Cloning And Functional Analysis Of The Gene Encoding 4,5-Dopa Dioxygenase In Suaeda Salsa L.

Suaeda salsa L. is a leaf succulent euhalophyte. The shoot of S. salsa plant is red-violet throughout the whole growth period in the intertidal zone, but green on highland and land far from the seaside. As a halophyte, physiological and molecular responses of S. salsa to salinity stress have been extensively studied. And it is proved that the red pigments accumulated in S. salsa are betacyanins. However, nothing is known about the biological functions of the red pigments in S. salsa. The molecular mechanism and the regulation of relevant genes involving betacyanin synthesis in S. salsa have not been elucidated so far. In this work, the cloning of the gene DODA coding for the 4,5-DOPA dioxygenase from S. salsa and its promoter were cloned, and their primary functions were determined. The results are showed as follows:1. cDNA cloning and sequence analyzing of 4,5-DOPA dioxygenase in S. salsaBased on sequence homologies to the DODA genes from other plants, we used reverse transcriptase RT-PCR amplification and 5'- and 3'-RACE amplification to obtain the full-length transcript sequences of SsDODA. The full-length cDNA of SsDODA was 1030 bp with an 813 bp open reading frame (ORF), which encoded a 271-amino-acid polypeptide with the predicted molecular mass of 30.4kDa. BLAST search results showed that the sequence of SsDODA was 80% similar to that of Beta vulgaris.2. Constuction of plant expression vector pROKII::SsDODABy using DNA recombinant technology, the binary expression vector (pROKII::SsDODA), which fused the open reading frame (ORF) of SsDODA, was constructed.3. Obtaining of transgenic Arabidopsis thaliana plantsThe transformation of Arabidopsis thaliana was mediated by Agrobacterium GV3101. Some transgenic Arabidopsis have been obtained after screened by application 50 mg/L Kan to the medium.4. Cloning and sequence analyzing of SsDODA promoterThe 600 bp promoter region of SsDODA gene from S. salsa was amplified by TAIL-PCR technology. Promoter sequence analysis showed that the cloned fragment contains lots of AT rich-regions, TATA-box, CAAT-box, which are the characteristic regions in all promoters. Meanwhile, there are some other regulation regions in this promoter sequence such as light-regulated region.5. Constuction of plant expression vector pBI121::SsDODA-PPlant transformation vector pBI121 was digested completely with Hind III and BamH I. The bigger fragment was isolated and ligated to the SsDODA promoter fragment which had also been cleaved with Hindâ…¢and BamH I to generate plant expression vector named pBI121::SsDODA-P harboring the fusion gene SsDODA promotor ::GUS.6. Histochemically GUS activity of the SsDODA promoterThe transformation of tobacco plants was mediated by Agrobacterium EHA105. And histochemically GUS activity detecting results demonstrated that the SsDODA promoter fragment was active to drive the expression of the GUS gene.