普通烟草生长素抑制因子NtIAA17的克隆与功能分析

Cloning and functional analysis of the auxin inhibitor gene NtIAA17 in common Nicotiana tabacum

【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。

[Background] Salt stress inhibits the growth and development of crops, thereby affecting the yield and quality of crops. Auxin/indole-3-acetic acid(Aux/IAA) protein is a key factor in response to auxin signals, which plays a role in regulating plant growth and development and responding to abiotic stress. [Methods] In order to study the function of tobacco auxin-responsive gene NtIAA17 in the process of tobacco growth and development, tobacco K326 was used as test material to obtain tobacco NtIAA17 gene by cloning, its sequence characteristics were analyzed by bioinformatics, and the qRT-PCR technology was used to analyze the expression pattern of NtIAA17 in different tobacco tissues under 0-12 h salt stress treatment. The NtIAA17-OX(NtIAA17-overexpression) transgenic tobacco was obtained by transgenic technology, and its function was identified in response to salt stress. [Results] The NtIAA17 gene fragment is 624 bp, encoding 207 amino acids;the relative molecular mass is 23.07 kD, and the theoretical isoelectric point is 8.24. The promoter region of this gene contains cis-acting elements that are related to hormones and stress, with four domains(I-IV) and conserved sequences of Aux/IAA family proteins.The genetic relationship between NtIAA17 gene and spinach auxin suppressor gene Mo IAA13 is the closest;subcellular localization analysis showed that NtIAA17 protein is a nuclear localization protein. The qRT-PCR results showed that the NtIAA17 gene was highly expressed in stems and old leaves, and the highest expression level was obtained under 2 h of salt stress. Under salt stress conditions, compared with wildtype NtIAA17-OX transgenic tobacco, the biomass increased significantly, the accumulation of proline content increased, while the content of MDA decreased, and the expression of antioxidant enzyme genes Nt SOD and Nt POD increased. [Conclusion] The NtIAA17 gene can respond to salt stress signals, resist the damage caused by salt stress by improving the antioxidant capacity of transgenic tobacco, and increase plant biomass. This study provides candidate genes and theoretical basis for subsequent salt-tolerant molecular breeding.