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Overexpression of a NAC transcription factor enhances rice drought and salt tolerance
过表达 NAC 转录因子可增强水稻耐旱和耐盐性

Xingnan Zheng 1 1 ^(1){ }^{1}, Bo Chen 1 1 ^(1){ }^{1}, Guojun Lu, Bin Han *
郑兴楠 1 1 ^(1){ }^{1} ， 陈 1 1 ^(1){ }^{1} 波 ， 卢国军， 韩斌 *National Center for Gene Research & Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 500 Caobao Road, Shanghai 200233, China
中国科学院上海生物科学研究院国家基因研究中心和上海植物生理生态研究所，地址为漕宝路500号，上海200233，中国

A R T IC L E IN F O

Article history:  文章历史：

Received 29 December 2008
收稿日期 2008 年 12 月 29 日
Available online 9 January 2009
2009 年 1 月 9 日在线提供

Keywords:  关键字：

Abiotic stress  非生物胁迫
Drought  干旱
Salt  盐
Transcriptional activator
转录激活剂
Oryza sativa  水稻

Abstract  抽象

The plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factors play diverse roles in plant development and stress responses. In this study, a rice NAC gene, ONACO45, was functionally characterized, especially with regard to its role in abiotic stress resistance. Expression analysis revealed that ONAC045 was induced by drought, high salt, and low temperature stresses, and abscisic acid (ABA) treatment in leaves and roots. Transcriptional activation assay in yeast indicated that ONACO45 functioned as a transcriptional activator. Transient expression of GFP-ONACO45 in onion epidermal cells revealed that ONAC045 protein was localized in the nucleus. Transgenic rice plants overexpressing ONACO45 showed enhanced tolerance to drought and salt treatments. Two stress-responsive genes were upregulated in transgenic rice. Together, these results suggest that ONACO45 encodes a novel stress-responsive NAC transcription factor and is potential useful for engineering drought and salt tolerant rice.
植物特异性 NAC （NAM， ATAF1/2， CUC2） 转录因子在植物发育和胁迫反应中起着多种作用。在这项研究中，对水稻 NAC 基因 ONACO45 进行了功能表征，特别是其在非生物胁迫抗性中的作用。表达分析显示，ONAC045 是由干旱、高盐和低温胁迫以及叶片和根中的脱落酸 （ABA） 处理诱导的。酵母中的转录激活测定表明 ONACO45 起转录激活剂的作用。洋葱表皮细胞中 GFP-ONACO45 的瞬时表达显示ONAC045蛋白位于细胞核中。过表达 ONACO45 的转基因水稻植株对干旱和盐处理表现出更强的耐受性。转基因水稻中 2 个胁迫反应基因上调。总之，这些结果表明 ONACO45 编码一种新的胁迫反应性 NAC 转录因子，并且可能对工程抗旱和耐盐水稻有用。

© 2009 Elsevier Inc. All rights reserved.
© 2009 爱思唯尔公司保留所有权利。
Being sessile, plants are frequently up against various environmental stresses such as drought, salt, and low temperature. To cope with these adverse conditions, numerous genes are induced in plant cells, which finally lead to physiological and metabolic changes that increase the chance of plant survival [1,2]. Among these genes, transcription factors play essential roles in stress responses by regulating their target genes through binding to the cognate cis-acting elements [3-5]. Some transcription factors, such as CBF1/DREB1B [6], OsbZIP72 [7], AtMYB44 [8] have been reported to be involved in plant stress responses. Transgenic plants overexpressing these genes could enhance their tolerance to various stresses.
作为无柄植物，植物经常面临各种环境压力，例如干旱、盐分和低温。为了应对这些不利条件，植物细胞中诱导了许多基因，最终导致生理和代谢变化，从而增加植物存活的机会 [1,2]。在这些基因中，转录因子通过与同源顺式作用元件结合来调节其靶基因，从而在应激反应中发挥重要作用 [3-5]。据报道，一些转录因子，如 CBF1/DREB1B [6]、OsbZIP72 [7]、AtMYB44 [8] 参与植物胁迫反应。过表达这些基因的转基因植物可以增强它们对各种胁迫的耐受性。
NAC family, which is one of the largest plant transcription factor families, is only found in plants to date [9]. Proteins of this family are characterized by a highly conserved DNA binding domain, known as NAC domain in the N-terminal region. In contrast, the C-terminal region of NAC proteins, usually containing the transcriptional activation domain, is highly diversified both in length and sequence [10]. More than 100 members of this family have been identified in both Arabidopsis and rice [ 10 , 11 ] [ 10 , 11 ] [10,11][10,11]. However, only a few of them have been functionally characterized, especially in rice. NACs play important roles in plant development, including pattern formation of embryos and flowers [12], formation of secondary walls [13], and development of lateral roots [14]. NACs
NAC 家族是最大的植物转录因子家族之一，迄今为止仅在植物中发现 [9]。该家族的蛋白质具有高度保守的 DNA 结合结构域，称为 N 末端区域的 NAC 结构域。相比之下，NAC 蛋白的 C 端区域通常包含转录激活结构域，在长度和序列上都高度多样化 [10]。在拟南芥和水稻 [ 10 , 11 ] [ 10 , 11 ] [10,11][10,11] 中已鉴定出该科的 100 多个成员。然而，它们中只有少数被进行了功能表征，尤其是在水稻中。NACs 在植物发育中起着重要作用，包括胚和花的模式形成 [12]、次生壁的形成 [13] 和侧根的发育 [14]。NAC
are also reported to participate in abiotic and biotic responses. In potato, StNAC was rapidly and strongly induced by wounding [15]. In Brassica napus, nine NACs were reported to be differently regulated by biotic and abiotic stresses [16]. Expression of GRAB1 and GRAB2 in cultured wheat cells inhibited DNA replication of the wheat dwarf geminivirus [17]. Three Arabidopsis NAC genes, ANAC019, ANAC055, and ANAC072, were shown to bind to the promoter region of ERD1 which was characterized as a stress-responsive gene [5]. Overexpressing these three genes in Arabidopsis resulted in enhanced tolerance to drought stress. Moreover, two other NAC genes in Arabidopsis, ATAF1 and ATAF2, played negative roles in response to drought and pathogen infection respectively [ 18 , 19 ] [ 18 , 19 ] [18,19][18,19]. Recently, the roles of two rice NAC genes in rice stress adaptation were characterized [20-22]. SNAC1 was induced mainly in guard cells under drought conditions, and overexpression of this gene in rice resulted in significant increase in drought resistance under field condition at the stage of anthesis [20]. Overexpression of another NAC gene OsNAC6/SNAC2 in rice resulted in enhanced tolerance to drought, salt, and cold during seedling development [21,22].
据报道，还参与非生物和生物反应。在马铃薯中，StNAC 是由伤口快速而强烈地诱导的 [15]。据报道，在甘蓝型油菜中，有 9 个 NAC 受生物和非生物胁迫的不同调节 [16]。GRAB1 和 GRAB2 在培养的小麦细胞中的表达抑制了小麦矮缩双子病毒的 DNA 复制 [17]。三个拟南芥 NAC 基因 ANAC019、 ANAC055 和 ANAC072 被证明与 ERD1 的启动子区结合，该区被表征为应激反应基因 [5]。在拟南芥中过表达这三个基因导致对干旱胁迫的耐受性增强。此外，拟南芥中的另外两个 NAC 基因 ATAF1 和 ATAF2 分别在响应干旱和病原体感染中起负作用 [ 18 , 19 ] [ 18 , 19 ] [18,19][18,19] 。最近，研究了两个水稻NAC基因在水稻逆境适应中的作用[20-22]。SNAC1 在干旱条件下主要在保卫细胞中诱导，在水稻中过表达该基因导致开花期田间条件下抗旱性显著增加 [20]。在水稻中过表达另一个 NAC 基因 OsNAC6/SNAC2 导致幼苗发育过程中对干旱、盐和寒冷的耐受性增强 [21,22]。
By analyzing the microarray data (GSE 6901) in public database at NCBI, we found that a NAC gene, designated as ONAC045 according to Ooka et al. [10], was highly induced by abiotic stresses. In this work, the expression profiles of this gene in different organs and under various stress treatments were studied. The transcriptional activation and the subcellular localization of ONAC045 were also investigated. Transgenic rice plants overexpressing ONAC045 showed enhanced drought and salt tolerance, indicating that
通过分析 NCBI 公共数据库中的微阵列数据 （GSE 6901），我们发现一个根据 Ooka 等人 [10] 命名为 ONAC045 的 NAC 基因受到非生物胁迫的高度诱导。在这项工作中，研究了该基因在不同器官和各种胁迫处理下的表达谱。还研究了 ONAC045 的转录激活和亚细胞定位。过表达 ONAC045 的转基因水稻植株表现出更强的耐旱性和耐盐性，表明
ONAC045 played an important role in abiotic stress response and may serve as a potential target for engineering stress tolerant rice.
ONAC045 在非生物胁迫响应中发挥重要作用，可能作为工程耐逆水稻的潜在靶点。

Materials and methods  材料和方法

Constructs and transformation. The full-length cDNA of ONAC045 was amplified using a cDNA clone (GenBank Accession No. CT829509) from a cDNA library of Guangluai 4 (Oryza sativa L. ssp. indica) [23] as template (for primers, see Supplementary Table S1). The sequencing-confirmed PCR fragment was ligated into the overexpression vector pCAMBIA1300S. The resultant construct, pCAMBIA1300S-ONAC045, was transformed into Nipponbare (Oryza sativa L. ssp. japonica) by Agrobacterium-mediated transformation method to generate transgenic rice plants [24].
构造和转换。使用 cDNA 克隆（GenBank Accession No.ONAC045CT829509）的 cDNA 文库 4 （Oryza sativa L. ssp. indica） [23] 作为模板（引物见补充表 S1）。将测序确认的 PCR 片段连接到过表达载体 pCAMBIA1300S 中。通过农杆菌介导的转化方法将所得构建体 pCAMBIA1300S-ONAC045 转化到 Nipponbare （Oryza sativa L. ssp. japonica） 中，生成转基因水稻植物 [24]。
Plant materials, growth condition and stress treatments. The rice seeds of Guangluai4 were germinated at 37 ∘ C 37 ∘ C 37^(@)C37^{\circ} \mathrm{C} for 2 days, and then grown in a growth chamber ( 14 h light 30 ∘ C / 10 h 30 ∘ C / 10 h 30^(@)C//10h30^{\circ} \mathrm{C} / 10 \mathrm{~h} dark 28 ∘ C 28 ∘ C 28^(@)C28^{\circ} \mathrm{C} cycles) to harvest the young leaves and young roots. Mature leaves, stems, and panicles after heading were prepared from the same staged plants. For expression analysis, seedlings at the three-leaf stage were subjected to various stress treatments. For salt and ABA treatments, seedlings were incubated in solutions containing 200 mM NaCl and 100 μ M ABA 100 μ M ABA 100 muMABA100 \mu \mathrm{M} \mathrm{ABA}, respectively. For drought treatment, whole seedlings were exposed to air under dim light. For cold treatment, seedlings were treated at 8 − 10 ∘ C 8 − 10 ∘ C 8-10^(@)C8-10^{\circ} \mathrm{C} under dim light. Leaves and roots were harvested at different time points as shown in Fig. 1. For the marker gene analysis, leaves of wild type rice and transgenic rice at the three-leaf-stage were harvested for RNA isolation.
植物材料、生长条件和胁迫处理。将广鸭庵4号的水稻种子发芽 37 ∘ C 37 ∘ C 37^(@)C37^{\circ} \mathrm{C} 2 d，然后在生长室中生长（14 h光 30 ∘ C / 10 h 30 ∘ C / 10 h 30^(@)C//10h30^{\circ} \mathrm{C} / 10 \mathrm{~h} 暗 28 ∘ C 28 ∘ C 28^(@)C28^{\circ} \mathrm{C} 循环）收获幼叶和幼根。抽穗后成熟的叶、茎和圆锥花序由相同阶段的植物制备。对于表达分析，三叶期幼苗经受各种胁迫处理。对于盐和 ABA 处理，将幼苗分别在含有 200 mM NaCl 和 100 μ M ABA 100 μ M ABA 100 muMABA100 \mu \mathrm{M} \mathrm{ABA} 的溶液中孵育。对于干旱处理，将整株幼苗暴露在昏暗光线下的空气中。对于冷处理，幼苗在昏暗的 8 − 10 ∘ C 8 − 10 ∘ C 8-10^(@)C8-10^{\circ} \mathrm{C} 光线下处理。叶子和根在不同的时间点收获，如图 1 所示。对于标记基因分析，收获野生型水稻和转基因水稻三叶期的叶片进行 RNA 分离。
Real-time RT-PCR analysis. Total RNA was extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The DNase-treated RNA was reverse-transcribed using M-MLV reverse transcriptase (TaKaRa). Real-time RT-PCR was performed on the Applied Biosystems 7500 real time PCR System using SYBR Premix Ex Taq TM TM ^(TM){ }^{\mathrm{TM}} (TaKaRa). The PCR thermal cycle conditions were as following: denature at 95 ∘ C 95 ∘ C 95^(@)C95^{\circ} \mathrm{C} for 10 s and 40 cy − 40 cy − 40cy-40 \mathrm{cy}- cles for 95 ∘ C , 5 s ; 60 ∘ C , 34 s 95 ∘ C , 5 s ; 60 ∘ C , 34 s 95^(@)C,5s;60^(@)C,34s95^{\circ} \mathrm{C}, 5 \mathrm{~s} ; 60^{\circ} \mathrm{C}, 34 \mathrm{~s}. Two rice genes used as internal reference genes for calculating relative transcript levels were UBQ5 (GenBank Accession No. AK061988) and eEF-1 α α alpha\alpha (GenBank Accession No. AK061464) [25]. The primer efficiency used for calculating the relative quantification was 2.0 [26].
实时 RT-PCR 分析。根据制造商的说明，使用 Trizol 试剂 （Invitrogen） 提取总 RNA。使用 M-MLV 逆转录酶 （TaKaRa） 对 DNase 处理的 RNA 进行逆转录。在 Applied Biosystems 7500 实时荧光定量 PCR 系统上使用 SYBR Ex Taq TM TM ^(TM){ }^{\mathrm{TM}} （TaKaRa） 进行实时荧光定量 RT-PCR。PCR 热循环条件如下：变性 95 ∘ C 95 ∘ C 95^(@)C95^{\circ} \mathrm{C} 10 s 和 40 cy − 40 cy − 40cy-40 \mathrm{cy}- cles 。 95 ∘ C , 5 s ; 60 ∘ C , 34 s 95 ∘ C , 5 s ; 60 ∘ C , 34 s 95^(@)C,5s;60^(@)C,34s95^{\circ} \mathrm{C}, 5 \mathrm{~s} ; 60^{\circ} \mathrm{C}, 34 \mathrm{~s} 用作计算相对转录水平的内部参考基因的两个水稻基因是 UBQ5 （GenBank Accession No.AK061988） 和 eEF-1 α α alpha\alpha （GenBank 登录号AK061464） [25]。用于计算相对定量的引物效率为 2.0 [26]。
Drought and salt tolerance assay. For drought tolerance test, transgenic seedlings were grown hydroponically for 15-18 days to three-leaf stage, and parallelly grown WT rice plants of the same stage were used as control. Whole plants were exposed to air for 9.5 h , and then rehydrated and recovered for an additional 10 days. The survival rates of transgenic lines and the WT control were calculated. For salt tolerant assay, three-leaf stage WT and transgenic seedlings were incubated in solution containing 100 mM NaCl for
耐旱和耐盐试验。对于耐旱性测试，转基因幼苗水培生长 15-18 天至三叶期，并使用同期平行生长的 WT 稻植株作为对照。将整株植物暴露在空气中 9.5 h，然后再水化并回收 10 d。计算转基因株系和 WT 对照的存活率。对于耐盐测定，将三叶期 WT 和转基因幼苗在含有 100 mM NaCl 的溶液中孵育
13 days(the salt solution was refreshed every two days), then all the plants were transferred into solution without NaCl and recovered for an additional 10 days. The survival rates of transgenic lines and the WT control were calculated.
13 d（盐溶液每两天更新一次），然后将所有植物转移到不含 NaCl 的溶液中，再回收 10 d。计算转基因株系和 WT 对照的存活率。
Transcriptional activation analysis in yeast. For transcriptional activation assay, the sequencing-confirmed PCR fragment of full ORF, N-terminal ORF with the NAC domain(1-159 amino acids), and C-terminal ORF with the potential activation domain (160-359 amino acids) were fused in frame with GAL4 DNA binding domain in pGBKT7 to construct pGBKT7-ONAC045, pGBKT7-ONAC045AC, and pGBKT7-ONAC045AN, respectively (for primers, see Supplementary Table S1). pGBKT7 was used as a negative control. These different constructs were transformed into yeast strain AH109. The transformants were streaked on the SD/ Trp- and SD/Trp-/His-/Ade- medium. After incubated at 28 ∘ C 28 ∘ C 28^(@)C28^{\circ} \mathrm{C} for 3 days, the growth status of the transformants was evaluated. The ß ß ßßß-galactosidase filter assay was carried out according to the manufacturer’s instructions (Clontech).
酵母中的转录激活分析。对于转录激活测定，将经测序证实的全 ORF、具有 NAC 结构域的 N 端 ORF（1-159 个氨基酸）和具有潜在激活结构域（160-359 个氨基酸）的 C 端 ORF 的 PCR 片段与 pGBKT7 中的 GAL4 DNA 结合结构域融合在框架中，分别构建 pGBKT7-ONAC045、pGBKT7-ONAC045AC 和 pGBKT7-ONAC045AN（引物见补充表 S1）。pGBKT7 用作阴性对照。将这些不同的构建体转化到酵母菌株 AH109 中。转化体在 SD/Trp- 和 SD/Trp-/His-/Ade- 培养基上划线。孵育 28 ∘ C 28 ∘ C 28^(@)C28^{\circ} \mathrm{C} 3 天后，评估转化体的生长状态。这 ß -半乳糖苷酶滤膜测定根据制造商的说明 （Clontech） 进行。
Subcellular localization analysis. The full open reading frame (ORF) of ONAC045 was amplified using the cDNA clone mentioned above as template (for primers, see Supplementary Table S1). The PCR product was ligated into the pA7-GFP vector, resulting in an in-frame fusion protein of GFP gene and the ONAC045 ORF. The construct (p35S:GFP-ONAC045) and the control vector (p35S:GFP) were transformed into onion epidermal cells by particle bombardment using a Biolistic PDS-1000/He gene gun system (BIO-RAD). After 24 h incubation of transformed onion epidermal cells, GFP signal was detected by a confocal fluorescence microscope (Zeiss, LSM510 Meta, Germany).
亚细胞定位分析。使用上述 cDNA 克隆作为模板扩增 ONAC045 的全开放阅读框 （ORF）（引物见补充表 S1）。将 PCR 产物连接到 pA7-GFP 载体中，产生 GFP 基因和 ONAC045 ORF 的框内融合蛋白。使用 Biolistic PDS-1000/He 基因枪系统 （BIO-RAD） 通过粒子轰击将构建体 （p35S：GFP-ONAC045） 和控制载体 （p35S：GFP） 转化到洋葱表皮细胞中。转化的洋葱表皮细胞孵育 24 小时后，通过共聚焦荧光显微镜 （Zeiss， LSM510 Meta， Germany） 检测 GFP 信号。

Results  结果

Expression profile of ONAC045
ONAC045 的表达谱

Expression pattern of ONAC045 in young leaves, young roots, mature leaves, stems, and panicles was investigated using realtime RT-PCR. It was shown that the expression level was higher in young roots than in other organs examined (Fig. 1A).
使用实时 RT-PCR 研究 ONAC045 在幼叶、嫩根、成熟叶、茎和圆锥花序中的表达模式。结果表明，幼根的表达水平高于检查的其他器官（图 1A）。
The expression pattern of ONAC045 under various stress treatments in leaves and roots was also investigated (Fig. 1B). Under ABA treatment, the expression level was peaked at 2 h in leaves and then decreased, while it could only be observed until 4 h after ABA treatment in roots and maintained at the similar level until 8 h . Under drought treatment, the expression of ONACO45 was peaked at 2 h in both roots and leaves, and then decreased at 4 and 8 h , respectively. When treated with 200 mM NaCl , the expression of ONAC045 was only slightly induced in leaves; however, the induction reached a much higher level in roots at 4,12 , and 24 h after NaCl treatment. Upon cold conditions, the most dramatic
还研究了 ONAC045 在不同胁迫处理下叶片和根系的表达模式（图 1B）。ABA 处理下，叶片表达量在 2 h 达到峰值后下降，而根系中表达量只能在 ABA 处理后 4 h 观察到，并维持在相似水平直至 8 h。干旱处理下，ONACO45 在根和叶片中的表达量在 2 h 达到峰值，然后在 4 h和 8 h 分别下降。当用 200 mM NaCl 处理时，ONAC045 的表达在叶片中仅略微诱导;然而，在 NaCl 处理后 4 、 12 和 24 h 时，根系的诱导水平要高得多。在寒冷的条件下，最戏剧性
Fig. 1. Expression pattern of ONACO45 in Guangluai 4 was detected by real-time RT-PCR. The experiments were repeated twice, and similar tendency was observed. The expression levels shown here were according to one measurement. Error bars are standard deviations of three technical repeats. (A) The organ specific expression of ONA045 in young roots (YRs), young leaves (YLs), mature leaves (MLs), stems (STs), and panicles (PAs). (B) Expression of ONAC045 in response to ABA ( 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} ), drought, NaCl ( 200 mM ) ( 200 mM ) (200mM)(200 \mathrm{mM}), and cold ( 8 − 10 ∘ C ) 8 − 10 ∘ C (8-10^(@)C)\left(8-10^{\circ} \mathrm{C}\right) treatments in young leaves and young roots.
图 1.实时 RT-PCR 检测广陆 4 ONACO45 的表达模式。实验重复两次，观察到类似的趋势。此处显示的表达水平是根据一项测量得出的。误差线是 3 次技术重复的标准差。（A） ONA045 在幼根 （YR）、幼叶 （YL）、成熟叶 （MLs）、茎 （STs） 和圆锥花序 （PAs） 中的器官特异性表达。（B） 响应 ABA （ 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} ）、干旱、NaCl ( 200 mM ) ( 200 mM ) (200mM)(200 \mathrm{mM}) 和冷 ( 8 − 10 ∘ C ) 8 − 10 ∘ C (8-10^(@)C)\left(8-10^{\circ} \mathrm{C}\right) 处理在幼叶和幼根中 ONAC045 的表达。
induction of ONAC045 was observed in both leaves and roots. The induction was peaked at 12 h in leaves and decreased at 24 h while it was continuously increased and peaked at 24 h in roots.
在叶和根中都观察到 ONAC045 的诱导。诱导在叶片中在 12 h 达到峰值，在 24 h 时降低，而在根中诱导持续增加并在 24 h 达到峰值。
ONAC045 had transcriptional activation and was localized in the nucleus
ONAC045具有转录激活并定位于细胞核
Yeast two-hybrid system was used to investigate the transcriptional activation of ONAC045. As shown in Fig. 2A, all transformants grew well on SD/Trp- medium. However, only transformants containing pGBKT7-ONAC045 and pGBKT7-ONAC045AN could grow on SD/Trp-/His-/Ade- medium and showed β β beta\beta-galactosidase activity while those containing pGBKT7 and pGBKT7-ONACO45DC could
酵母双杂交系统用于研究 ONAC045 的转录激活。如图 2A 所示，所有转化体在 SD/Trp- 培养基上生长良好。然而，只有含有 pGBKT7-ONAC045 和 pGBKT7-ONAC045AN 的转化体可以在 SD/Trp-/His-/Ade- 培养基上生长并显示出 β β beta\beta 半乳糖苷酶活性，而含有 pGBKT7 和 pGBKT7-ONACO45DC 的转化体可以
Fig. 2. Transcriptional activation assay and nuclear localization assay of ONACO45. (A) Fusion proteins of pGBKT7-ONAC045, pGBKT7-ONAC045AC, and pGBKT7ONAC045AN and pGBKT7 were expressed in yeast strain AH109. The transformant carrying pGBKT7 vector was used as a negative control. The transformants were incubated on SD/Trp- and SD/Trp-/His-/Ade- to examine their growth and tested for β β beta\beta-galactosidase activity. (B) Nuclear localization of OANC045. p35S:GFP (as a control) and p 35 S :GFP-ONAC045 were transiently expressed in onion epidermal cells. The photographs were taken in the dark field for green fluorescence (a and d), under bright light for the morphology of the cell ( b b b\mathbf{b} and e) and in combination ( c c cc and f), respectively for p35:GFP control plasmid (a-c) and p35S:OsDREB1F-GFP plasmid (d-f).
图 2.ONACO45的转录激活测定和核定位测定。（A） pGBKT7-ONAC045、pGBKT7-ONAC045AC 和 pGBKT7ONAC045AN 和 pGBKT7 的融合蛋白在酵母菌株 AH109 中表达。携带 pGBKT7 载体的转化体用作阴性对照。将转化体在 SD/Trp- 和 SD/Trp-/His-/Ade- 上孵育，以检查其生长并检测 β β beta\beta -半乳糖苷酶活性。（B） OANC045的核定位。p35S：GFP （作为对照） 和 p 35 S ：GFP-ONAC045 在洋葱表皮细胞中瞬时表达。照片是在暗场中拍摄的绿色荧光 （a 和 d），在强光下拍摄细胞形态 （ b b b\mathbf{b} 和 e） 和组合 （ c c cc 和 f），分别用于 p35：GFP 对照质粒 （a-c） 和 p35S：OsDREB1F-GFP 质粒 （d-f）。
not. These results indicated that ONAC045 functioned as a transcriptional activator and the activation domain was located in the C-terminal region.
不。这些结果表明，ONAC045 起转录激活剂的作用，激活结构域位于 C 端区域。
To determine the subcellular localization of ONAC045, p35S:GFP-ONAC045 and p35S:GFP were transiently expressed in onion epidermal cells. As shown in Fig. 2B, the onion cells transformed with p35S:GFP vector displayed fluorescence throughout the whole cells (Fig. 2B, a-c). In contrast, fluorescence in the onion cell transformed with p35S:GFP-ONAC045 was detected exclusively in the nucleus (Fig. 2B, d-f), indicating that ONAC045 encoded a nuclear localized protein.
为了确定ONAC045的亚细胞定位，p35S：GFP-ONAC045 和 p35S：GFP 在洋葱表皮细胞中瞬时表达。如图 2B 所示，用 p35S：GFP 载体转化的洋葱细胞在整个细胞中显示荧光（图 2B，a-c）。相比之下，用 p35S：GFP-ONAC045 转化的洋葱细胞中的荧光仅在细胞核中检测到（图 2B，df），表明 ONAC045 编码了核定位蛋白。
Overexpression of ONAC045 in transgenic rice improved drought and salt tolerance
转基因水稻中过表达 ONAC045 提高了耐旱性和耐盐性
In order to characterize the in vivo function of ONACO45, transgenic rice plants overexpressing this gene were generated. The T2 generations of two homozygous transgenic lines, overexpression line 2 (OE2) and overexpression line 3 (OE3), were used for stress tolerance assay (for overexpression level, see Supplementary Fig. S1).
为了表征 ONACO45 的体内功能，产生了过表达该基因的转基因水稻植物。两个纯合转基因系，过表达系 2 （OE2） 和过表达系 3 （OE3） 的 T2 代用于胁迫耐受性测定（过表达水平，见补充图 S1）。
We tested the effect of ONAC045 overexpression on drought tolerance. As shown in Fig. 3A and B, more than 90% of OE2 and more than 70 % 70 % 70%70 \% of OE3 remained vigorous respectively after recovery, while only about 35 % 35 % 35%35 \% of wide type survived, suggesting that overexpression of ONAC045 could improve drought tolerance in transgenic rice ( t t tt test, P < 0.01 P < 0.01 P < 0.01P<0.01 ).
我们测试了 ONAC045 过表达对耐旱性的影响。如图 3A 和 B 所示，恢复后分别超过 90% 的 OE2 和超过 70 % 70 % 70%70 \% OE3 保持活力，而只有大约 35 % 35 % 35%35 \% 宽型存活下来，表明 ONAC045 过表达可以提高转基因水稻的耐旱性（ t t tt 测试， P < 0.01 P < 0.01 P < 0.01P<0.01 ）。
The effect of ONAC045 overexpression on salt tolerance was also investigated. As shown in Fig. 3C and D, the survival rates of OE2 and OE3 were more than 60%, significantly higher than that of WT plants (16%), suggesting that overexpression of ONAC045 could improve salt tolerance in transgenic rice ( t t tt test, P < 0.01 P < 0.01 P < 0.01P<0.01 ).
还研究了 ONAC045 过表达对耐盐性的影响。如图 3C 和 D 所示，OE2 和 OE3 的存活率超过 60%，显著高于 WT 植株的存活率（16%），表明过表达 ONAC045 可以提高转基因水稻的耐盐性（ t t tt 测试， P < 0.01 P < 0.01 P < 0.01P<0.01 ）。
Overexpression of ONAC045 induced expression of two stressresponsive genes
ONAC045过表达诱导的两个应激反应基因的表达
To better understand the mechanisms of drought and salt tolerance conferred by overexpressing ONAC045, we investigated the expression of several known drought and salt induced genes in transgenic rice plants. As shown in Fig 4, the expression levels of a late embryogenesis abundant (LEA) gene (OsLEA3-1[27], GenBank Accession No. Z68090), and a homologue gene of wheat plasma membrane protein (WPM-1) [28] (termed as OsPM1 here, GenBank Accession No. NM_001061933) were strongly induced in transgenic rice compared with that in wild type rice under normal growth condition.
为了更好地了解过表达 ONAC045 赋予的耐旱和耐盐机制，我们研究了几种已知的干旱和盐诱导基因在转基因水稻植物中的表达。如图 4 所示，晚期胚胎发生丰度 （LEA） 基因（OsLEA3-1[27]，GenBank 登录号 Z68090）和小麦质膜蛋白 （WPM-1） 同源基因 （WPM-1） [28]（此处称为 OsPM1，GenBank 登录号。NM_001061933） 在正常生长条件下，转基因水稻与野生型水稻相比具有很强的诱导性。

Discussion  讨论

Rice is one of the most important crops in the world. The growth and productivity of rice are often threatened by environmental factors, such as drought, salt, cold, and biotic stresses. Many efforts have been undertaken to generate stress tolerant rice by manipulating the expression of stress-responsive genes [29,30].
水稻是世界上最重要的农作物之一。水稻的生长和生产力经常受到环境因素的威胁，例如干旱、盐分、寒冷和生物胁迫。通过操纵胁迫反应基因的表达，已经采取了许多措施来产生耐逆水稻[29,30]。
Some members of NAC family have been shown to be involved in plant stress responses [15-19]. In this study, we functionally characterized a novel rice stress-responsive NAC gene ONAC045. Expression analysis showed that ONAC045 was highly induced by drought, salt, cold, and ABA in leaves and roots (Fig. 1B). Interestingly, the expression pattern was different between leaves and roots. For example, after salt treatment, the induced expression was much higher in roots than that in leaves at all three examined time points (Fig. 1B), suggesting that the expression of ONAC045 was differently regulated in leaves and roots. A previous study showed that ONAC045 was not induced under drought treatment
研究显示，NAC 家族的一些成员参与植物的逆境反应 [15-19]。在这项研究中，我们对一种新的水稻胁迫响应性 NAC 基因ONAC045进行了功能表征。表达分析表明，ONAC045 受干旱、盐、寒冷和 ABA 在叶片和根中的高度诱导（图 1B）。有趣的是，叶子和根之间的表达模式不同。例如，盐处理后，在所有三个检查时间点，根中的诱导表达都远高于叶片中的诱导表达（图 1B），这表明 ONAC045 的表达在叶和根中的调节不同。先前的一项研究表明，在干旱处理下不会诱发ONAC045
  1. E-mail address: bhan@ncgr.ac.cn (B. Han).
    电子邮件地址：bhan@ncgr.ac.cn （B. Han）。
    1 1 ^(1){ }^{1} These authors contributed equally to this work.
    1 1 ^(1){ }^{1} 这些作者对这项工作做出了同样的贡献。