Study On Regulating Salt Tolerance Of OsCBL5 In Rice

Calcium is a ubiquitous intracellular secondary messenger in eukaryotes cells and plays a vital role in various environmental and developmental signaling pathways in plant cells.Under stress treatment,cytosolic Ca²⁺ fluctuation could be sensed and decode by calcineurin B-like proteins?CBLs?,which further regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases?CIPKs?to relay the signal and induce cellular responses.In our previous,the transgenic rice of overexpression of OsCBL5 is highly sensitive to high concentrations of NaCl and enhances tolerance to abscisic acid.A series of physiological experiments and ion tolerance tests have found that overexpression lines is only sensitive to Na+ in NaCl,indicating that OsCBL5 may be involved in the negative regulation of salt stress in rice.Therefore,in order to further clarify regulation the molecular mechanism of tolerance of OsCBL5 in rice,the study of japonica rice variety ZH11 as the research material,mainly to carry out the following experiments:?1?The latest CRISPR/Cas9 system gene editing technology was used to construct two different target loci-specific Cas9/gRNA expression vectors of rice OsCBL5 gene.Then,the transformation of Japonica rice was introduced into wild type ZH11 by Agrobacterium tumefaciens-mediated transformation method.And 27 transgenic seedlings knocked out from two target sites of OsCBL5 gene were obtained.?2?According to the principle of CRISPR/Cas9 system editing target,the transgenic seedlings were screened by PCR amplification and sequencing and identify four homozygous OsCBL5 gene knockout lines.?3?In field,some traits such as plant height,tiller number,effective panicle number,filled grain number per panicle,secondary branch and blade width showed significant difference between the OsCBL5 gene knockout lines and wild-type ZH11.?4?To investigate the function of OsCBL5 in response to salt stress,the seeds of wild type?WT?and OsCBL5 gene knockout lines were germinated and grown in medium plus NaCl for 7 days.The results showed that OsCBL5 gene knockout lines showed enhangced tolerance to salt stresses.The researches on OsCBL5 in rice will not only help us 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.