Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Plant Cell. 2009 Sep;21(9):2672-87. doi: 10.1105/tpc.108.060079. Epub 2009 Sep 25.
We have isolated and characterized the cDNA encoding the ornamental tobacco (Nicotiana langsdorffii X N. sanderae) homolog of the antirrhinum (Antirrhinum majus) MYB305. This transcription factor was robustly expressed at Stage 12 of nectary development but was only weakly expressed in the earlier Stage 6 nectaries. The ornamental tobacco MYB305 contains a conserved R2R3 MYB DNA binding domain with 76 amino acids in the activation domain. A green fluorescent protein-MYB305 fusion localized to nucleus of tobacco protoplasts and yeast one-hybrid assays demonstrated that it functions as a transcription activator. A conserved 23-amino acid C-terminal domain is required to activate gene expression. The coding region of the myb305 cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and was purified to homogeneity. This protein shows binding to two consensus MYB binding sites on the ornamental tobacco Nectarin I (nec1) promoter as well as to the single site located on the Nectarin V (nec5) promoter. Deletions of either of the binding sites from the nec1 promoter significantly reduced expression in nectary tissues. Temporally, MYB305 expression precedes that of nec1 and nec5, as would be expected if the MYB305 factor regulates expression of the nec1 and nec5 genes. Ectopic expression of MYB305 in foliage was able to induce expression of both nec1 and nec5, as well as two flavonoid biosynthetic genes in the foliage. Finally, RNA interference knockdown of MYB305 resulted in reduced expression of both nectarins and flavonoid biosynthetic genes. We conclude that expression of MYB305 regulates expression of the major nectarin genes in the floral nectary.
我们已经分离并鉴定了观赏烟草（Nicotiana langsdorffii X N. sanderae）同源物的 cDNA，该基因编码拟南芥（Antirrhinum majus）MYB305。该转录因子在蜜腺发育的第 12 期强烈表达，但在早期的第 6 期蜜腺中仅微弱表达。观赏烟草 MYB305 含有一个保守的 R2R3 MYB DNA 结合域，在激活域中有 76 个氨基酸。绿色荧光蛋白-MYB305 融合蛋白定位于烟草原生质体的细胞核中，酵母单杂交试验表明它作为转录激活因子起作用。保守的 23 个氨基酸 C 末端结构域是激活基因表达所必需的。myb305 cDNA 的编码区在大肠杆菌中作为谷胱甘肽 S-转移酶融合蛋白表达，并被纯化至均一性。该蛋白显示与观赏烟草 Nectarin I（nec1）启动子上的两个共识 MYB 结合位点以及位于 Nectarin V（nec5）启动子上的单个位点结合。从 nec1 启动子中删除任一个结合位点都会显著降低蜜腺组织中的表达。如果 MYB305 因子调节 nec1 和 nec5 基因的表达，则 MYB305 的表达先于 nec1 和 nec5 的表达。在叶组织中异位表达 MYB305 能够诱导 nec1 和 nec5 以及叶组织中两个类黄酮生物合成基因的表达。最后，RNA 干扰敲低 MYB305 导致 nectarins 和类黄酮生物合成基因的表达降低。我们得出结论，MYB305 的表达调节花蜜腺中主要 nectarin 基因的表达。
