Cucumber CsSAMs Gene Clone And Interacts Validation With The Transcription Factor CsGT-3b

The S-adenosyl-L-methionine synthase(SAMs)gene is among the functional genes induced during environmental stress.The protein synthesis product S-adenosine methionine synthase(SAM)is a kind of important active metabolic substances,which serves as a methyl group donor in the trans-methylation of DNA,RNA,protein,lipids,sterols,volatiles,pectin,lignin,and flavonoids.Also it acts as a precursor to PAs and ethylene.Previous studies have demonstrated that the SAMs gene is significantly induced in many plants by NaCl stress.However,little is known about the molecular mechanisms and upstream factors regulating this salt-inducible gene in cucumber plants.In the present study,the salt-sensitive cucumber ’Jinchun No.2’ was used as the material to analysis the gene’ function by qRT-PCR method.Different exogenous substances or conditions were treated on cucumber seedling to simulate stress environments.In addition,the promoter of CsSAMs gene was cloned and further studied to explore the functional elements response to salt stress.Finally,a yeast one-hybrid method was used to identified the transcription factors interacted to CsSAMs gene.The main results were as follows:1.The qRT-PCR analysis showed that CsSAMs gene expression was up-regulated in response to various stimuli,such as salt stress,drought stress,ABA,MeJA,except SA.In addition,the 1742 bp sequence upstream of the initiation codon(indicated with +1)was isolated and analyzed using the PLACE and PlantCARE databases.Bioinformatics analysis revealed many TATA-box and CAAT-box existed in this region.Moreover,multiple important elements modulated stress and defense genes expression were found such as GT-1,MYB-related,HSE and other functional response elements.Both qRT-PCR analysis and promoter function prediction results showed that the cucumber CsSAMs gene belonged to stress response gene,and its transcription could be regulated by various hormones.2.The tobacco Nicotiana tabacum.’NC89’was used to analysis promoter characteristics.The full promoter and truncation mutations segments were cloned into the pCAMBIA1303 plasmid to replace the CaMV 35S promoter,and then transformed into tobacco leaf to regenerate the transgenic plants.GUS stain and activity analysis revealed that the full-length promoter displayed maximal promoter activity,whereas the P4 promoter,showed no basal promoter activity.In addition,the CsSAMs promoter exhibited stress-inducible regulation rather than tissue specific activity in transgenic tobacco.GUS staining stronger in leaves,stems,and roots,especially in the veins of leaves,the vascular bundle of stems,and root tip zones following NaCl stress.A transient expression assay confirmed that the 242 bp region(-1742 to-1500)was sufficient for the NaCl-stress response.3.The amino acid sequence of the Arabidopsis transcription factor AtGT-3b was used as a probe to explore the transcription factors that interact with the GT-1 cis-element within the CsSAMs promoter.By constructing a phylogenetic tree,we preliminarily identified three proteins that were homologous to AtGT-3b.The qRT-PCR analysis showed that the transcription of CsGT-3b increased by 10.13-fold in leaves and 35.87-fold in roots at 30 min and reached a maximum(approximately 108.39-fold in leaves and 112,26-fold in roots)at 1 h during NaCl stress.Protein structure analysis showed that the CsGT-3b has an SANT domain and multiple phosphorylation sites were detected in this domain,which may play roles in regulating down-stream gene transcription.4.The CsGT-3b protein can specifically bind to the GT-1 cis-element within the CsSAMs promoter in Y1H yeast selection system.The subcellular localization indicated that the CsGT-3b protein likely localizes to the nucleus,whereas the CsSAMs protein might be localized to the cytoskeleton and cytoplasm.