The Cloning, Transformation And Homology Modeling Of Betaine Aldehyde Dehydrogenase Gene From Liqusticum Churning Franch Hort

Glycine betaine, the most effective and non-toxic osmoprotectant for the plant to respond to osmotic stress, can improve plant stress tolerance. To study the relationship between glycine betaine and stress resistance of Liqusticum churning Franch Hort, the content of glycine betaine in Liqusticum churning Franch Hort under drought stress was measured by HPLC. Betaine aldehyde dehydrogenase is the key enzyme in the betaine biosynthetic pathway in Liqusticum churning Franch Hort. To study the biosynthetic mechanism of glycine betaine in Liqusticum churning Franch Hort and to control its metabolism and biosynthesis at the molecular level, the transcription level of betaine aldehyde dehydrogenase gene under different stress conditions was determined by Semi quantitative RT-PCR. The cDNA of betaine aldehyde dehydrogenase gene was cloned and employed to construct the tobacco transformation system. Homology modeling and molecular docking were applied to reveal the possible catalytic mechanism of betaine aldehyde dehydrogenase. Our study provides a theoretical basis for the control of the metabolism and biosynthesis of betaine in Liqusticum churning Franch Hort at the molecular level.The experimental results were as follows:The content of glycine betaine in the leaves of Liqusticum churning Franch Hort under drought stress initiated with PEG solution reached a maximum at 24h. The rapid accumulation of glycine betaine proved that it was one of the osmoprotectants in Liqusticum churning Franch Hort.The transcript level of betaine aldehyde dehydrogenase gene of Liqusticum churning Franch Hort was up-regulated under different stress conditions tested. The expression level of the gene under stress conditions from high to low was drought, low temperature, salt.The gene of betaine aldehyde dehydrogenase was successfully cloned from the leaves of Liqusticum churning Franch Hort under drought stress condition, and verified by sequencing. The cloned sequence of the gene could be translated into a polypeptide with 508 amino acid residues. The eukaryotic expression system of the gene was constructed to transform the ordinary Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. The transformed tobacco was identified by PCR amplification with specific primers. Positive plants tested verified the successful construction of tobacco transformation system.Three dimensional structure of betaine aldehyde dehydrogenase of Liqusticum churning Franch Hort with a typical coenzyme binding domain and a substrate binding domain was constructed by homology modeling. On the channel linked the two domains locate a highly conserved catalytic cysteine residue of Aldehyde dehydrogenase family. The key amino acid residues involved in substrate binding mainly through hydrogen bonds and Van der Waals force were identified by the analysis of molecular docking. A bloated isoleucine residue on substrate binding pocket was considered to decrease the affinity of the enzyme to betaine aldehyde by the analysis of the substrate binding mode.