Analysis Of NCED1Gene Promoter And Prokaryotic Expression In Caragana Korshinskii

Abscisic acid (ABA) is a phytohormone and mediates the response and adaptation of higher plants to various environmental stresses in life cycle. Oxidative cleavage of cis-epoxycarotenoids by9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. The primary research which focused on gene promoter and protein function will build a foundation for further illustration of NCED1gene function and resistance regulation mechanism in forage crops.Genomic walking technique was used to clone promoter sequence of CkNCED1gene from C. korshinskii. The PlantCARE cis-element analysis revealed that, beside the basic elements, the promoter sequence also included two ABA responsive elements (ABRE) and many other stress-responsive elements.The two binary vector P::GUS were constructed by fusing the promoter GUS gene originated from PBI121and pGWB533. Histochemical staining of transgenic Arabidopsis showed that GUS reporter gene expressed in the tissues of leaf, root, stem, flower and silique.The expression level is especially higher in leaves. The above results identified the localization and expression intensity of CkNCED1gene in vivo, and suggested that this gene play an important role in ABA involved stress-resistance regulation.In order to further verify the gene function, the research analysis the subcellular location and prokaryotic expression of CkNCEDl protein. A recombinant plasmid was constructed by fusing NCED1and green fluorescent protein gene, then, it was transformed into Arabidopsis mesophyll protoplast by PEG-mediated method. Transient expression analysis indicated that pCAMBIA1302-NCED1-GFP fusion protein was localized in the plastid. By using a protein reconstruction method, the prokaryotic expression of the CkNCED1protein was studied. SDS/PAGE and western blot result demonstrated that the highly purified fusing protein was obtained, and the protein has the immunogenicity. This work paved way for further functional study of NCED1gene in C. korshinskii...