普通烟草生长素抑制因子NtIAA17的克隆与功能分析

【背景和目的】盐胁迫会抑制作物的生长发育,从而影响作物的产量和品质。生长素/吲哚-3-乙酸(Aux/IAA)蛋白是响应生长素信号的关键因子,参与调控植物的生长发育和响应非生物胁迫。【方法】为了研究烟草生长素反应基因NtIAA17在烟草生长发育过程中的功能,以烟草K326为试验材料,通过克隆获得烟草NtIAA17基因,利用生物信息学分析其序列特征,qRTPCR技术分析NtIAA17在烟草不同组织及盐胁迫处理0~12 h的表达模式,利用转基因技术获得过表达NtIAA17-OX(NtIAA17-overexpression)转基因烟草,对其进行响应盐胁迫的功能鉴定。【结果】NtIAA17基因片段为624 bp,编码207个氨基酸;相对分子质量为23.07 kD,理论等电点为8.24,该基因启动子区含有顺式作用元件与激素及胁迫相关;具有Aux/IAA家族蛋白的四个结构域(I-IV)和保守序列。NtIAA17基因与菠菜生长素抑制基因MoIAA13亲缘关系最近;亚细胞定位分析表明,NtIAA17蛋白是核定位蛋白。qRT-PCR结果表明,NtIAA17基因在茎和老叶中高表达,盐胁迫2 h时表达量最高。在盐胁迫条件下,NtIAA17-OX转基因烟草与野生型相比,生物量显著增加,脯氨酸含量积累增加,而MDA含量减少,抗氧化酶基因NtSOD、NtPOD表达量增加。【结论】NtIAA17基因可以响应盐胁迫信号,通过提高转基因烟草的抗氧化性来抵御盐胁迫带来的伤害,增加植株的生物量;本研究为后续耐盐胁迫分子育种提供候选基因和理论依据。

烟草硝酸盐转运蛋白NtNRT1.5基因的克隆及功能分析

【背景和目的】低亲和硝酸盐转运蛋白NRT1家族基因在植物吸收和转运硝态氮过程中发挥重要作用,为研究烟草NRT1家族基因在烟草氮素利用中的作用。【方法】从栽培烟草品种K326中克隆获得NtNRT1.5的全长cDNA序列,利用生物信息分析、qRT-PCR技术和转基因技术系统分析NtNRT1.5基因的序列特征、表达模式及在提高烟草氮素利用效率方面的功能。【结果】NtNRT1.5全长序列包含1800bp开放阅读框、编码599个氨基酸,编码的蛋白质具有NRT基因家族的共有结构特征,与拟南芥AtNRT1.5相似性达到59.28%。亚细胞定位结果表明NtNRT1.5蛋白定位在细胞膜上,其启动子中包含多个与硝酸盐(A(C/G)TCA)、胁迫(MYB)、根系特异表达(ROOT MOTIF BOX)相关的响应元件。表达模式分析结果表明NtNRT1.5主要在根部表达,其次是衰老叶片和茎,新叶基本不表达;低氮抑制其在根系中的表达,说明NtNRT1.5主要负责正常条件下的硝酸盐的吸收;低氮条件下,NtNRT1.5过表达显著提高了转基因烟株的氮素利用率。【结论】NtNRT1.5基因在烟草氮素的吸收和转运上行使重要功能,通过提高过表达烟草的氮素利用率,增加植株的生物量。本研究为后期培育烟草氮高效利用新品种提供了理论依据。

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烟草硝酸盐转运蛋白NtNRT1.7基因的克隆、亚细胞定位及表达模式分析

为研究硝酸转运蛋白(Nitrate transporter,NRT)在烟草吸收和转运硝酸盐中的作用,以烟草K326的cDNA为模板,克隆了一个全长1839 bp、编码612个氨基酸的硝酸盐转运蛋白基因,命名为NtNRT1.7。生物信息学分析表明烟草NtNRT1.7具有典型跨膜结构域。同源性分析结果显示,NtNRT1.7与拟南芥AtNRT1.7的同源性达57.23%。亚细胞定位结果表明该蛋白定位在细胞膜上,是一个典型的膜蛋白。启动子分析和不同胁迫处理的qRT-PCR结果显示,NtNRT1.7主要在成熟叶中高表达,并受低氮诱导抑制表达,受干旱、低温、高盐等胁迫诱导增强表达,表明NtNRT1.7基因可能参与非生物胁迫条件下硝酸盐的再利用。

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