Preliminary Studies On3Stress-resistance Genes (BADH、CMO And NECD) Of Lycium Barbarum

Drought and salinity is a serious problem all over the world. Betaine will be acculumated in plants in response to drought and salinity stress. Betaine plays an important role in improving osmotic adjustment, and gets involved in the protection of macrocomponents of plant cells, such as function and structure of protein. Several enzymes are involved in the betaine synthesis, as phosphoethanolamine N-methyltransferase(PEAMT) catalyzes the phosphoethanolamine to choline which is vital for betaine synthesis, choline is then catalyzed by choline monooxygenase (CMO) to glyeine betaine aldehyde, and finally catalyzed by betaine aldehyde dehydrogenase (BADH) to betain. Abscisic acid (ABA) regulates the essential physiological and developmental processes of plants and plays imporant roles in plant responses to various environmental stresses.9-cis-epoxycarotenoid dioxygenase (NCED) is the key regulatory enzyme in the biosynthesis pathway of ABA in higher plants.Lycium barbarum L, a deciduous perennial bush and the sole halophyte genus of Solanaceae, has a very developed root system, which has the character of high resistance to drought, salinization, cold and barren. L. barbarum grows maily in mild salinization areas, which is an important pioneer species during salinization land improvement. During the western ecological construction and Chinese herbal medicine base construction, L. barbarum has the very high ecological, economic and social value.In this paper, we overexpressed the key genes involved in the betaine biosynthetic pathway, CMO and BADH in transgenic Arabidopsis thaliana plants by using genetic engineering method. Meanwhile we cloned and analysed the expression of ABA biosynthetic gene NCED in L. barbarum, and further to study the resistance mechanism of L. barbarum, increased its stress-resistance and provide theoretic basis to breed new stress-resistant varieties. The main results are as follows:1) In this experiment, CMO and BADH was transformed into A.thaliana using Agrobacterium tumefaciens-mediated approach. The genes which were integated in the genomes of transgenic A.thaliana plants were verified by southern blot. The relative electronic conductivity rate of transgenic A.thaliana were relatively lower than those of control respectively. The content of proline and content of betaine of transgenic A.thaliana increased more than those of control, while the content of MDA of transgenic A.thaliana increased relatively lower than those of control. The result indicated that great difference had been found between the physiological and biochemical indexes of transfornied A.thaliana seedling and control, which proved that the transformation of stress tolerance genes could improve the tolerance of A.thaliana.2) In the study, a full-lengh cDNA of NCED gene(LbNCED) was fristly isolated and characterized from the leaves of L.barbarum. LbNCED has2,316bp, containing a1,824-bp ORF and encoding607amino acids. Comparative and bioinformatics analyses revealed that the deduced protein of LbNCED was highly homologous to NCEDs from other plant species. At the N-terminus of the LbNCED located a15-aa putative chloroplast transit peptide. Southern blot analysis revealed that it was a low-copy gene in the genome of L.barbarum. The RT-QPCR analysis revealed that dehydration, salt stress signficantly enhanced LbNCED transcript expression and ABA content accumulation.