Cloning,Construction Of Plant Expression Vector And Transformation Of GmNCED1Gene And Promoter From Glycine Max

Soybean (Glycine max) is an important global crop, providing oil and protein. It belongs to non-halophytes, which was limitied by salt and dehydration.The plant hormone abscisic acid (ABA) plays important roles in seeds maturation and dormancy and in adaptation to a variety of environmental stresses. The oxidative cleavage of cis-epoxycarotenoids catalyzed by9-cis-epoxycarotenoid dioxygenase (NCED) is considered to be the ratelimiting step in abscisic acid (ABA) biosynthesis.Here we demonstrate that the expression of GmNCED1gene in soybean plants is significantly up-regulated by dehydration and high salinity. We report the isolation and characterization of the NCED1gene from the leaves of Glycine max. Multiple alignments and phylogenetic tree of NCED1show that GmNCEDl was homologous to other all NCEDs. In addition,That stress tolerance of soybean plants can be increased by expression of the GmNCED1gene causing accumulation of ABA level.The results of this study are as follows:1. The GmNCED1had a1836ORF, which encodes a peptide of611amino acids. With the aid of the prediction algorithm ChloroP and online software ChoroP1.1Server-prediction it was predicted that an73-aa putative chloroplast transit polypeptide was located at the N-terminus of the GmNCED1.2. Phylogenetic tree analysis of the GmNCED1in Glycine max with NCEDs in17other plant species. The results shows that the GmNCED1genes has highest homology with dicotyledonous legume plants(peanuts, pea,etc)and the homology is as high as85%.It has hinher homology with other dicotyledonous(arabidopsis,ect) and the homology is between55%and75%. It has low homology with others such as rice, wheat, corn, barley, the homology is under50%.3. In this study, qRT-PCR analysis showed that expression of NCED1gene was enhanced in roots and leaves under treatment with salt, cool, NAA, endogenous ABA and drought. The increased transcript levels of GmNCED1were much higher in shoots than roots under osmotic stress.4. We isolated the GmNCED1promoter from soybean genomic DNA. The promoter sequence was analyzed for potential cis-elements using the programs PlantCare. A number of motifs were found in the GmNCED1promoter upstream of two putative basic cis-elements, TATA box and CAAT box.And included two copes of ABRE motif involved in drought and ABA responsiveness, and one TC-rich motif in involved in defense and stress responsiveness, and BoxI. The fact many cis-elements related to various stresses are present in the promoter region suggests that the GmNCED1gene is controlled by a complicated regulatory mechanism and that it can respond to various stress.5. Using Gateway Technology, NCED1gene was ligated into the pCB35SR1R2-NCED1binary vector. Wild-type soybean was transformed with Agrobacterium harboring pCB35SR1R2-NCED1construct by cotyledon dip method, generating five transgenic lines by PCR detection. The transgenic soybean plants were subsequently growth in soil.6. Construct pBI121-pGmNCED1-GUS fusion gene expression vector. Using agrobacterium mediated leaf disc transformation, generating many transgenic tobacco lines by PCR detection.