Functional Research Of Receptor-like Protein Kinase SgLRPK1 In Stylosanthes Guianensis

Stylosanthes guianensis is an important forage legume widely cultivated in tropical and subtropical areas.It has high yield,rich nutritional value and wide use,but cold resistance has limited the extension of planting to the north.RLK(Receptor-Like protein Kinase)is a kind of important plant protein kinase,which is located in the cell membrane.They act as a receptor of signal molecules,perceiving and receiving extracellular signals,and then transfering them to cells to turn off downstream target proteins by phosphorylation or dephosphorylation.RLKs are involved in plant development,hormone-signal transduction and stress tolerance.However,there are few studies on receptor-like protein kinases in S.guianensis.LRR-RLK(Leucine-rich repeats receptor-like protein kinase)is the largest subfamily of RLKs.Studies on some members of LRR-RLKs genes have showed that they play very important roles in the regulation of plant development and stress resistance.In this research,we have initially analyzed the function of a leucine-rich repeats receptor-like protein kinase gene SgLRPK1.This is a reference for studying the mechanisms of stress resistance and improving yield of the S.guianensis.The SgLRPK1 gene was cloned from S.guianensis.And SgLRPK1 gene contains a 1800 bp ORF and the polypeptide is 599 amino acids long.There is a N-terminal signal peptide,a extracellular Leucine-rich repeats domain,a transmembrane domain and a Serine/Threonine cytoplasmic protein kinase domain in the SgLRPK1 amino acid sequence by bioinformatics analysis.This suggested that the SgLRPK1 is a typical LRR-RLKs.Based on the phylogenetic relationships of kinase domains,LRR-RLK genes were classified into 15 groups in Arabidopsis thaliana.Phylogenetic analyses revealed that SgLRPK1 belongs to LRR VI subfamilies.The expression pattern of the SgLRPK1 gene under nomal and cold treatment were examined by Quantitative real-time PCR(qRT-PCR).The relative expression of SgLRPK1 gene was simulated under a photoperiod at normal temperature.The expression of SgLRPK1 gene was detected before and after nomal and cold treatment.Under normal temperature,the expression level of SgLRPK1 in 2h was maximum level,with values more than 7 times higher than control.Under cold treatment,the expression level of SgLRPK1 in 2h was maximum level,with values more than 13 times higher than control.Then,the expression level of SgLRPK1 always higher than control.Therefore,we concluded that the SgLRPK1 gene might play fundamental roles in response to cold stress.The PYL323-SgLRPK1-GFP expression vector was constructed and transformed into rice protoplast,while only green fluorescence can be seen in the cytomembrane that is same as the location of the red fluorescence of membrane localization marker gene(OsRac3).As control,transformation of rice protoplast cell by vector with only green fluorescent protein(GFP).The green and red fluorescence can be seen in the whole protoplast cell.This result indicates that the SgLRPK1 protein is located on the cell membrane.SgLRPK1 coding sequence was cloned in a pCAMBIA3301 expression vector which carry Bar marker.Besides,356 bp long specific sequences were selected to construct the pCAMBIA3301-RNAi vector which carry Bar marker.Then,we selected two target sites in SgLRPK1 gene’s ORF 5’and protein kinase domain to connect to pYLCRISPR/Cas9-DB to construct the targeting vector.Then,several vectors were carried into S.guianensis by Agrobacterium mediated method to get transgenic plants.This provides a theoretical basis for studying the cold resistance mechanism of S.guianensis.