The Expression Of BADH Gene Driven By The BADH Promoter In Tobaccos

Betaine aldehyde dehydrogenase (BADH) is the key enzyme for betaine synthesis. Betaine is regarded as being a kind of small, non-toxic osmoprotectant and plays an important role in plant salt-tolerance. BADH expression is induced by salinity and its expression is related to its promoter (pB:-300~+62bp). pB is a salt-induced promoter. Compared to noninductive leaves, the GUS enzyme activity driven by pB promoter was enhanced 6.3-fold in transgenic tobacco leaves in the presence of 400 mmol/L NaCl.In this paper,the plant expression vector,pCAMBIA1301-pB-BADH was constructed and then transferred into Agrobacterium tumefaciens. pB-BADH and CaMV 35S-BADH gene were transferred into tobaccos via Agrobacterium-mediated leaf disk transformation respectively.The expression of BADH and the content of betaine were analyzed in the two types transgenic plants .The main results are as follows:1. The 1.5 kb coding region of BADH gene was amplified from cloning vector pMD18- T-BADH through PCR. The new expression vector pCAMBIA1301-pB-BADH was constructed by replacing the GUS report gene in pCAMBIA1301- pB-GUS. Then it was transferred into E.coli competent cell-DH5αby liquid nitrogen freeze thaw method.2. The plant expression vector, pCAMBIA1301-pB-BADH, was transferred into Agrobacterium tumefaciens strain LBA4404.3. pB-BADH and CaMV 35S-BADH were transferred into tobaccos via Agrobacterium- mediated leaf disk transformation. PCR analysis showed that hygromycin-resistant transgenic tobaccos were obtained. RT-PCR detection showed that the expression of BADH in CaMV35S-BADH tobaccos was higher than that in pB-BADH type tobaccos. Betaine content analysis showed that the betaine content in pB-BADH type tobaccos was similar to that in wild type tobaccos, and lower than that in CaMV35S-BADH tobaccos.4. After 24 h under 0.1mol/L NaCl stress, RT-PCR detection showed that the expression of BADH in pB-BADH tobaccos was higher than that in CaMV35S-BADH type tobaccos. Betaine content analysis showed that the betaine content in pB-BADH tobaccos was enhanced when compared to noninductive condition, further more, higher than that in wild type and CaMV35S-BADH type tobaccos.