烟草NtMYB4a基因酵母双杂交诱饵载体的构建及互作蛋白的筛选

为探究NtMYB4a是否与其他蛋白质发生互作,并共同影响烟草次生代谢产物的合成和响应非生物胁迫,通过构建NtMYB4a基因酵母双杂交诱饵载体,从烟草cDNA文库中筛选出与NtMYB4a互作的候选蛋白,并利用实时荧光定量PCR初步验证低温胁迫下候选蛋白和NtMYB4a的共表达关系。结果表明,从cDNA文库中初步筛选出39个与NtMYB4a互作的蛋白,经回交验证排除假阳性,最终得到6个与NtMYB4a具有互作关系的蛋白,分别是RALF-like 22蛋白、锌指A20/AN1结构域蛋白、液泡分选蛋白、天冬氨酸蛋白酶、B2蛋白和应激增强蛋白。在低温胁迫下,6种互作蛋白的基因表达量变化趋势与NtMYB4a相似,均呈升高—降低—升高的趋势。相关性分析发现,液泡分选蛋白、B2蛋白、RALF-like 22蛋白和应激增强蛋白基因的表达与NtMYB4a的表达呈正相关关系,锌指A20/AN1结构域蛋白和天冬氨酸蛋白酶基因的表达与NtMYB4a的表达呈较弱的负相关关系,推测NtMYB4a可能与这些蛋白互作参与烟草低温胁迫的防御调控。本研究结果为进一步探究NtMYB4a参与响应非生物胁迫的分子调控机制提供了依据。

Construction of Yeast Two-hybrid Bait Vector and Identification of Proteins Interacting With NtMYB4a in Tobacco

In order to explore whether NtMYB4a interacts with other proteins to jointly affect the synthesis of tobacco secondary metabolites and response to abiotic stress, in the study, yeast two-hybrid bait vector of NtMYB4a gene was constructed and screened the candidate proteins interacting with NtMYB4a were screened from tobacco cDNA library then their co-expression under low temperature stress were verfied preliminarily by real-time quantitative PCR to preliminatively verify their co-expression under low temperature stress. The results showed 39 proteins interacted with NtMYB4a were initially screened from the cDNA library, after backcross verification to exclude false positives, 6 protein interacted with NtMYB4a were obtained, namely Ralf-like 22 protein, A20/AN1 zinc-finger domain protein, vacuolar sorting protein, aspartic protease, B2 protein and stress enhanced protein. Under low temperature stress, the expression of the six interacting proteins were similar to those of NtMYB4a, which showed an increase-decrease-increase trend. Correlation analysis showed that the expressions of vacuolar sorting protein, B2 protein, Ralf-like 22 protein and stress enhanced protein were positively correlated with those of NtMYB4a, while A20/AN1 zinc-finger domain protein and aspartic protease were weakly negatively correlated with those of NtMYB4a. It is speculated that NtMYB4a may interact with these proteins to participate in the regulation of tobacco responding to low temperature stress. The results provide a basis for further exploring the molecular regulation mechanism of NtMYB4a in response to abiotic stress.