NtMYB68基因对烟草苗期紫黄质和脱落酸生物合成及烟株耐旱性的影响研究

Effect of NtMYB68 Gene on Tobacco Seedlings Violaxanthin and Abscisic Acid Biosynthesis,and Drought Tolerance Effects of NtMYB68 gene on biosynthesis of flaxanthin and abscisic acid and drought tolerance of tobacco plants at seedling stage

【目的】为探究烟草NtMYB68基因在类胡萝卜素和脱落酸合成生物合成中的作用及其对烟株抗旱性的影响。【方法】从烤烟品种K326中同源克隆NtMYB68基因,采用生物信息学分析NtMYB68基因序列特征,利用RT-PCR分析其在烟草不同组织的表达模式。利用基因编辑手段创制NtMYB68敲除材料,并分析NtMYB68基因敲除后对烟株类胡萝卜素合成及抗旱性的影响。【结果】NtMYB68基因有2个拷贝,NtMYB68-1和Nt MYB68-2。NtMYB68在烟草根、茎、叶、花中均有表达,NtMYB68-2的表达水平显著高于NtMYB68-1。NtMYB68敲除材料与对照相比,紫黄质合成基因NtZEP显著上调,紫黄质含量显著增加,脱落酸(ABA)合成基因Nt NCED5表达量达到对照植株的4倍。干旱胁迫下,敲除材料幼苗与对照相比,叶片萎蔫程度更轻,长势更优,复水处理后生长状态恢复的更好。干旱处理前后,敲除植株中ABA的含量均显著提高。【结论】Nt MYB68转录因子能抑制NtZEP和Nt NCED5基因表达,敲除该基因可以提高烟叶中紫黄质的积累,促进脱落酸的合成,从而提高烟株抗旱性。

[Purpose] This study aims to explore the effect of Nt MYB68 gene on the biosynthesis of carotenoids and abscisic acid and the drought resistance of tobacco plants at seedling stage. [Methods] The NtMYB68 gene was cloned from flue-cured tobacco variety K326.The NtMYB68 gene sequence characteristics were analyzed by bioinformatics, and its expression pattern in different tobacco tissues was analyzed by RT-PCR. Gene editing technology was used to create a homozygous mutant line of the NtMYB68 gene, and the effects of NtMYB68 gene knock-out on tobacco carotenoid synthesis and drought resistance were analyzed. [Results] There are 2 copies of Nt MYB68gene, including NtMYB68-1 and NtMYB68-2. Nt MYB68 is expressed in tobacco roots, stems, leaves and flowers, and the expression level of NtMYB68-2 is significantly higher than that of NtMYB68-1. Compared with the control, the violaxanthin synthesis gene Nt ZEP was significantly up-regulated in the NtMYB68 knock-out material, and the violaxanthin content was significantly increased. The expression level of the abscisic acid(ABA) synthesis gene Nt NCED5 was 4 times that of the control material. Under drought stress, compared with the control, the knock-out material seedlings showed lower degree of leaf wilting, better growth, and better growth recovery after rewatering.Before and after drought treatment, the content of ABA in the knock-out material was significantly increased. [Conclusion] Nt MYB68transcription factor can inhibit the expression of NtZEP and Nt NCED5 genes, knocking out this gene can increase the accumulation of violaxanthin in tobacco leaves and promote the synthesis of abscisic acid, thereby improving the drought resistance of tobacco.