Browsing by Author "Zhang, Xinxin"

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    Characterization of cis-acting elements of NtPMT3 and trans-acting factors of NtQPT1 in Nicotiana tabacum
    (2002-04-12) Zhang, Xinxin; Dr. Stephanie E. Curtis, Chair; Dr. Mark A. Conkling, Member; Dr. Michael D. Purugganan, Member
    The primary focus of this thesis is the study of the regulation of NtPMT3 and NtQPT1 gene expression. Their spatial and temporal expression has been demonstrated to be regulated coordinately. In this work, a 2.05kb of 5' flanking region of NtPMT3 has been cloned from N. tabacum. Then a series of nested 5'-deletion of the NtPMT3 promoter driving a GUS reporter gene were generated. The chimeric reporter gene constructs, as well as a 35S CaMV promoter::GUS were introduced into tobacco N. tabacum cv. D-174 using Agrobacterium-mediated leaf disc transformation. Several independent transgenic lines for each construct were regenerated. Samples of leaf, stem and root were collected from different transformants and GUS activities were quantified by fluorometric assays. These deletion analyses showed that 320 base pairs from the site of transcription initiation is sufficient to direct its root-specific expression. A negative regulatory element that suppressed expression of the reporter gene in roots is located between -2.048kb to -1.632kb 5' of the transcription initiation site. In this 417bp region, six Auxin Response Elements (AuxREs) or AuxRE-like elements were found. Auxin is known to down regulate PMT transcription and this part of sequence might play an important role in its auxin response. Sequential deletion analyses also defined three positive regulatory elements, -1631bp to -1335bp, -601bp to -318bp and -317bp to -116bp. Deletion of these regions resulted in a decrease in their GUS expression. Previous studies of the NtQPT1 have identified a Nic gene response element located between -731bp and -1056bp from the transcription initiation site. To isolate the trans-acting factors binding to this sequence, I screened a tobacco root lambda-gt11 cDNA expression library. I obtained 5 cDNA clones encoding three different proteins that bind to the target sequence. DNA sequence of one protein (~790bp) shows high homology to N. tabacum DNA-binding protein of HMG protein family gene (ID AF002226). DNA sequence of the second protein (550bp) is about 99% identical to an A. thaliana small nuclear ribonucleoprotein-like protein gene (ID AY042797). Two cDNAs encoding the third protein are about 2.3kb. 500bp of both 3' and 5' ends were sequenced and showed high homology to a putative jasmonate inducible gene of A. thaliana (ID AY035108). The homology of 3' untranslated region indicates that the N. tabacum root expression library was contaminated by Arabidopsis thaliana cDNA.