Phosphorylated Proteomics Analysis And SiRLK35 Function Identification Of Foxtail Millet(Setaria Italica.L) Under Salinity

Salt stress affects the crop yield and quality.Phosphorylation as an important post-translational modification,is widely involved in various metabolic processes of life activities.Phosphorylation omics is widely used as a novel method of plant stress research.There are almost a thousand foxtail millet germplasms were collected in our lab,and the salt tolerance abilities were screened and assessed by hydroponic cultivation combined with Na Cl treatment.Several foxtail millet varieties were obtained,and the YG2 is defined as salt tolerant and AN04 is defined as salt-sensitive cultivars.The seedlings of YG2 and AN04 at germination stage were used for further phosphorylation modification analysis under salt stress,and the mechanisms of millet participating in salt stress response to the protein modification level were uncovered.Meanwhile,the receptor like protein kinase gene SiRLK35 was heterogenously transformed from rice,and the SiRLK35 over expressed rice lines was used for the phenotype,physiological indicators and salt responsive marker genes expression patterns detected under salinity,and the possible responsive mechanisms of SiRLK35 to salt were.The results are listed as follows:1.Four foxtail millet varieties of JG11,YG2,YG11 and AN04 were preliminarily screened out from nearly a thousand collected germplasms.YG2 was identified as salt-tolerant and AN04 was defined as salt-sensitive after further verification.Considering the variation in pH is related to stress resistance abilities of varieties,the BCECF-AM fluorescent dyes and laser confocal microscope were used,and the intracellular pH values in root cells of YG2 and AN04 were detected before and after salt stress.The results indicated that the variation on pH in YG2 is less than that of AN04 under salinity,and salt tolerant trait of YG2 is confirmed.2.The phosphorylation omics was carried out using the roots of YG2 and AN04seedlings before and after 150 mmol·L^-11 NaCl treatment.A total of 5925 phosphorylated proteins and 19148 phosphorylated sites were identified,and among which 86%of serine（Ser）,10%of threonine（Thr）,and 4%of tyrosine（Tyr）were phosphorylated.And 14 of significant enrichment phosphorylation motifs,which were mainly recognized by MAPK,CDK,CDOK,AGC,CaMK-II,PKA and PKC,were identified.By GO and COG analysis of the differential phosphorylation groups,the phosphorylated proteins were mainly classified into the processes of regulation of metabolism,transcription,translation,protein processing and other processes.Among them,ABA signal transduction pathway,Ca²⁺signal transduction pathway,antioxidant stress signal pathway and ascorbic acid synthesis pathway were the main processes.3.The SiRLK35 was previously identified as a drought-responsive receptor-like protein kinase of foxtail millet by iTRAQ analysis.The further expression pattern indicated that SiRLK35 could also be highly induced to express by salt.By constructing the dual plant expression vector of pCAMBIA1301P-SiRLK35,the heterogonous transgenic rice lines were obtained,and the OE lines of OE-1,OE-2 and OE-3 were identified respectively.The SiRLK35 expression,phenotype observation,physiological indexes measurements and expression patterns of certain salt responsive genes were analyzed using control and OE transgenic rice lines under 150mmol/L NaCl treatment.The results are listed as follows:（1）The rice line of OE-1 was detected with the highest SiRLK35 relative expression level.After treated with 150mmol/L NaCl,the root length,plant height,dry weight,mortality and leaf mortality of the OE rice lines and zhonghua11 were detected,and the results showed that the growth inhibition of OE lines were less than that of control under salinity,and the inhibition degree of OE-1,which with the highest SiRLK35 expression level,was the least.The SiRLK35 could improve salt resistance abilities of plants.（2）The NBT and DAB dyeing,and antioxidant enzyme activities detection of SOD and POD under salt stress all showed that lower accumulation of O^2-and H₂O₂ in OE-1strain than that of control,which showed higher scavenging capacity of reactive oxygen species.（3）Analysis of the relative expression levels of some salt responsive genes in SiRLK35 OE rice strains showed that partial genes were up-regulated after salt treatment.Among them,the relative expression level of OsLEA3 at 24h of salt treatment was 1.9times than that of control,while the expression level of OsHKT1 was up-regulated at 6h under salt treatment,and then decreased with the extension of salt treatment.Therefore,SiRLK35 of foxtail millet may participate in improving the salt tolerance of rice mainly by influencing the scavenging mechanism of the peroxide,regulating the expression and signaling pathways of downstream stress responsive genes such as OsLEA3 and OsHKT1.