Functional Analysis Of A LRR Receptor-like Kinase Gene OsRK2 Involved In Drought And Salt Stress Tolerance In Rice

Drought and salt stresses are the main factors affecting the growth and development of crops.Under the threat of abiotic stresses,plants perceive and transmit the stress signal through receptors,activate the expression of a series of stress-related genes,and finally alleviate or resist the harm caused by abiotic stresses.Among them,protein kinase as upstream components of signal pathway,often plays a key role as the "switch".Through the perception of external environment stress signal,then activates intracellular different protein phosphorylation pathway,and then regulates the transcriptional expression of downstream resistant genes,starting the corresponding physiological and biochemical process of reverse tolerance makes it an important part of the mechanism involved in the plant response to reverse tolerance.Via genevestigator analysis,the rice protein kinase gene OsRK2 can be induced by drought and salt stress,suggesting that OsRK2 may be involved in abiotic stress response.In order to clarify the biological function of protein kinase gene OsRK2,this study was carried out.We used rt-PCR and qrt-PCR to detect the expression levels of OsRK2 gene at different time after stress treatment.The results showed that OsRK2 was significantly induced by salt and drought stress.In addition,GUS staining in leaf tissues of OsRK2pro::GUS transgenic lines treated with drought was significantly deepened.The subcellular localization of OsRK2 indicated that the gene was located on the cytoplasmic membrane.The rk2 mutant and OsRK2 overexpression lines in Arabidopsis thaliana were subjected to salt and drought stress treatment,it was found that the germination rate,root length,shoot length of rk2 were inhibited,and rk2 adult plants was obviously less tolerant to stress conditions than that of the wild type,while the Arabidopsis overexpression lines showed enhanced tolerance to stress.From the physiological and biochemical data such as DAB,NBT and Evans blue staining and water loss rate,it is also found that rk2 mutant was more sensitive to drought and salt,which proved that OsRK2 gene may be involved in regulating the plant response to drought and salinity.In order to further explore the mechanism of OsRK2 in response to abiotic stress,we used the yeast system to screen the rice cDNA library,and obtained a zinc finger transcription factor OsZF1,which belongs to the Drought induced 19 family.Further assays with Y2H and BiFC confirmed the interaction of OsRK2 and OsZF1.Moreover,it is detected by qRT-PCR that OsZFl was also significantly induced by abiotic stress.It is decuced that OsRK2 might mediate plant tolerance to abiotic stressesvia phosphorylation of OsZF1.In addition,we also conducted transcriptome analysis of rk2 mutants and found that many genes related to drought and salt stress had significant changes,such as NF-YA8,ABCA5,OsSCP,OsRMC,OsNAC106,Salt,YUCCA6,OsHKTl and so on.The expression level of these genes in rk2 mutants was significantly lower than that in wild-type rice.The promotors of these genes were analyzed with bioxin software,and we found that NF-YA8,ABCB14,OsNAC106,Saltl and YUCCA6,contained TACAA/GT,which is OsZF1's binding site.Next,Yeast mono-hybrid experiment will be used to verify whether OsZFl can participate in the regulation of the transcription expression of these gene.It is proved that OsRK2 regulates the expression of genes related to downstream stress through the interaction with OsZF1,thus enhances the tolerance of plants to abiotic stress.By performing the above experiments,our studyclarified that OsRK2 plays an important role in mediating the plant tolerance to drought and salt stress,which might be via phosphorylation to OsZFl.It is deduced that under the phosphorylation of OsRK2,the transcriptional activity of OsZFl is activated in order to activate the expression of downstream stress-related genes,which thus endow the plants abiotic stress tolerance.There is hope that our study gets through a new signaling pathway in mediating stress tolerance in plants.The findings will not only enrich the present theory but also provide thoughts and strategies for cultivating stress-tolerant rice,which is of great value in application.