Identification Of S-Adenosylmethionine Synthetase Gene And Its Salt Stress Response Function In Cucumber

Cucumber(Cucumis sativus L.)is one of the worldwide cultivated vegetables.However,various abiotic and biotic stresses cause great adverse effects on its yield and quality.S-adenosylmethionine(SAM),catalyzed by S-adenosylmethionine synthetase(SAMS,EC 2.5.1.6)is an important methyl donor for methylation and is also a common precursor for the synthesis of polyamines and ethylene.Plant polyamines and ethylene are widely involved in plant growth,development and response to different stresses.However,the function of cucumber SAMS is still unclear,and its mechanism in response to salt stress remains to be elucidated.In this study,the SAMS members were explored with the released data of cucumber genome,and their expression patterns were analyzed.In addition,the prokaryotic expression system and transgenic techniques were applied to further charify the function of cucumber SAMS in response to salt stress.Finally,the yeast two-hybrid system was used to screen for the interacting partners of cucumber SAMS.The key findings were as follows:1.Two putative CsSAMS genes(CsSAMSl and CsSAMS2)were identified in cucumber genome.They were located on chromosome 7 and chromosome 6,respectively.The coding sequence(CDS)size of CsSAMS1 and CsSAMS2 were 1182 bp and 1173 bp,encoding 393 and 390 amino acids,respectively.The amino acid sequences of CsSAMS1 and CsSAMS2 were highly conserved and were close to AtMAT1 and NtSAMS2,respectively.The analysis of phosphorylation site revealed that there were 35 and 31 phosphorylation sites with a score higher than 0.5 in the CsSAMS1 and CsSAMS2 polypeptide sequences,respectively.A total of 26 and 14 putative cis-regulatory elements involved in hormone and abiotic stress response were predicted in the promoter sequences of CsSAMSl and CsSAMS2,respectively.The qRT-PCR analysis showed that CsSAMS1 was predominantly expressed in the stem,male flower,and young fruit,whereas CsSAMS2 was preferentially accumulated in stem and female flower.Additionally,salinity,drought,low temperature,and various hormones induced the transcriptions of CsSAMS1 and CsSAMS2.2.To elucidate the function of cucumber SAMS,the full-length CDS of CsSAMS1 was cloned,and prokaryotic expression system and transgenic materials were constructed.The results showed that the recombinant strain expressed specific protein by the induction of 0.1 mM IPTG,and exhibited strong tolerance to salt stress compared to non-recombinant bacteria.Down-regulating SAMS led to transgenic tobacco plants late flowering,fewer seeds,and low seed fertility.On the contrary,overexpressing CsSAMSl in tobacco plants produced more biomass,early germination,and rapid growth rate.Under normal conditions,overexpression of CsSAMS1 significantly increased MDA content,H2O2 content,and POD activity in transgenic lines.MDA content,H2O2 content and POD activity in the leaves of overexpressing CsSAMS1 tobacco seedlings were significantly higher than wild type.However,after three days under salt stress,the MDA content was lower than that of the wild type,the H2O2 content remained high,and the performance of Na+/K+ uptake,polyamine metabolism,and ACC synthesis in transgenic lines exhibited a CsSAMS1-expressed dependent way.These results implied that a proper increase in plant expression level of CsSAMS1 is benificial to enhance the salt tolerance.3.The yeast two-hybrid cDNA library was constructed using Gateway technology.The titer of the primary library and the yeast two-hybrid library were 6.3 × 106 cfu mL-1 and 6.0× 106 cfu mL-1,respectively.The reorganization rates were both close to 100%.The average insert size of yeast two-hybrid library ranged from 0.3 to 2.5 kbp.Simultaneously,the bait expression vector pGBKT7-CsSAMS1 was constructed and introduced into Y2H Gold yeast.The recombinant Y2H Gold yeast grew well on SD/-Trp medium and could not grow on SD/-Leu/-Trp/-His/-Ade medium,indicating that the bait protein was not toxic to yeast cells and could not activate the transcription of reporter genes.Yeast mating was performed to screen cucmber cDNA library.The sequencing results of positive clones showed that they were partial sequences of WD40,AOS,CDPK6,and ATG8,respectively.4.The full-length CDS of CsCDPK6 and CsSAMS2 were cloned from the cucumber leaves and female flowers,respectively.The CDS size of CsCDPK6 is 1632 bp,encoding 543 amino acids.The yeast two-hybrid assay revealed that the CsSAMSl and its N-terminal domain could interact with CsCDPK6.It was further verified by bimolecular fluorescence complementation that CsSAMS1 and CsSAMS2,as well as their N-terminal,C-terminal,and central domains,were able to interact with CsCDPK6.The qRT-PCR test showed higher relative expression of CsCDPK6 in the stem,male flower,and the female flower was higher.Salt stress treatment significantly stimulated the expression of CsCDPK6 in cucumber leaves but inhibited the expression of CsCDPK6 in roots.Furthermore,the expression of CsCDPK6 in cucumber leaves and roots was induced by ABA,SA,MeJA,PEG and low temperature treatment.