Studies On Cloning Of CDNA Encoding Betaine Synthetase From Suaeda Liaotungensis And Salt Tolerance Of Transgenic Tobacco

Betaine is a very effective osmopretectant existed in many organisms. In higher plants, betaine is synthesized by a two-step oxidation of choline: choline - betaine aldehyde -betaine. The first step is catalyzed by choline monooxygenase (CMO). The second step is catalyzed by betaine aldehyde dehydrogenase (BADH). Suaeda liaotungensis Kitag is a halophyte with high salt tolerance ability. This research performed a systemic study on betaine, betaine synthetase and the genes encoding betaine synthetase in Suaeda liaotungensis.Betaine concentration in Suaeda liaotungensis was determined by Chemistry Colorimetry. It is up to 95.5 ug/g.FW leaves which is lower than that in Suaeda ussuriensis, but higher than that in other plants.The purified BADH, which became a single band in SDS-PAGE, has been obtained by a combination of ammonium sulfate fractionation, ion exchange chromatography and gel filtration chromatography. The specific activity of BADH is 2363nmol/min.mg. BADH from Suaeda liaotungensis, whose molecular weight is 129kD, is composed of two same subunit. IEF study showed one band with pi value of 5.70. The optimum pH is 7.80 and the optimum temperature is 30癈. Estimated values of Kmand Fmax at pH 8.0 and 25 癈 are 67.7 umol/L and 78.9umol/min for betaine aldehyde. K+, Na+, Ca2+ and Mg2+ can effect the relative activity of purified BADH.The full length CMO and BADH cDNA were obtained from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. CMO cDNA (1820 bp) included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1329 bp ORF encoding a 442-amino-acid polypeptide which was 77%, 72% and 74% to CMO sequences of spinach, sugar beet and Atriplex hortensis in amino acid homology respectively. BADH cDNA (1901bp) included a 66 bp 5' UTR, a 329 bp 3' UTR and a 1506 bp ORF encoding a 501-ammo-acid polypeptide which showed 88% sequence identity to BADH from spinach, sugar beet and Atriplex hortensis respectively. The deduced amino acid sequence included a decapeptide sequence "VTLELGGKSP", which is highly conserved among general aldehyde dehydrogenases (ALDH), and a Cysteine residue.The CMO and BADH ORF were obtained and the plant expression vector pBI121-CMO,inpCAMBIA1301-BADH were constructed. The resultant plasmids pBI121-CMO, pCAMBIA1301-BADH were transferred into Agrobacterium tumefaciens (LBA4404) by the liquid nitrogen freeze thaw method.The CMO and BADH genes were transferred into tobaccos (Nictiana tabacum L. cv. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO and BADH genes were integrated into tobaccos' genome. Betaine accumulation in transformed tobaccos was higher than that in control plants. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type. The transformed tobacco with CMO gene could survive on MS medium containing 1.5% NaCl. The transformed tobacco with BADH gene can survive on MS medium containing 1.2% NaCl. However the normal tobacco cann't grow on MS medium containing 1.0% NaCl.