Analysis Of The Interaction Of A Transcription Factor (AtGT-3b) With CMO And BADH Gene Promoters

Choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are the two key enzymes for betaine synthesis. Betaine is regarded as being a kind of small, non-toxic osmoprotectant and plays an important role in plant salt-tolerance. Our preliminary work showed that CMO gene promoter (pC: -267 ~ +1bp) and BADH gene promoter (pB:-300 ~ +1bp) are salt-induced promoters. Compared to noninductive leaves, the GUS enzyme activity driven by these two promoters was enhanced 5-fold and 6.3-fold respectively in transgenic tobacco leaves in the presence of 400mmol/L NaCl. Both of the two promoters contain basic elements, salt-induced element, GT-1 cis-element (GAAAAA), and other stress-induced elements.AtGT-3b is a NaCl- and pathogens-induced GT-1-like transcription factor in Arabidopsis thaliana. It can interact with GT-1 cis-element and enhance the expression of the downstream gene. Both CMO and BADH gene promoters contain the recognition sequence of AtGT-3b, GAAAAA. In this paper, we will study the interaction of AtGT-3b with GT-1 cis-element and analyze the regulation mechanisms of CMO, BADH transcription.In this paper, AtGT-3b gene was cloned from Arabidopsis thaliana and transferred into the transgenic tobaccos (C5A,B5A) containing pC, pB. The exogenous gene expression and GUS activity were analyzed in AtGT-3b transgenic plants. The main results are as follows:1. AtGT-3b gene were obtained through RT-PCR, including a 870bp ORF, encoding a 289-amino-acid polypeptide. The cloning vector, pMD19T- AtGT-3b, was constructed and transferred into E.coli competent cell-DH5αby liquid nitrogen freeze thaw method.2. The plant expression vector, pBI121-AtGT-3b, was constructed and transferred into Agrobacterium tumefaciens strain LBA4404.3. AtGT-3b gene was transferred into C5A and B5A tobaccos via Agrobacterium mediation. PCR analysis showed that kanamycin-resistant AtGT-3b transgenic tobaccos were obtained. RT-PCR detection showed that AtGT-3b gene has expressed in transgenic tobacco. GUS histochemical staining and fluorescence quantitative analysis in the transgenic tobacco leaves showed that the GUS activity in transgenic tobaccos is higher than that in control tobacco. It suggests that AtGT-3b transcription factor can interact with pC and pB promoters, and enhance the expression of their downstream genes.