谷子转录因子SiNAC18通过ABA信号途径正向调控干旱条件下的种子萌发

doi:10.3864/j.issn.0578-1752.2017.16.002

中国农业科学 ›› 2017, Vol. 50 ›› Issue (16): 3071-3081.doi: 10.3864/j.issn.0578-1752.2017.16.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

谷子转录因子SiNAC18通过ABA信号途径正向调控干旱条件下的种子萌发

窦祎凝¹，秦玉海^1,2，闵东红²，张小红²，王二辉²，刁现民¹，贾冠清¹，徐兆师¹，李连城¹，马有志¹，陈明¹

¹中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部麦类生物学与作物遗传育种重点实验室，北京100081；²西北农林科技大学农学院/旱区作物逆境生物学国家重点实验室，陕西杨凌712100

收稿日期:2017-02-15 出版日期:2017-08-16 发布日期:2017-08-16
通讯作者: 陈明，Tel：010-82108750；E-mail：chenming02@caas.cn
作者简介:窦祎凝，E-mail：1614520157@qq.com。秦玉海，E-mail：263820084@qq.com。窦祎凝和秦玉海为同等贡献作者。
基金资助:
国家转基因生物新品种培育科技重大专项（2016ZX08002002）、中国农业科学院科技创新工程

Transcription Factor SiNAC18 Positively Regulates Seed Germination Under Drought Stress Through ABA Signaling Pathway in Foxtail Millet (Setaria italic L.)

DOU YiNing¹, QIN YuHai^1,2, MIN DongHong², ZHANG XiaoHong², WANG ErHui², DIAO XianMin¹, JIA GuanQing¹, XU ZhaoShi¹, LI LianCheng¹, MA YouZhi¹, CHEN Ming¹

¹Institute of Crop Science, Chinese Academy of Agricultural Sciences/National Key Facility For Crop Gene Resource and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crop, Ministry of Agriculture, Beijing 100081; ²College of Agronomy, Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi

Received:2017-02-15 Online:2017-08-16 Published:2017-08-16

摘要/Abstract

摘要： 【目的】谷子（Setaria italica L.）具有显著耐旱性。研究旨在通过反向遗传学方法分析并鉴定在干旱条件下影响植物萌发过程的重要调控因子，为研究作物干旱条件下种子萌发的调控机制创造条件。【方法】使用ClustalX 2.0和MEGA 5.05软件对谷子SiNAC18蛋白序列及其同源序列进行多序列比对，并构建系统进化树；利用real-time PCR方法检测SiNAC18在不同胁迫条件下的表达模式；通过瞬时转化的方法分析SiNAC18蛋白亚细胞定位；在拟南芥中过表达SiNAC18，分析SiNAC18的生物学功能；分析SiNAC18在转基因拟南芥中可能控制的下游基因。【结果】SiNAC18全长1 074 bp，编码由357个氨基酸组成的亲水性蛋白，分子量约为38.8 kD；系统进化树分析表明SiNAC18属于NAC转录因子家族第Ⅰ组的NAP亚组，与拟南芥基因AtNAC29同源性最高；氨基酸序列比对结果显示，SiNAC18与其他物种包括水稻、拟南芥、大豆和玉米中同源性最高的NAC类转录因子蛋白的N端都具有A、B、C、D和E这5个保守结构域，蛋白C端具有高度多态性，证明SiNAC18的N端序列与其结合下游基因启动子元件相关；real-time PCR结果显示，SiNAC18在干旱（PEG）、ABA、高盐（NaCl）及过氧化氢（H₂O₂）处理条件下的表达量明显上升；亚细胞定位结果表明SiNAC18蛋白定位于细胞核中；基因功能分析结果显示，在ABA和PEG胁迫处理下，SiNAC18转基因拟南芥与野生型种子的萌发率存在明显差异：在正常生长条件下，野生型拟南芥WT和SiNAC18转基因拟南芥的萌发率基本一致，在PEG浓度为10%和15%的MS培养基上，SiNAC18转基因拟南芥的萌发率显著高于WT。在2和5 μmol·L^-1 ABA处理条件下，转基因拟南芥的萌发率显著低于WT；下游基因表达分析结果显示，ABA信号途径相关基因AtRD29A，脯氨酸合成相关基因AtP5CR和AtPRODH以及过氧化物酶基因AtPRX34在SiNAC18转基因株系中的表达量高于WT中的表达量，表明SiNAC18通过调控这些下游基因影响转基因植物在干旱条件下的萌发率。【结论】谷子NAC类转录因子基因SiNAC18可能通过ABA信号途径、氧化胁迫调控等途径正向调控植物在干旱条件下的萌发过程。

关键词: 谷子, NAC类转录因子, 种子萌发, 干旱胁迫, ABA信号途径

Abstract: 【Objective】 Foxtail millet (Setaria italica L.) has strong tolerance to drought stress. The objective of this research is to screen key regulatory factors affecting the germination process under drought conditions in plants through reverse genetics method, which will contribute for further research of regulation mechanism of seed germination under drought condition. 【Method】 Multiple sequence alignment of SiNAC18 protein sequences and millet homologous sequences were made by using ClustalX 2 and MEGA 5.05 softwares, and the phylogenetic tree was constructed. The expression patterns of SiNAC18 under the stress condition were analyzed using the real-time PCR method. SiNAC18 protein subcellular localization was analyzed by transient transfection method. Biological function of SiNAC18 was analyzed by using overexpression of SiNAC18 in Arabidopsis thaliana. The expression of downstream genes of SiNAC18 was analyzed by real-time PCR. 【Result】 The length of SiNAC18 is 1 074 bp encoding a hydrophilic protein with polypeptide of 357 amino acids, and its molecular weight is about 38.8 kD. Phylogenetic tree analysis indicated that SiNAC18 belongs to NAP subgroup of group I in the NAC transcription factors family and has the highest homology with Arabidopsis gene AtNAC29. The amino acid sequence alignment results show that the N-terminal of SiNAC18 and the highest homology transcription factors of SiNAC18 in other species, including rice, Arabidopsis thaliana, soybean and maize, has A, B, C, D and E five conserved domains. The C-terminal of the protein has a high degree of polymorphism, demonstrating that the N-terminal sequence of SiNAC18 is associated with its downstream promoter. Real-time PCR results showed that SiNAC18 were induced by drought (PEG), high salt (NaCl) and hydrogen peroxide (H₂O₂) treatment. Subcellular localization results showed that SiNAC18 protein is localized in the nucleus. Gene function analysis showed that in the ABA and PEG stress treatments, the germination rate of SiNAC18 transgenic Arabidopsis thaliana and wild type seeds was significantly different. Under normal growth conditions, germination rate of the wild type Arabidopsis WT and SiNAC18 transgenic Arabidopsis was the same, and when the concentration of PEG was increased to 10% and 15% on MS medium, the germination rate of SiNAC18 transgenic Arabidopsis was significantly higher than WT. Under the conditions of 2 and 5 μmol·L^-1of ABA treatment, the germination rate of SiNAC18 transgenic Arabidopsis was significantly lower than that of WT. Analysis results showed that the expression of downstream genes of ABA related genes AtRD29A, proline synthesis related genes AtP5CR and AtPRODH and peroxidase gene AtPRX34 in SiNAC18 transgenic plants was higher than that in WT, which suggesting that SiNAC18 affects the germination rate of transgenic plants under drought conditions by affecting the expression of those downstream genes. 【Conclusion】 NAC like transcription factor gene SiNAC18 positively regulates the germination of plants under drought conditions through ABA and oxidative stress response signaling pathway in foxtail millet.

Key words: foxtail millet (Setaria italic L.), NAC transcription factors, seed germination, drought stress, ABA signaling pathway

窦祎凝，秦玉海，闵东红，张小红，王二辉，刁现民，贾冠清，徐兆师，李连城，马有志，陈明. 谷子转录因子SiNAC18通过ABA信号途径正向调控干旱条件下的种子萌发[J]. 中国农业科学, 2017, 50(16): 3071-3081.

DOU YiNing, QIN YuHai, MIN DongHong, ZHANG XiaoHong, WANG ErHui, DIAO XianMin, JIA GuanQing, XU ZhaoShi, LI LianCheng, MA YouZhi, CHEN Ming. Transcription Factor SiNAC18 Positively Regulates Seed Germination Under Drought Stress Through ABA Signaling Pathway in Foxtail Millet (Setaria italic L.)[J]. Scientia Agricultura Sinica, 2017, 50(16): 3071-3081.

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谷子转录因子SiNAC18通过ABA信号途径正向调控干旱条件下的种子萌发

Transcription Factor SiNAC18 Positively Regulates Seed Germination Under Drought Stress Through ABA Signaling Pathway in Foxtail Millet (Setaria italic L.)

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