The Study On Drought Resistance And Functional Analysis Of Receptor-like Protein Kinase In Castor(Ricinus Communis L.)

Drought stress is one of an important abiotic stress factor endangering the growth,development and yield of castor(Ricinus communis L.).Many researches reported that protein kinase(Receptor-like protein kinase,RLKs)plays a key role in alleviating abiotic stress in plants.RLKs in plants is an extracellular receptor domain(ECLB),a transmembrane domain(TM)and an intracellular kinase domain(PKC).Stress signals caused by environmental changes are sensed by ECLB and transmitted into cells to make plants respond accordingly.Castor contains a large amount of RLKs,however the molecular mechanism of alleviating drought stress using the RLKs is not clear.In this study,the RLKs induced by drought in castor roots were screened by transcriptome and proteomic techniques,and the RLKs involved in the molecular mechanism were studied.The results were as followed:(1)Six castor lines including to 2017001,2017002,2017003,2017004,2017005 and 2017006 were treated by drought.The results showed that the 2017005 castor line had the strongest drought resistance,and this line was selected with follow-up transcriptome and proteomic studies.Under PEG-6000,the H2O2 and MDA contents in the roots of the 2017005 castor line increased along with the time of stress.At the same time,antioxidant enzymes,such as POD,GR,SOD,APX and CAT also increased under stress.(2)Transcriptome analysis of castor root under PEG-6000 was carried out using RNA-seq technology.A total of 2926 differential genes(DEGs)were screened out under stress at 3 d,1516 DEGs were found at 7 d after rehydration,and 111 DEGs were detected after rehydration at 4 days.GO analysis showed that DEGs were mainly enriched in biological processes such as stress response,signal transduction,antioxidation.KEGG pathway analysis suggested that the main metabolic pathways involved in DEGs included phenylpropane biosynthesis,flavonoid biosynthesis diterpenoid biosynthesis,plant hormone signal transduction,glutathione metabolism and carbon sequestration of photosynthetic organisms.(3)The iTRAQ technique were selected to analyze the proteomics of castor root under PEG-6000,a total of 318 differential proteins(DAPs)were screened out after stress at 3 d,190 DAPs were found at 7 d after rehydration,and 100 DAPs were detected after rehydration at 4 d.COG functional analysis showed that DAPs classified into 20 categories,among them the largest group was protein synthesis and modification,followed by carbohydrate metabolism,intracellular transport,synthesis and catabolism of secondary products.KEGG pathway analysis showed that DAPs was mainly involved in carbohydrate metabolism,phenylpropane biosynthesis,terpenoid skeleton biosynthesis,flavonoid biosynthesis,plant hormone signal transduction,amino acid synthesis and catabolism.(4)By combined analysis of transcriptome and proteomics,a total of 95 differential genes were obtained,of which DEGs were positively correlated with DAPs,but their expression abundances were different.The combined analysis showed that differential genes were involved in many different metabolic pathways,including carbohydrate metabolism,phenylpropane biosynthesis,flavonoid biosynthesis,oxidative phosphorylation,amino acid synthesis and catabolism.The results showed that in addition to primary metabolism involved in stress response,secondary metabolism including phenylpropane biosynthesis metabolism and flavonoid biosynthesis metabolism also played an important role in castor in response to drought stress.(5)Four RLK genes(SPKATPK2,CDPK33,CDPK26,SPKBSK7)were screened based on omics data,and their gene expressions changed under PEG-6000 stress.The subcellular localization of these four genes showed that SPKATPK2,CDPK33 and CDPK26 were located in the nucleus and cell membrane,while SPKBSK7 was located in the nucleus,cell membrane and cytoplasm.At the same time,the drought resistance of overexpression of Arabidopsis thaliana of these four genes was studied.Accordingylly,the transgenic plants with SPKBSK7-3 gene had the strongest drought resistance and were more sensitive to ABA and mannitol stress.Additionlly,the castor plants with overexpression of SPKBSK7 gene had stronger drought resistance than the wild type.In addition,the interaction of the SAP3 protein with SPKBSK7 protein was screened by yeast two-hybrid technique,and the affinity of these two proteins was KD=18.8 nM.Our results speculation that transgenic castor overexpressed with SPKBSK7 had enhanced drought resistance ability due to the interaction with SAP3.