Molecular Cloning And Founctional Analysis Of SsNCED1 Gene In Suaeda Salsa

Abscisic acid (ABA) is a phytohormone which plays an important role in plant growth and development, seeds dormancy, and plant responses to environmental stresses. Because many of these physiological progresses are correlated with the endogenous ABA level, the regulation of ABA biosynthesis is the key elment facilitating our understanding of these characteristics. In higher plants, the ABA biosynthesis pathway proceeds from carotenoids to xanthoxin via several steps catalyzed by zeaxanthin epoxidase, 9-cis-epoxycarotenoid dioxygenase. and Moco-requiring aldehyde oxidase.There are nine genes encoding the 9-cis-epoxycarotenoid dioxygenase in Arabidopsis genome (table 1), named AtNCED1-9, respectively. AtNCED3 was demonstrated to be the key factor involved in ABA biosynthesis under environmental stresses, while AtNCEDl may not involved in ABA biosynthesis because it catalyze the 9-10 (9'-lCF) cleavage reactions but not 11-12 cleavage reactions. In our experiments, out of the 1,000 ESTs obtained from large sequencing the random-selected cDNA clones from a NaCl-treated cDNA library of Suaeda salsa, we found two ESTs encoding 9-cis-epoxycarotenoid dioxygenase. We obtained its full-length cDNA sequences by PCR methods. The 1, 972 bp full-length cDNA has an open reading frame of 1,698 bp and encode a 62.5 KDa protein with 566 aa. The amino acid sequences of SsNCEDl has a high identities of 77% with AtNCEDl from Arabidopsis. By northern blotting analysis, we found that the mRNA level of SsNCEDl increased by NaCl and mannitol treatment in root and in arieal part tissues, and high temperature also up-regulate the expression of SsNCEDJ both in arieal part tissues of 5. salsa.To study the function of SsNCEDl, we produced the transgenic arabidopsis with overexpression of SsNCEDl gene mediated by Agrobacterium tumefaciens. PCR and Southern blotting analysis indicated that all lines had been intergrated of SsNCEDl, and Northern blotting analysis revealed the presence of expression of SsNCEDl in transgenic lines, except 9-23 transgenic line. After we measured some physiological characteristics of transgenic arabidopsis, we found that overexpression of SsNCEDl did not increase the plant response TO NaCl and mannitol stresses, nor did the endogenous ABA content change in root, arieal part and seeds. But interestingly, transgenic plants showed more sensitive to extrogemous ABA and less carotenoid content than wild type arabidopsis. All these data showed that although SsNCEDl belongs to NCED genes family, it may not be involved in ABA biosynthesis, but involved in carotenoid catabolism. With increasing numbers of gene encoding the homology of AtNCEDJ in GeneBank database, more and more studies should be carried on it.