Cloning And Functional Analysis Of The Ss DODA Promoter Of Suaeda Salsa L.

Suaeda salsa L. is an annual leaf succulent euhalophyte. The shoot of S. salsa plant is red-violet throughout the whole growth period in the intertidal zone, but green on highland and land far from the seaside. As a halophyte, physiological and molecular responses of S. salsa to salinity stress are very complicated. Up to now, the mechanism of S. salsa responses to stresses, especially to salinity, has been extensively studied at biochemical and molecular level in our laboratory. Our earlier results showed that the red pigments accumulated in S. salsa are betacyanins. However, the molecular mechanism and the regulation of relevant enzymes involving in betacyanin synthesis in S. salsa have not been elucidated. In particular, nothing is known about how environmental stressors such salinity, low temperature and flooding regulate gene expressions encoding enzymes involving in betacyanin synthesis and their activities leading to formation of different phenotypes of S. salsa. In our laboratory, the gene DODA coding for the 4, 5-DOPA dioxygenase which is one of key enzymes controlling betacyanin biosynthesis from S. salsa was cloned. In the present study, the promoter of SsDODA from S. salsa was cloned, and SsDODA expression and their relation to betacyanin content of S. salsa seedlings were determined. The results are as follows:1. Cloning and functional analysis of the SsDODA promoter of S. salsaThe 1219 bp promoter segment of SsDODA gene from S. salsa was amplified by TAIL-PCR. BLAST analysis showed that the 3,-end of this segment has a 98% homology with the 5,-end of SsDODA. Therefore, this segment is dentified as the promoter of SsDODA. Sequence analysis with the Plant CARE and PLACE program revealed that promoter of SsDODA contains a typical motifs are found in most eukaryotes such as TATA-motif and CAAT-motif. In addition, other cis-elements such as light-regulated regions (GA-motif,GATA-motif GT1-motif) and low tempreture-regulated region (LTR) were also found in the SsDODA promoter.A series of 5,-deleted plant transformation vectors were constucted to clarify the functions of different regulatory elements in the SsDODA promoter. Each segment was digested with Pst I/BamH I and subcloned into Pst I/BamH I-digested pCAMBIA 1391 to generate six promoter deletion derivatives. All constructs were verified by PCR and enzymolysis and correctly inserted, named full-length, GA, GATA, GT1, LTR and SP1 respectively.Each promoter-GUS fusion construct was introduced into Agrobacterium tumefaciens strain GV3101 via electroporation. Tobacco leaves were infilltrated with Agrobacterium harboring the constructs. GUS staining demonstrated that all constructs containing different SsDODA promoter segments were to drive the expression of the GUS gene. To further examine the functions different SsDODA promoter elements, Arabidopsis thaliana were infilltrated with Agrobacterium harboring these constructs. It was found that GUS gene was expressed in roots, stem and leaves of T1 transgenic lines.2. Expression analysis of DODA in S. salsaTo evaluate the effect of environment factors on SsDODA expression and its relation to betacyanin accumulation, the expression of SsDODA and betacyanin accumulation in different parts of S. salsa under different light and temperature treatments were determined.S. salsa seeds were sown in sand and grown in a chamber in dark. When the seedlings were three days old, radicals, hypocotyls and cotyledons were harvested to determine the SsDODA expression and the betacyanin accumulation. Surprisingly, SsDODA expression in hypocotyls and cotyledons were significantly lower than that in the radicals while the betacyanin accumulation in the shoot were much higher than in the radicals.S. salsa seeds were sown in sand and grown in a chamber in dark. Three days later, plants were grown in a room at 200μmol/m2·s illumination under a 14-h-light/10-h-dark period. The shoots were harvested at 2nd and 10th day and used to determine the SsDODA expression and the betacyanin accumulation. The results demonstrated that the SsDODA expression and the betacyanin accumulation increased up to 2 d and decreased after the plants were transferred from dark to light.S. salsa seeds were sown in sand and grown in a chamber at 25℃. 30 days later, the plants were treated with low temperature at 10℃/6℃(day/night) for 7 d and then the plants grow under normal condition for another 7 d. The shoots were harvested at different treat time and used to determine the SsDODA expression and the betacyanin accumulation. The results indicated that low temperature treatment increased the SsDODA expression and the betacyanin accumulation. However, both the SsDODA expression and the betacyanin decreased after the low-temperature-treated plants moved back to the normal temperature (25℃) condition.