抑制NtVTC1基因的表达延缓烟草生长发育

Suppression of Nt VTC1 gene expression delays tobacco growth and development

目的研究VTC1通过调控细胞内抗坏血酸(Ascorbic acid, AsA)的合成参与植物生长发育和响应逆境的重要功能。方法构建烟草VTC1基因的RNAi干扰表达载体RNAi-NtVTC1并转化烟草,通过抑制烟草VTC1基因的表达分析其影响植物发育的重要功能。结果将构建的干扰载体RNAi-NtVTC1转化烟草,成功获得了转基因株系RI。半定量PCR(RT-PCR)和实时荧光定量PCR(qRT-PCR)检测结果表明转基因烟草株系RI中NtVTC1基因的表达显著被抑制。AsA含量测定结果表明:与野生型烟草相比,转基因烟草株系RI的AsA含量均显著降低,为野生型的30%～40%。对烟草的表型观察发现,转基因烟草株系RI的发育显著受到抑制,尤其是在植株整体发育、叶片延展发育、开花等方面表现出显著延迟的表型。烟草抗氧化系统酶活性测定结果表明:转基因烟草株系RI中抗氧化系统酶活性如抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、过氧化物酶(CAT)和谷胱甘肽还原酶的酶活性(GR)都显著降低。结论 VTC1基因在植物的生长发育过程中发挥着重要作用,为进一步深入研究VTC1基因的功能提供了良好参考。

Objective VTC1 gene plays important roles in plant growth and development and in response to biotic and abiotic stresses through controlling the biosynthesis of ascorbic acid(AsA) in plant cells. M ethods To further investigate the function of VTC1, RNA interfering vector RNAi-NtVTC1 of tobacco NtVTCl gene was constructed and was transformed into tobacco by leaf disk method. Results The resulted transgenic tobacco plants were used to analyze the VTC1 function in regulating plant growth and development. Semiquantitative RT-PCR and quantitative real-time PCR(qRT-PCR) showed that the expression of NtVTC1 gene was significantly inhibited in transgenic tobacco lines RI. Compared with wild type tobacco, the AsA content in the transgenic lines RI was significantly reduced, which was about 30-40% of wild type tobacco. Phenotype observation demonstrated that the RI tobacco plants showed significantly retarding growth, especially in the growth and development of whole plant, leaf expansion and flowering time. Measurement of enzyme activities showed that the activities of antioxidant such as ascorbate peroxidase(APX), dehydroascorbate reductase(DHAR), catalase(CAT) and glutathone reductase(GR) in RI transgenic tobacco lines were obviously decreased. Conclusion These results indicated that VTC1 gene played an important role in plant growth and development, and provided a reference for further investigating the function of VTC1.