Cloning CDNA Of Choline Monooxygenase Gene From Spinach And Prokaryotic Expression

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Many important crop plants, such as rice, potato and tobacco, are sensitive to drought and salinity. Drought and salinity frequently cause the severe production losses of crops due to lack of effective tolerance mechanism. Improvement of crops and breeding of new varieties to increase their drought and salinity tolerance is the most economic and effective ways for using arid and salty soils. With the rapid development of modem molecular biology, the great progress has been achieved in improving crop drought and salinity tolerance by genetic engineering. One of the fundamental physiological mechanisms of high plant to cope with environmental stresses is osmotic adjustment, and glycine betaine is one of the most important of osmolytes. Glycine betaine was regarded as one of the most promising osmolytes because it possessed simple biosynthesis pathways, non-toxic to cells, and non-osmotic functions of stabilizing enzyme activities and membrane structures, reduction the peroxidation of membrane lipids.Some crop plants, such as rice, potato, tobacco and tomato, are unable to synthesize glycine betaine. Introduction of genes related to glycine betaine synthesis and establishing a glycine betaine biosynthetic pathway in glycine betaine-deficient crop plants through genetic engineering, which could be enhanced drought and salinity tolerance of crop plants. Until now, many transgenic plants were obtained by introducing the related genes of glycine betaine synthesis and increased the ability of plants at the various levels to tolerate drought and salinity stresses.The main purpose of the study is to isolate a choline monooxygenase (CMO) gene which is one of the key enzymes for glycine betaine synthesis from spinach (Spinacia oleracea L.), construct the expression vectors of CMO gene under the control of the promoters CaMV 35S and rd29A, and to induce expression of CMO gene in Escherichia coll. The main results as following: 1. Total RNA was extracted from leaf of spinach. The cDNA of choline monooxygenase (CMO) gene was obtained using the reverse transcription-polymerase chain reaction (RT-PCR) method, and then cloned into the vector pBluescript SK＋. The sequence analysis demonstrated that the full-length CMO cDNA was 1424 bp, and the open reading frame was 1320 bp which encoded a 440-amion acid polypeptide. Homology analysis of the nucleotide sequence and the deduced amino acid sequence showed that the spinach CMO gene shared 99.8% and 99.6% identity with the published sequence respectively.2. The expression vectors pBI-CMO and pBIr-CMO were constructed respectively by fusing CMO gene with the constitutive promoter CaMV 35S and the stress-induced promoter rd29A. The expression vectors were then introduced into Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method and proved by enzyme digestion and PCR methods.3. Using DNA recombination technology, the CMO gene was subcloned into the expression vector pET28a and was recombinant plasmid pET28a-CMO, which was transformed into Escherichia coli BL21(DE3). Expression induced by IPTG and SDS-PAGE analysis showed that the CMO gene could express in E. coli, and induced time had less effect on protein expression.