Transgenic Plants Expressed BADH From Halophyte And Using Strong Promoters

Many diverse species of plants accumulate glycine betaine (Glybetaine) in response to various stresses,and the biosynthesis and accumulation of Glybetaine are thought to contribute to various stress. Glybetaine is found in a large variety of microorganisms,higher plants and animals. At high concentrations,Glybetaine does not interfere with cytopasmic function and many macromolecules. Thus it belongs to non-toxiic osmoprotectants. Glybetaine appears to be a critical determinant of stress tolerance in plants. The accumulation of Glybetaine is induced under stress conditions,and the level of Glybetaine is correlated with the degree of enhanced tolerance to stress. However,Glybetaine does not accumulate in many important crop plants such as potato,tomato or rice. Among plants Glybetaine biosynthetic pathway has been thoroughly characterized in sugar beet and spinach,and biosynthesis is localized in the chloroplast by a two-step oxidation of choline via the intermediate betaine aldehyde. The first enzyme,choline monooxygenase (CMO) and the second enzyme are all localized in chloroplast. The simplicity of the Glybetaine biosynthetic pathway makes it amenable to genetic engineering. Many scientists have introduced BADH gene to plants and got transgenic plants which overexpressed BADH,but the accumulations of Glybetaine are too low to function effectively. It is possible that there is lack of substrate of BDAH and /or BADHs is not localized in chloroplast. The BADH protein and mRNA synthesis are induced by drought,saline,cold stress and ABA treatment,in parallel with an increase in the betaine level. However,the exact mechanisms of transcriptional activation of the BADH gene leading up to such activation events remain poorly understood.Up to date,the cauliflower mosaic virus (CaMV) 35S promoter and its derivatives are used commonly. No report show transgenic plants expressedBADH from halophyte and using its own promoter or other strong promoters. We isolated the BADH cDNA (AcBADH) from the halophyte Atriplex centralasiatica Iljin using RT-PCR and the deduced protein sequences showed high similarity with other plants. Analysis of AcBADH expression in leaves showed that the expression levels of AcBADH increased about 10-fold after plants were treated with NaCl and ABA for 48 hours when compared to control plants respectively. These data suggested that stresses can induce the AcBADH gene expression and some cis-elements in the upstream of AcBADH must participate in this reaction. A genomic DNA library of Atriplex centralasiatica Iljin was constructed using Lambda EMBL3/BamH I as vector. The library consisted of 1.3 X 106 clones with an average insert size of about 18Kb .The capacity of this library was about 20 times the equivalent of the genome of Atriplex centralasiatica Iljin. Screening the genomic library with a 400bp probe located at the 5'end of the BADH gene obtained by RT-PCR,we got four positive clones. One of them contained the entire BADH genomic sequences. BADH gene extends 7.7kb containing 15 exons with the open reading frame of SOOaa,1.2kb putative promoter region. The transcription start site was localized 81bp upstream of the start ATG by primer extension. Besides the TATA-box and CAAT-box,several transcription binding motifs which are related with stress response are found in the promoter,such as GC-motif,EIRE,MRE,WUN-motif,ABRE and HSE. To investigate the molecular basis of the stresses-induced gene regulation,the AcBADH promoter- /?-glucuronidase chimeric gene constructs containing six deletions were introduce into tobacco by agrobacterium-mediated transformation. These results demonstrated the AcBADH promoter is a strong salt-stress-induced promoter. The AcBADH 5'-flanking region contains two salt-responsive enhancer regions localized between -1114 and -892,-464 and -234 and one sliencer region localized between -892 and -643.