Identification And Expression Analysis Of Potential Salt-Sensitive Protein ScSrlk And Its Downstream Interaction Protein ScMPK6 In Root Of Stellera Chamaejasme

Stellera chamaejasme is one of the steppe poisonous grasses.It spreads on the steppe by its strong resistance and destroys the ecological balance of steppe.Based on the observation of the salt tolerance of S.chamaejasme in the early stage of the laboratory,and the related literature reports,We chose the S.chamaejasme,which is dependent on the mechanism of efficient salt removal from its roots,as the research material.Because the receptor-like protein kinase Srlk protein can sense salt signal in Medicago truncatula and Medicago sativa,Receptor-like protein kinases generally use MAPK family proteins as intracellular signal transduction proteins,in which MPK6 protein can activate SOS1 protein in response to salt stress.so we hypothesized a salt signal transduction pathway in the root of S.chamaejasme.We did the following research:(1)Compared the reported target gene sequences with the trancriptome database of S.chamaejasme,The gene of sequence number c39384.graph_c0,c73211.graph_c1,c59271.graph_c0_seq6 are identified as the target gene,named ScMPK6?ScSrlk?ScSOS1,respectively.And primer was designed by using Primer 5,Oligo 7 software.Then the target gene was amplified from the cDNA reverse transcripted from the root of S.chamaejasme root,and the target gene was successfully constructed on the sequencing vector with the help of the kit for sequencing comparison.(2)Then we analyzed the bioinformatics of ScMPK6,ScSrlk,ScSOS1,The results of the prediction are as follows: The relative molecular mass of ScMPK6,ScSrlk and ScSOS1 protein is 44.2467 K Da,66.83682 K Da,127.13664 K Da,respectively;ScMPK6 and ScSrlk protein has hydrophilicity;ScSOS1 protein has hydrophobicity;ScMPK6 protein has no transmembrane domain and no signal peptide,It has the activation ring of TEY motif And CD domain;ScSrlk protein sequence is mainly divided into extracellular domain,transmembrane domain and intracellular domain.The extracellular domain includes signal peptide and 5 leucine-rich repeat,and contains 1 transmembrane domain,The intracellular domain includes ATP binding sites and PKclike superfamily;The N-terminal of ScSOS1 protein consists of 11 transmembrane domains,which are Na+/H+exchange region.The C-terminal consists of 3 conserved functional domains: cyclicnucleotide binding domain,SOS1 protein self-inhibition region and phosphorylation site of SOS2 protein.consists of three conserved functional domains: cyclicnucleotide binding domain,SOS1 protein self-inhibition region and phosphorylation site of SOS2 protein;ScSrlkhe,ScSOS1 protein was anchored on the plasma membrane,ScMPK6 protein was free in nucleus and cytoplasm;ScMPK6 protein is relatively conserved,The origin of ScSOS1 protein is consistent with that of known salt-expelling plants.(3)Quantitative Real-time PCR was used to determine the expression of 3 gene under salt stress.ScSrlk gene first responded to the signal of salt stress,and the expression reached the peak value for a period of time.The expression of ScMPK6 gene began to rise slowly,then increased to the maximum,and then decreased to the lowest rapidly and no longer showed an upward trend.After a period of stress treatment,the expression of ScSOS1 gene began to increase,and then slowly recovered to the initial level after rising to the highest value.(4)Bimolecular fluorescence complementation technique was used to verify the interaction between ScMPK6 and ScSrlk protein in vivo,acting on the surrounding cells;The specificprotein was successfully induced and expressed in vitro,the target fusion protein ScMPK6:: His,GST: ScSrlk was identified by Western blotting technique.We speculate that ScSrlk protein acts as a salt signal sensing protein.When it senses the salt stress signal in the environment,it transmits the signal to ScMPK6 protein and activates the salt stress response protein ScSOS1 in the cell as an intracellular signal transduction protein to resist the stress.Our results are consistent with the roles of the 3proteins in the analog signaling pathway.These studies provide a good reference for our further work.