Cloning And Analysis Of The Gene Encoding 5-ugt In Suaeda Salsa L.

As a leaf succulent euhalophyte, Suaeda salsa L. has a high salt resistance,and high nutritional health value.At the same time, planting Suaeda salsa in the saline land can significantly improve the soil properties, reduce soil salinity,increase soil organic matter and it is beneficial to eliminate bare saline land, prevent soil erosion, and plays a very important role in maintaining and reconstructing the ecology of saline soil.The shoot of S. salsa plant is red-violet throughout the whole growth period in the intertidal zone or in the flooding area, but green on highland and land far from the seaside. Our earlier results showed that the red pigments accumulated in S. salsa are betacyanins. The physiological and molecular responses of S. salsa to salinity stress have been extensively studied. However, the molecular mechanism and the regulation of relevant enzyme involving in betacyanin synthesis in S. salsa have not been elucidated. In our laboratory, the gene DODA coding for the 4,5-DOPA dioxygenase from S. salsa was cloned. On this basis,the gene SsUGT coding for 5-O-glucosyltransferase was cloned in this work, and its primary functions were determined. The results are showed as follows:1.cDNA cloning and sequence analyzing of 5-O-glucosyltransferase in S. salsaBased on sequence homologies to the 5-UGT from other plants,we used reverse transcriptase RT-PCR amplification to obtain the intermediate fragment of SsUGT,which is 992bp.BLAST analysis showed that this fragment has a high homology with the UGTs in other plants, initially identified as SsUGT.Design specific primers accoding to the sequence for 5'-and 3'-RACE amplification, and we obtained the 5'-sequence about 631bp and the 3'-sequence about 206bp. We spliced the three sequences and obtained the full-length transcript sequence of SsUGT. The full-length cDNA of SsUGT was 1653 bp with an 1482 bp open reading frame (ORF), which encoded a 493-amino-acid polypeptide with the predicted molecular mass of 56.7kDa. BLAST search results showed that the sequence of SsUGT was 68%,64% 和61% similar to that of Phytolacca americana,Beta vulgaris, and Dorotheanthus bellidiformis.2. Expression analysis of 5-UGT in S. salsaUsing RT-PCR method, we detected the expression of SsUGT in leaves and stems in S. salsa under inundation treatment and cold treatment. The results showed that SsUGT expression in leaves and stems were upregulated by and cold treatment, and they reched the maximum in 2 h/d and 4 h/d respectively.3. The effects of inundation and low temperature on the betacyanin accumulation of S. salsaThe effects of inundation and low temperature on the betacyanin accumulation of S. salsa were investigated. The results indicated that when S. salsa plants were treated with flooding for 2 h daily for 14 d and treated with low temperature for 4 h daily for 14 d, the contents of betacyanin reached the highest in both stems and leaves of them.4. The correlation of SsUGT expression and betacyain contentThe correlation of SsUGT expression and betacyain content was analysised. The results showed that the correlation in stem under cold condition is higher, but the correlation in leaf under cold condition and correlation of leaf and stem under inundation is lower.