Functional Analysis Of BADH Gene Promoter From Suaeda Liaotungensis K.

Betaine aldehyde dehydrogenase (BADH) is the key enzyme to synthesize betaine which is a non-toxic osmopretectant and plays an important role in plant salt-tolerance. BADH expression is induced by salt and the amount increases with the increasing of salt concentration . The expression of BADH must be concerned with its promoter. This study is about BADH promoter from Suaeda liaotungensis K., and it is important to learn the molecular mechanism of BADH. On the condition that the salt-induced promoter fragment is applied to genetic engineering, it will be very valuable to enhance the salt tolerance of transgenic plant.Sequence analysis of the 1993 bp 5' upstream region of BADH from Suaeda liaotungensis K. using the PLACE and PlantCARE database revealed that there are several putative cis-elements, such as TATA-box and CAAT-box. Meanwhile, several stress-induced elements were found in this promoter. Three GT-1 motifs (GAAAAA) were believed to be involved in NaCl-responsive gene expression. Six MYC recognition sites (CANNTG) were observed for dehydration, ABA, cold and freezing responsiveness . Respectively, nine WUN-motifs ANATTNCNN, described as the wound inducing elements and eleven heat shock elements (HSE) were responsible for heat stress responsiveness.Using the designed five forward and one reverse primers, the different length of promoter fragments, that is, P1(-1993~+62bp), P2(-1466~+62bp), P3(-1084~+62bp), P4(-573~+62bp) and P5(-300~+62bp) were amplified by PCR. Through restriction digestion, purification and ligation, the amplified fragments were inserted into a plant expression vector containing GUS gene to substitute the CaMV 35S promoter and the resulted recombinant plasmids were designated as pCAMBIA1301-P1, pCAMBIA 1301-P2, pCAMBIA1301-P3, pCAMBIA1301-P4 and pCAMBIA1301-P5.These expression vectors were transferred to Nicotiana tabacum L.cv.89 by Agrobacterium tumefaciens-mediated leaf discs transformation. The plants resistant to Hygromycin being analyzed by PCR and GUS gene expression showed that the genes had been transferred into tobaccos and could drive GUS gene to express. The functional properties of each promoter fragment were examined by GUS histochemical staining and fluorescence quantitative analyses in the transgenic tobacco leaves treated with different NaCl concentrations for 48h. The results showed that induction levels were tightly proportional to concentration of the NaCl treatment. GUS enzyme activity was enhanced 6.3-fold in transgenic tobacco leaves containing P5 (-300bp to +62bp) promoter fragment in the presence of 400mmol/L NaCl when compared to noninductive leaves. It suggests that the smallest promoter fragment (-300bp to +62bp) possesses all the essential cis-acting elements and is sufficient for NaCl induction.