Cloning And Characterization Of A PEAMT Gene And Its Promoter From Halophyte Salicornia Europaea

Salicornia europaea is a dicot halophyte,which could grow in marsh with salinities greater than 3.5%and the major osmotica in its fleshy stems was Na~+ and g lycine betaine (GlyBet).In higher plants,glycin betaine is synthesis from choline by choline monooxygenase(CMO)and betaine aldehyde dehydrogenase(BADH).CMO and BADH genes have been engineered into many plants that lack GlyBet,but the accumulation of GlyBet are low and the increments in stress tolerance are commensurately small.It has been shown that the low levels of GlyBet in the engineered plants are due in large part to an inadequate capacity for choline synthesis.The choline is synthesized from phosphoethanolamine through three steps methylation,which is catalyzed by phosphoethanolamine N-methyltransferase(PEAMT).In order to increase the choline content in transgenic plants,a PEAMT was isolated from S.europaea by RT-PCR and RLM-RACE.The full-length of SePEAMT cDNA was 1,971 bp with 301 bp 5' untranslated region(UTR),185 bp 3'UTR and a possible polyA signal.The 1,485 bp open read frame (ORF)encoded a 494 amino acid polypetide,which included a conserved Adot-Met binding motif in the N and C end.The SePEAMT showed 94%,84%and 85%identity with that from Suaeda liaotungensis,Spinacia olerace and Atriplex nummularia.Using Agrobacterium- mediation,the recombinant plasmid was transferred into tobacco (Nictiana tabacum 89).PCR,Southern blotting and RT-PCR analysis indicated the PEAMT gene was inserted in tobacco genome as well as expressed on the level of transcription.The choline content in the transgenic tobacco plants was almost 10 times of the control and the salt stress was also improved.Promoter is an important element for the expression and regulation of a gene and so far the PEAMT promoter was only cloned from Zea mays and without any further expression reports.The Northern bloting indicated that SePEAMT was induced by salt,low tempreture (LT)and abscisic acid(ABA).A 5' flanking region of PEAMT(1,249 bp)was isolated from S.europaea by anchored PCR.In this region,many potential cis-acting elements were predicted by PlantCARE and PLACE programs.Aside from the basal transcriptional elements TATA-box and CAAT-box,some stress-responsive motifs such as ABRE,HSE and LTR were found.In addition,some pollen-specific activation-related elements were also present in this region.The full-length and 5'-truncated fragment of this sequence were fused to a GUS reporter gene,then introduced to tobacco via Agrobacterium-mediated transformation.The histochemical analysis showed that the SePEAMT promoter not only expressed in leaves and stems,but also had strong activity in the pollen.Fluorometric GUS analysis indicated that SePEAMT promoter activity was enhanced under the stresses of salt,low temperature and ABA.