloning from rice, based on the amino acid sequence of homologous CIPKs in Arabidopsis. It was found that the structures of intron and ex on were greatly distinct in these12OsCIPKs, and there existed two alternative-splicing isoforms in both OsCIPK3and OsCIPK24. The sequence analysis showed that their identity was high at amino acid level (41.2-78.5%) in12OsCIPKs proteins. All of these proteins had a serine/threonine kinase domain in N terminal and a NAF motif in C terminal essential for protein interaction. Their expression patterns and induction by multiple stresses were analyzed in rice. Expression patterns of these genes were distinct in treatments with high salt, low K+, ABA, Hg2+, cold and Xanthomonas oryzae pv. Oryzae, respectively, and OsCIPK2,10,11and14were induced by6above-mentioned stresses. These4genes may be upstream regulators in stress-response pathways and involved in multiple resistance-related signaling pathways.In previous study, AtCIPK24/SOS2was identified as a crucial factor in the salt-resistance pathway in Arabidopsis. To explore the function of OsCIPK24under stresses, its two alternative open reading frames (ORFs) were cloned into the vector pCABMIA1304and transformed into the calli of rice variety Nipponbare by Agrobacterium tumefaciens-mediated method. The transgenic regenerated seedlings screened by Hygromycin have been confirmed through PCR assay, and their functional identification will be carried out.Transgenic plants (T3) overexpressing sense or anti-sense OsCLB8gene have been obtained from previous studies. Here, expression patterns of exogenous gene, agronomic traits and tolerance to the abiotic stresses including salt, drought and cold were further analyzed in these sense or anti-sense transgenic lines. Semi-quantitative RT-PCR analysis showed that the expression of OsCBL8was dramatically increased in sense transgenic plants; on the contrary, that of endogenous OsCBL8was inhibited to some extent in antisense transgenic ones. In field, some traits such as plant height, tiller number, effective panicle number, panicle length and100-seed weight showed no significant difference in both these16transgenic and control lines, however, the filled grain number per panicle and seed set rate in these16transgenic lines were significant or extremely significant lower than those in the control. Of these16transgenic lines,5were extremely significant lower in the filled grains number per panicle and seed set rate, compared with the control. One line8F12with high salt resistance was screened out from sense transgenic plants. Compared with the antisense transgenic plants and wide type, sense transgenic plants8F12had a higher K+/Na+ratio in roots10days after treated with150mM NaCl, but this high ratio was not observed in shoots and leaves. Another line8R14screened out from antisense transgenic plants displayed high drought resistance. After2-day simulative drought treatment with20%PEG6000, increased levels of both malondialdehyde and proline were much less in the8R14plants than in the sense transgenic plants and wide type. Under low temperature stress, plant growth or the levels of malondialdehyde and proline did not show visible difference among sense or anti-sense transgenic and non-transgenic plants.