microRNA1917调控番茄乙烯信号负调控因子CTR4sv3的表达

摘要

In order to understand the expression pattern of Sly-miR1917 and its target SlCTR4sv3 during tomato growth and development in response to hormone and stress, we performed real-time qRT-PCR to detected the expression pattern of Sly-miR1917 and targeted SlCTR4sv3 in various organs of tomato cultivar 'Zhongshu 6', second combined with cis-acting elements analysis of promoter sequences of Sly-miR1917 and SlCTR4sv3 taking advantage of bioinformatics to explore the effects of corresponding plant hormone and abiotic stress treatments on the expression level of Sly-miR1917 and SlCTR4sv3 in tomato seedlings, third introduced SlCTR4sv3 and Sly-miR1917-resistant mutant SlCTR4sv3(mSlCTR4sv3) into the over-expressing vector pB7YWG2 via Gateway recombinant technology, and detected the expression level of obtained positive transgenic tomatoes. Our results showed a negative correlation of Sly-miR1917 and targeted SlCTR4sv3 appeared in tomato seedling and leaf, pedicel, flower and ripe fruit of adult plant. SlCTR4sv3 expression was induced by jasmonic acid, gibberellin, salicylic acid, ethylene and drought treatments, while the expression level of Sly-miR1917 increased under jasmonic acid treatment. The SlCTR4sv3 accumulation in 35S::SlCTR4sv3 lines were slightly higher than that in wildtype plants. While the expression of SlCTR4sv3 was significantly induced in 35S::mSlCTR4sv3 lines, which indicated the expression level of SlCTR4sv3 was strictly controlled by Sly-miR1917, and the increased expression level of SlCTR4sv3 induced Sly-miR1917 accumulation, especially in 35S:: mSlCTR4sv3 lines, which implied the negative feedback regulation of SlCTR4sv3 on Sly-miR1917. The exquisite and complex regulationpattern between SlCTR4sv3 and Sly-miR1917 provides guarantee for the accurate response to ethylene in plant.%为弄清Sly-miR1917以及其靶基因SlCTR4sv3在番茄生长发育和响应外界刺激中的表达模式,以中蔬6号番茄(Solanum lycopersicum L.Zhongshu 6)为试材,实时荧光定量PCR检测Sly-miR1917和SlCTR4sv3在番茄不同组织部位的表达,并结合生物信息学分析Sly-miR1917和SlCTR4sv3启动子顺式作用元件,调查了二者对相应激素和非生物胁迫的响应模式.此外,为进一步明确靶基因是否对Sly-miR1917存在反馈调控机制,应用Gateway重组技术将SlCTR4sv3和点突变后抗Sly-miR1917降解的mSlCTR4sv3连入超表达载体pB7YWG2,获得35S::SlCTR4sv3和35S::mSlCTR4sv3转基因阳性植株,检测相应植株中的Sly-miR1917表达变化.结果显示:在番茄幼苗,成株叶片、花柄、花、成熟果实中Sly-miR1917与靶基因SlCTR4sv3表达模式呈负相关;茉莉酸、赤霉素、水杨酸、乙烯,以及干旱处理诱导SlCTR4sv3的表达量上调,而Sly-miR1917只在茉莉酸处理的幼苗中上调表达;35S::SlCTR4sv3株系中SlCTR4sv3表达量略高于野生型,而在抗Sly-miR1917降解的35S::mSlCTR4sv3转基因株系中显著升高,说明SlCTR4sv3表达水平受到Sly-miR1917的严格调控;此外,SlCTR4sv3的高表达会诱导Sly-miR1917的表达量上调,这一现象在35S::mSlCTR4sv3株系中尤为明显,暗示着SlCTR4sv3能够反馈调节Sly-miR1917的表达,二者严格而精确地调控模式为植物能够正确的响应乙烯提供保证.