转<italic>TiERF1-RC7</italic> 双价基因小麦的鉴定及其全蚀病抗性

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刘菲, 杨丽华, 王爱云, 刘欣, 马小飞, 杜丽璞, 李盼松, 张增艳, 马翎健. 转TiERF1-RC7 双价基因小麦的鉴定及其全蚀病抗性 . 作物学报, 39(11): 2094-2098[LIU Fei, YANG Li-Hua, WANG Ai-Yun, LIU Xin, MA Xiao-Fei, DU Li-Pu, LI Pan-Song, ZHANG Zeng-Yan, MA Ling-Jian. Molecular Detection and Take-all Response Assays ofTiERF1-RC7 Transgenic Wheat . 作物学报, 39(11): 2094-2098] 复制到剪切板

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转TiERF1-RC7 双价基因小麦的鉴定及其全蚀病抗性

刘菲¹, 杨丽华^1,², 王爱云³, 刘欣², 马小飞¹, 杜丽璞², 李盼松¹, 张增艳^2,^*, 马翎健^1,^*

¹西北农林科技大学农学院, 陕西杨凌 712100

²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遗传育种重点实验室, 北京 100081

³中南林业科技大学生命科学与技术学院, 湖南长沙 410004

^* 通讯作者(Corresponding authors): 张增艳, E-mail:zhangzengyan@caas.cn; 马翎健, E-mail:malingjian@nwsuaf.edu.cn

收稿日期:2013-06-24

基金:本研究由国家科技支撑计划项目(2011BAD35B03), 国家转基因生物新品种培育科技重大专项(2013ZX08002001-004, 011ZX08002-001)和西北农林科技大学基础科研业务费项目(ZD2012001)资助。

摘要

为探讨TiERF1 和RC7 基因对小麦抗全蚀病的防御反应, 本研究对转TiERF1-RC7 双价基因小麦进行了分子检测以及全蚀病抗性的室内和田间鉴定。结果表明, 转入的TiERF1 和RC7 基因在转基因小麦中可以遗传和转录; 与受体扬麦18相比, 5个转TiERF1-RC7 小麦株系在整个生育期抗病性显著提高, 苗期的全蚀病严重度在10%以下, 成熟期的白穗率在13%以下, 而扬麦18的严重度为62.98%, 白穗率为26.09%。电子显微镜观察结果表明抗病转基因小麦根表的全蚀病原菌菌丝数量及生长势明显低于感病材料。上述结果说明, 转入的TiERF1 和RC7 基因抑制了全蚀病原菌的侵染及在转基因小麦中繁殖, 进而提高了转基因小麦对全蚀病的抗性。

关键词: 全蚀病; Gaeumannomyces graminis var. tritic; 转基因小麦; 扫描电镜

Molecular Detection and Take-all Response Assays ofTiERF1-RC7 Transgenic Wheat

LIU Fei¹, YANG Li-Hua^1,², WANG Ai-Yun³, LIU Xin², MA Xiao-Fei¹, DU Li-Pu², LI Pan-Song¹, ZHANG Zeng-Yan^2,^*, MA Ling-Jian^1,^*

¹ College of Agronomg, Northwest A&F University, Yangling 712100, China

² National Key Facility of Crop Gene Resources and Genetic Improvement / Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture / Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

³College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410004, China

Abstract

The aim of this study was to understand the role ofTiERF1 andRC7 genes in wheat defense response to take-all. Both genes were simultaneously transformed into wheat cultivar Yangmai 18. The resistance to talk-all in five transgenic lines were analyzed by PCR and RT-PCR methods, and evaluated by artificial inoculation in greenhouse and field tests. Scanning electron microscope technique was used to observe the morphology and number of the pathogen mycelia on the root surfaces of resistant transgenic and susceptible receptor wheat plants. The PCR assay showed that bothTiERF1 andRC7 genes were integrated into the transgenic lines and inherited to the T₃ generation. The RT-PCR assay confirmed expressions of both genes in the transgenic lines. The results showed that five transgenic lines exhibited stable and efficient resistance to take-all with disease severity lower than 10% at seedling stage in greenhouse and rate of white spike lower than 13% at maturity in field. In the contrast of Yangmai 18, the severity was 62.98%, rate of white spike was 26.09%. Electron microscope observation revealed that the number and growth tendency of hyphae on roots of the resistant transgenic wheat were significantly lower and weaker than those of the wild type. The expressions ofTiERF1 andRC7 inhibit the growth of pathogen hyphae in the transgenic wheat plants, leading to improved resistance to take-all.

Keyword: Take-all; Gaeumannomyces graminis var. tritici; Transgenic wheat; Scanning electron microscope

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引言

小麦全蚀病是由禾顶囊壳小麦变种( Gaeumannomyces graminis var. tritici )引起的一种小麦根部土传病害, 可造成小麦减产40%~60%^[1]。在我国河南、河北、陕西、内蒙古、山东、宁夏、甘肃等多个麦区均有发生, 目前已成为河南、山东、陕西、河北等地小麦优质高产的严重威胁。选育和种植抗病小麦品种是防治全蚀病最有效、最直接的途径^[2], 但迄今, 常规育种中还没有抗全蚀病小麦品种的相关报道。采用转基因技术培育抗全蚀病小麦新种质成为拓宽小麦全蚀病抗源的重要途径。

ERF转录因子即乙烯答应因子, 是具有单个ERF/AP2 DNA结合域的调控蛋白。一些ERF转录因子与植物抗病反应有关,如超量表达拟南芥 ERF1 的转基因拟南芥植株对坏死营养型真菌和土传病原菌的抗性显著提高^[3]。中间偃麦草ERF转录因子基因 TiERF1 受病原菌诱导, 该基因转入烟草后提高了植株对烟草赤星病菌和烟草花叶病毒的抗性^[4]。现已发现 TiERF1 具有正向调控防御反应的作用, 其过表达可以提高转基因小麦对纹枯病抗性^[5]。几丁质酶可通过水解真菌菌丝生长点初生细胞壁中的几丁质, 引起真菌细胞壁变薄, 导致真菌细胞溶解, 从而达到抑菌的效果。Anuratha等^[6]从接种立枯纹枯菌( Rhizoctonia solani )的水稻中分离出的一个几丁质酶基因 RC7 , Datta等^[7]将 RC7 转入水稻, 发现 RC7 超量表达显著提高了转基因水稻对纹枯病的抗性; 刘宝业等^[8]通过体外抑菌实验证明 RC7 基因表达产物可显著抑制水稻纹枯病菌、小麦纹枯病菌、小麦赤霉病菌、棉花黄萎菌、烟草赤星菌的菌丝生长。为了增强转基因小麦的抗病效果, 我们构建了 TiERF1 和 RC7 双价基因的表达载体pTiERF1-RC7, 并通过基因枪法将 TiERF1-RC7 基因转入小麦(张增艳等未发表)。本试验对转 TiERF1-RC7 双价基因小麦进行分子检测和全蚀病的抗性鉴定, 并对其抗性机制进行初步探讨。

1 材料与方法

1.1 材料

转基因小麦的受体为小麦推广品种扬麦18 (江苏里下河农业科学研究所程顺和院士提供)。转 TiERF1-RC7 扬麦18的T₂代和T₃代10个株系。小麦全蚀病菌( Gaeum annomyces graminis var. tritici , Ggt )由西北农林科技大学植保学院提供。

1.2 转基因植株的PCR检测

采用改良的CTAB法^[9], 从T₂至T₃代植株叶片中提取基因组DNA。

根据转基因载体pTiERF1-RC7 (图1)的序列, 设计2对特异引物(RC7-U/TNOS-23L和RSS1P-1038U/TIET- 2088L)用于检测 RC7 和 TiERF1 基因, 预期扩增片段分别为260 bp和1050 bp。RC7-U (5′-GGCAACCTCGACTG CTACAA-3′)位于 RC7 读码框3′端, TNOS-23L (5′-TGTA TAATTGCGGGACTCTAAT-3′)位于Tnos终止序列上; RSS1P-1038U (5′-CATCCCCAACCAGTCCTTTT-3′)位于RSS1P启动子序列上, TIET-2088L (5′-CTCGGTAGTGC TTCCCCCT-3′)位于 TiERF1 序列上^[10]。

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	图1 pTiERF1-RC7双价基因的植物表达载体框架图箭头指示区为用特异引物PCR检测转基因的片段。Fig. 1 Scheme of pTiERF1-RC7 expression vector.The arrows indicate the regions amplified by PCR assays.

1.3 转基因植株的RT-PCR分析

利用TRIZOL试剂(Invitrogen)提取PCR阳性植株和未转基因扬麦18的叶片总RNA, 纯化后采用RNA kit ver.3.0 cDNA合成试剂盒(TaKaRa)合成第1条链cDNA。以小麦cDNA为模板, 利用小麦 actin 基因(ActA: 5′-CA CTGGAATGGTCAAGGCTG-3′, ActB: 5′-CTCCATGTC ATCCCAGTTG-3′)进行模板均一化; 然后用 TiERF1 特异引物(TIE1-317U: 5′-CGTCGTGCTTCGGTTTCCT-3′, TIE1- 743L: 5′-TCGCCTCTCTTCCTTCTTTT-3′)和 RC7 特异引物(RC7Q-649U: 5′-ATCGGGGTGGACCTGCTGA-3′, RC7Q- 855L: 5′-GCCGTTGACGATGTTGGTG-3′)进行半定量RT-PCR 分析。

1.4 转基因小麦全蚀病抗性鉴定

1.4.1 苗期人工接种鉴定利用PDA培养基在25℃下培养小麦全蚀病菌12~15 d, 待菌丝布满培养基后切成直径 1 cm的圆形菌饼, 按照Penrose^[11]菌饼法接种。小麦种子经1% H₂O₂消毒24 h并催芽, 待露白后将其接入菌饼, 置含灭菌沙的塑料盆中, 并用细沙覆盖, 每2~3 d浇水一次, 保持沙子湿润。每盆接种5粒, 在人工气候培养箱中培养28 d (18~22℃、12 h光照), 然后按0~6级记录反应型。其中, 0级为无病; 1~6级分别为黑根面积占总根面积的<5%、5%~10%、11%~25%、26%~40%、41%~65%和66%~100%。

1.4.2 田间成株抗病鉴定选出苗期花盆鉴定出的抗性好的转 TiERF1-RC7 基因小麦10个株系T₃代植株及其受体对照, 单株播种, 每株系30个单株进行全蚀病病圃鉴定。参照高小宁等^[12]的方法制备接种体, 将谷粒与水按照1.0∶1.5的比例置1000 mL锥形瓶中, 连续3 d高压灭菌1 h, 待冷却后在无菌操作室中接种小麦全蚀病菌。接种后, 室内放置培养4周晾干备用。

在西北农林科技大学全蚀病专用病圃(陕西杨凌)进行田间鉴定。每小区10行, 行长1.5 m, 播种深度15 cm; 每行均匀播撒6 g上述接种体, 小麦种子每行30粒, 间隔5 cm; 同时设受体感病对照(未转基因扬麦18), 小麦生长期的管理与大田一致。以苗期死苗率及成株期的死亡率和白穗率反映其抗病性。

1.5 小麦全蚀病菌的电镜观察

选择室内鉴定抗性好的转基因小麦材料和感病对照各8粒, 经消毒、催芽后, 按苗期室内鉴定方法接种小麦全蚀病菌, 分别在接种后的48、60和120 h切取穿过菌饼的幼根7~8段, 每段长约7 mm; 用4%戊二醛固定24 h, 再经CO₂干燥和喷金处理, 于SEM6360LV扫描电镜下观察菌丝及小麦细胞生长情况。

2 结果与分析

2.1 转基因小麦的PCR检测和外源基因转录分析

在T₂和T₃代植株中, 特异引物PCR检测出10个转 TiERF1-RC7 双价基因小麦株系, 这些转基因株系均呈现260 bp ( RC7 )和1050 bp ( TiERF1 基因)的预期扩增片段, 而未转基因的受体扬麦18无此扩增条带(图2-A, B)。抗病T₃代植株中目标基因的阳性检出率是100%, 说明这10个T₃代株系中外源转 TiERF1-RC7 基因已基本纯合。

选择5个全生育期抗病的转基因株系T₃代植株进行半定量RT-PCR分析, 结果这些植株中 TiERF1 与 RC7 基因的转录水平均明显高于未转基因扬麦18 (图2-C)。

2.2 转基因小麦材料的苗期盆栽全蚀病抗性鉴定

苗期人工接种鉴定结果表明, 转 TiERF1-RC7 基因小麦T₂代10个株系的各项抗病指标均好于受体扬麦18。对T₃代转 TiERF1-RC7 基因小麦的10个株系继续鉴定, 其抗性显著增强, 其反应等级均在1~2级, 根部、茎基部的感病程度明显减轻, 且根系长度和根系面积均显著大于受体扬麦18 (表1)。说明转基因小麦苗期的抗病性显著高于受体小麦, 并增强了转基因小麦根系的生长势。

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图2 转 TiERF1-RC7 基因扬麦18的T₃代PCR及RT-PCR分析A: RC7 特异引物扩增结果; B: TiERF1 特异引物扩增结果; C: RT-PCR结果。P: 质粒; WT: 受体扬麦18; CK: 水对照; 1~10: 转 TiERF1-RC7 双价基因小麦株系, 依次是10ZGY-14、10ZGY-49、10ZGY-75、10ZGY-92、10ZGY-34、10ZGY-113、10ZGY-152、10ZGY-15、10ZGY-157和10ZGY-165。Fig. 2 PCR and RT-PCR assays on T₃plants of TiERF1-RC7 transgenic wheat Yangmai 18A: amplification of RC7 specific primers; B: amplification of TiERF1RC7 specific primers; C: RT-PCR. P: plasmid; WT: Yangmai 18; CK: water control; 1-10: transgenic bivalent genes TiERF1-RC7 wheat lines: they are 10ZGY-14, 10ZGY-49, 10ZGY-75, 10ZGY-92, 10ZGY-34, 10ZGY-113, 10ZGY-152, 10ZGY-15, 10ZGY-157, and 10ZGY-165.

表1 转基因小麦及其对照苗期对全蚀病的抗性评价 Table 1 Resistance evaluation of transgenic wheat to take-all disease at seedling stage

2.3 转基因小麦材料的病圃抗性鉴定

在病圃抗性鉴定中, 受体扬麦18苗期死苗率达30%, 成株期死亡率达45%, 白穗率达26.09%; 而5个转基因株系的苗期死苗率为15%~25%, 成株期死亡率为20~30%, 白穗率为0.00~12.5%, 均显著低于受体扬麦18 (表2), 说明在整个生育期中转基因小麦对全蚀病抗性均显著提高。

表2 病圃中转基因小麦材料的全蚀病抗性评价 Table 2 Resistance evaluation on transgenic wheat to wheat take-all disease at nursery (%)

2.4 小麦全蚀病菌侵染的扫描电镜观察

以感病受体扬麦18为对照, 转基因株系10ZGY-92为试材, 进行全蚀病菌侵染过程的电镜对比分析。接种全蚀病病菌48 h, 根表面形成了平行菌丝, 菌丝数量较少, 且匍匐于根细胞表面, 没有侵入到皮层细胞, 10ZGY-92根细胞表面上菌丝比感病受体的菌丝细(图2-A, B), 说明抗性好的材料中菌丝生长更慢。在接种60 h小麦根部细胞表面布满大量菌丝, 且多缠绕在根毛周围, 说明根毛是菌丝侵入皮层的重要途径, 同样看到10ZGY-92中的菌丝数量明显少于感病对照(图3-C, D), 菌丝侵入感病受体的皮层细胞, 且菌丝形成“∧”形。接种120 h感病受体的中柱层已有菌丝出现(图3-E), 说明此时菌丝已经进入中柱, 这与刘常宏和商鸿生^[13]的研究结果一致, 而10ZGY-92的中柱中并未发现菌丝(图3-F)。

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图3 小麦全蚀病菌菌丝侵染转基因小麦及受体扬麦18根表的扫描电镜观察A和B: 接种48 h转基因小麦10ZGY-92和扬麦18根表面有少量菌丝(48 h); C和D: 接种60 h菌丝在10ZGY-92和扬麦18的根毛上缠绕, 与受体小麦扬麦18 (D)相比, 抗病转基因小麦10ZGY-92细胞表面的 Ggt 菌丝数量少且菌丝细(C); E: 接种120 h菌丝未进入转基因小麦10ZGY-92的中柱; F: 接种120 h后菌丝穿过扬麦18的中柱。Fig. 3 Scanning electron microscope observation of invasion on the root surfaces of the transgenic and Yangmai 18 plants by take-all pathogenA and B: a small amount of hyphae was on the root surfaces of transgenic wheat 10ZGY-92 and Yangmai 18 at inoculation for 48 h; C and D: mycelium winded root hairs in transgenic wheat 10ZGY-92 and Yangmai 18 at inoculation for 60 h; Ggt was less and thinner in resistant transgenic wheat 10ZGY-92 than in Yangmai 18; E: no mycelium invaded columns in transgenic wheat 10ZGY-92 at inoculation for120 h; F: mycelium invaded column layer in Yangmai 18 at inoculation for 120 h.

3 讨论

小麦全蚀病菌是由根到茎依次侵染的, 侵染后根系的发育情况也可以说明不同小麦材料对全蚀病的耐病性。因此, 本研究依据温室接种后小麦材料的黑根面积、总根面积、严重度(黑根面积与总根面积的比值)^[14]、黑茎面积、黑茎率等指标鉴定苗期的抗病性, 并结合病圃评价小麦苗期、成株期抗性, 同时对导入转 TiERF1-RC7 基因小麦进行分子检测及利用扫描电镜观察病原菌丝情况, 进一步探讨了转 TiERF1-RC7 基因小麦抗全蚀病机制。

本研究表明, 转入的 TiERF1 和 RC7 基因在10个转基因小麦株系中可以遗传, 并显著提高了转基因小麦株系苗期对全蚀病的抗性; 这10个转 TiERF1 和 RC7 基因小麦株系的苗期反应型均在2级以下, 而受体扬麦18的反应型等级为5级; 在病圃抗性鉴定中, 其中5个株系 (10ZGY-15、10ZGY-49、10ZGY-92、10ZGY-113和10ZGY- 152)的苗期死苗率、成株期死亡率和白穗率均显著低于受体扬麦18对照。对这5个株系抗病植株中 TiERF1 与 RC7 基因的RT-PCR分析表明, 这些转基因植株中 TiERF1 与 RC7 基因的转录水平明显高于未转基因小麦扬麦18。扫描电镜(SEM)表明, 全蚀病菌的侵入部位大多集中在根毛区, 少数从茎侵入, 这与王裕中等^[15]及郝祥之等^[16]的报道结果相同; 抗病转基因小麦根表附着的全蚀病菌丝明显少于感病小麦的, 且在同一时期抗病转基因小麦根表的菌丝更细。上述结果说明, 转 TiERF1-RC7 基因的转录表达抑制了全蚀病菌侵入与发育, 从而提高了转基因小麦对全蚀病的抗性。

致谢:

衷心感谢中国农业科学院作物科学研究所徐惠君研究员、庄洪涛、李钊和祝秀亮等研究生在载体构建、转基因小麦材料创制与早代(T₀和T₁)分子检测工作中的贡献。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

参考文献

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[1]	Daval S, Lebreton L, Gazengel K, Boutin M, Guillerm-Erckelboudt A Y, Sarniguet. The biocontrol bacterium Pseudomonas fluorescens Pf29Arp strain affects the pathogenesis-related gene expression of the take-all fungus Gaeumannomyces graminis var. tritici on wheat roots. Mol Plant Pathol, 2011, 12: 839-854 [本文引用:1] [JCR: 3.899]
[2]	Wei F-Q(魏芳勤), Wu J(武军), Zhao J-X(赵继新), Chen X-H(陈新宏), Liu S-H(刘淑会), Pang Y-H(庞玉辉). Genetic analysis of resistance to take-all fungus of wheat line h9021 derived from wheat-Psathyrostachys huashannica. J Triticeae Crops(麦类作物学报), 2009, 29(1): 153-156 (in Chinese with English abstract) [本文引用:1]
[3]	Berrocal-Lobo M, Molina A, Solano R. Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi. Plant, 2002, 29: 23-32 [本文引用:1]
[4]	Liang H X, Lu Y, Liu H X, Wang F D, Xin Z Y, Zhang Z Y. A novel activator-type ERF of Thinopyrum intermedium, TiERF1, positively regulates defence responses. TiERF1, positively regulates defence responses. J Exp Bot, 2008, 59: 3111-3120 [本文引用:1] [JCR: 5.364]
[5]	Chen L, Zhang Z Y, Liang H X, Liu H X, Du L P, Xu H J, Xin Z Y. Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat. J Exp Bot, 2008, 59: 4195-4204 [本文引用:1] [JCR: 5.364]
[6]	Anuratha C S, Zen K C, Cole K C. Induction of chitinases and β-1, 3-glucanases in Rhizoctonia solani-infected rice plants: isolation of an infection-related chitinase cDNA clone. Physiol Plant, 1996, 97: 39-46 [本文引用:1] [JCR: 6.535]
[7]	Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew T W, Muthkrishnan S, Datta S K. Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars. Plant Sci, 2001, 60: 405-414 [本文引用:1] [JCR: 2.945]
[8]	Liu B-Y(刘宝业), Liang G-L(梁国鲁), Zhang Z-Y(张增艳). Prokaryotic expression, refolding and antifungal acticity of a rice chitinase in vitro. J Nucl Agric Sci(核农学报), 2009, 23(3): 369-374 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.9108]
[9]	Cook R J, Thomashow L S, Weller D M, Fujimoto D, Mazzola M, Bangera G, Kim D. Molecular mechanisms of defense by rhizobacteria against root disease. Proc Natl Acad Sci USA, 1995, 92: 4197-4201 [本文引用:1] [JCR: 9.737]
[10]	Li Z(李钊), Zhuang H-T(庄洪涛), Du L-P(杜丽璞), Zhou M-P(周淼平), Cai S-B(蔡士宾), Xu H-J(徐惠君), Li S-S(李斯深), Zhang Z-Y(张增艳). Utilization of tissue specific expressing promoter RSS1P in TiERF1 transgenic wheat. Acta Agron Sin(作物学报), 2011, 37(10): 1897-1903 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.8267]
[11]	Penrose L. Evidence for resistance in wheat ultivars grown in sand culture to the take-all pathogen, Gaeumnnomyces graminis var. tritici. Ann Appl Biol, 1985, 107: 105-108 [本文引用:1] [JCR: 2.179]
[12]	Gao X-N(高小宁), Liu Q-L(刘起丽), Huang L-L(黄丽丽), Wei G-R(魏国荣), Kang Z-S(康振生). Resistance assessments of high yield wheat cultivars to take-all disease. J Yunnan Agric Univ(云南农业大学学报), 2004, 19(4): 384-386 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.7181]
[13]	Liu C-H(刘常宏), Shang H-S(商鸿生). The infection process of wheat seminal root by Gaeumannomyces graminis var. tritici. Acta Phytopathol Sin(植物病理学报), 2000, 30(1): 19-24 (in Chinese with English abstract) [本文引用:1]
[14]	Wang D-B(王殿波), Wang M-N(王美南), Jing J-X(井金学), Shang H-S(商鸿生), Li F-X(李方向). Study of resistance in distant hybridigation descendant of wheat to the take-all. Acta Bot Boreali-Occident Sin(西北植物学报), 2003, 23(9): 1717-1620 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.1098]
[15]	Wang Y-Z(王裕中), Shen S-W(沈素文), Chen H-G(陈怀古), Shi J-R(史建荣). Observations on infection of Gaeumannomyces graminis var. tritici in wheat roots. Jiangsu J Agric Sci(江苏农业学报), 1997, 13(1): 18-21 (in Chinese with English abstract) [本文引用:1]
[16]	Hao X-Z(郝祥之), Duan J-Y(段剑勇), Li L(李林). Wheat Take-All and Its Prevention and Control (小麦全蚀病及其防治). Shanghai: Shanghai Scientific and Technical Publishers, 1982. pp68-70 (in Chinese with English abstract) [本文引用:1]

2011

3.899

0.0

Mol. Plant Pathol.. 2011, 12(9):839-54 DOI:10.1111/j.1364-3703.2011.00715.x

The biocontrol bacterium Pseudomonas fluorescens Pf29Arp strain affects the pathogenesis-related gene expression of the take-all fungus Gaeumannomyces graminis var. tritici on wheat roots.

Daval S, Lebreton L, Gazengel K, Boutin M, Guillerm-Erckelboudt AY, Sarniguet A

INRA, Agrocampus Ouest, Université Rennes 1, UMR1099 BiO3P (Biology of Organisms and Populations Applied to Plant Protection), BP 35327, Le Rheu, France. stephanie.daval@rennes.inra.fr

The main effects of antagonistic rhizobacteria on plant pathogenic fungi are antibiosis, fungistasis or an indirect constraint through the induction of a plant defence response. To explore different biocontrol mechanisms, an in vitro confrontation assay was conducted with the rhizobacterium Pseudomonas fluorescens Pf29Arp as a biocontrol agent of the fungus Gaeumannomyces graminis var. tritici (Ggt) on wheat roots. In parallel with the assessment of disease extension, together with the bacterial and fungal root colonization rates, the transcript levels of candidate fungal pathogenicity and plant-induced genes were monitored during the 10-day infection process. The bacterial inoculation of wheat roots with the Pf29Arp strain reduced the development of Ggt-induced disease expressed as attack frequency and necrosis length. The growth rates of Ggt and Pf29Arp, monitored through quantitative polymerase chain reaction of DNA amounts with a part of the Ggt 18S rDNA gene and a specific Pf29Arp strain detection probe, respectively, increased throughout the interactions. Bacterial antagonism and colonization had no significant effect on root colonization by Ggt. The expression of fungal and plant genes was quantified in planta by quantitative reverse transcription-polymerase chain reaction during the interactions thanks to the design of specific primers and an innovative universal reference system. During the early stages of the tripartite interaction, several of the fungal genes assayed were down-regulated by Pf29Arp, including two laccases, a β-1,3-exoglucanase and a mitogen-activated protein kinase. The plant host glutathione-S-transferase gene was induced by Ggt alone and up-regulated by Pf29Arp bacteria in interaction with the pathogen. We conclude that Pf29Arp antagonism acts through the alteration of fungal pathogenesis and probably through the activation of host defences.

PMID: 21726382

... tritici )引起的一种小麦根部土传病害, 可造成小麦减产40%~60%^[1] ...

2009

0.0

... 选育和种植抗病小麦品种是防治全蚀病最有效、最直接的途径^[2], 但迄今, 常规育种中还没有抗全蚀病小麦品种的相关报道 ...

2002

0.0

... 一些ERF转录因子与植物抗病反应有关,如超量表达拟南芥ERF1 的转基因拟南芥植株对坏死营养型真菌和土传病原菌的抗性显著提高^[3] ...

2008

5.364

0.0

J. Exp. Bot.. 2008, 59(11):3111-20 DOI:10.1093/jxb/ern165

A novel activator-type ERF of Thinopyrum intermedium, TiERF1, positively regulates defence responses.

Liang H, Lu Y, Liu H, Wang F, Xin Z, Zhang Z

National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.

Thinopyrum intermedium is resistant to many different pathogens. To understand the roles of ethylene response factors (ERFs) in defence responses, the first member of the ERF family in T. intermedium, TiERF1, was characterized and functionally analysed in this study. The TiERF1 gene encodes a putative protein of 292 amino acids, belonging to the B3 subgroup of the ERF transcription factor family. Biochemical assays demonstrated that the TiERF1 protein is capable of binding to the GCC box, a cis-element present in the promoters of pathogenesis-related (PR) genes, and possessing transactivation activity, as well as localizing to the nucleus. The transcript of TiERF1 in T. intermedium is rapidly induced by infection with Rhizoctonia cerealis, Fusarium graminearum, or Blumeria graminis, and ethylene, jasmonic acid, and salicylic acid treatments. More importantly, the ectopic expression of TiERF1 in tobacco activated the transcript of the PR genes of tobacco with a GCC box cis-element, and ACO and ACS genes key to ethylene synthesis, and in turn improved the resistance level to Alternaria alternata and tobacco mosaic virus, as well as causing some phenotypic changes associated with ethylene response in the transgenic tobacco plants. Taken together, TiERF1 protein as an ERF transcription activator positively regulates defence responses via the activation of some defence-related genes.

PMID: 18611911

... 中间偃麦草ERF转录因子基因TiERF1 受病原菌诱导, 该基因转入烟草后提高了植株对烟草赤星病菌和烟草花叶病毒的抗性^[4] ...

2008

5.364

0.0

J. Exp. Bot.. 2008, 59(15):4195-204 DOI:10.1093/jxb/ern259

Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat.

Chen L, Zhang Z, Liang H, Liu H, Du L, Xu H, Xin Z

National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.

Wheat sharp eyespot, primarily caused by a soil-borne fungus Rhizoctonia cerealis, has become one of the most serious diseases of wheat in China. In this study, an ethylene response factor (ERF) gene from a wheat relative Thinopyrum intermedium, TiERF1, was characterized further, transgenic wheat lines expressing TiERF1 were developed, and the resistance of the transgenic wheat lines against R. cerealis was investigated. Southern blotting analysis indicated that at least two copies of the TiERF1 gene exist in the T. intermedium genome. Yeast one-hybrid assay indicated that the activation domain of TiERF1 is essential for activating the transcript of the reporter gene with the GCC-box cis-element. The TiERF1 gene was introduced into a Chinese wheat cultivar, Yangmai12, by biolistic bombardment. Results of PCR and Southern blotting analyses indicated that TiERF1 was successfully integrated into the genome of the transgenic wheat, where it can be passed down from the T0 to T4 generations. Quantitative reverse transcription-PCR analysis demonstrated that TiERF1 could be overexpressed in the stable transgenic plants, in which the expression levels of wheat pathogenesis-related (PR) genes primarily in the ethylene-dependent signal pathway, such as a chitinase gene and a beta-1,3-glucanase gene, were increased dramatically. Disease tests indicated that the overexpression of TiERF1 conferred enhanced resistance to sharp eyespot in the transgenic wheat lines compared with the wild-type and silenced TiERF1 plants. These results suggested that the overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat lines by activating PR genes primarily in the ethylene-dependent pathway.

PMID: 18953072

... 现已发现TiERF1 具有正向调控防御反应的作用, 其过表达可以提高转基因小麦对纹枯病抗性^[5] ...

1996

6.535

0.0

... Anuratha等^[6]从接种立枯纹枯菌(Rhizoctonia solani )的水稻中分离出的一个几丁质酶基因RC7 , Datta等^[7]将RC7 转入水稻, 发现RC7 超量表达显著提高了转基因水稻对纹枯病的抗性 ...

2001

2.945

0.0

2009

0.0

0.9108

J Nucl Agric Sci. 2009, 23(3):369 - 374

Prokaryotic Expression, Refolding and Antifungal Activity of a Rice Chitinasein vitro

水稻几丁质酶的原核表达、复性及体外抑菌活性的研究

National Key Science Facility of Crop Gene Resources and Gene Improvement，Institute of Crop Science，Chinese Academy of Agricultural Sciences，Beijing 100081

The full open-reading frame (ORF) sequence of a rice chitinase gene RC7 was isolated from the rice leaf cDNA induced by Rhizoctonia solani through subclone method. The RC7 was fused in frame to the ORF of Glutathione S-transferase （GST）in the prokaryotic expression vector pGEX-4T-1, in which the new recombinant vector pGST-RC7, containing the recombinant protein GST-RC7, was constructed. Then the pGST-RC7 vector was transformed into cells of Escherichia coli. After induced by isopropyl-β-D-thiogalactoside (IPTG), the recombinant protein GST-RC7 was highly expressed in the form of inclusion bodies. The protein GST-RC7 in the inclusion bodies could be solubilized, renatured and purified by a serial of treatments, including washing, denaturing and renaturing as well as purification. The renaturing protein possessed chitinase activity. The results of antifungal assayin vitro showed that the chitinase RC7 exerted significant inhibition to the mycelia growth of Rhizoctonia solani (rice sheath blight), Rhizoctonia cerealis (wheat sharp eyespot), Verticillium dahliae (cotton wilt), Fusarium graminearum (wheat scab) and Alternaria longipes (tobacco brown spot). RC7 may be useful for enhancing fungal resistance in several crop species.

利用RT-PCR反应，从纹枯菌诱导的水稻叶片cDNA中克隆了1个水稻几丁质酶基因RC7的全长编码序列（ORF）。通过亚克隆方法，RC7与原核表达载体pGEX-4T-1上的GST融合，构建成GST-RC7重组蛋白的原核表达载体pGST-RC7，然后，将其转化到大肠杆菌细胞中。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，获得了以包涵体形式高效表达的GST-RC7。经过洗涤、溶解（变性）、复性及纯化等处理，包涵体中GST-RC7得到溶解、复性和纯化。纯化的复性GST-RC7蛋白具有几丁质酶活性。对6种重要作物病原真菌进行体外抑菌活性分析的结果表明， RC7可显著抑制水稻纹枯病菌、小麦纹枯菌、小麦赤霉菌、棉花黄萎菌、烟草赤星菌的菌丝生长，说明RC7可作为上述植物病害抗性基因育种的重要基因。

... 刘宝业等^[8]通过体外抑菌实验证明RC7 基因表达产物可显著抑制水稻纹枯病菌、小麦纹枯病菌、小麦赤霉病菌、棉花黄萎菌、烟草赤星菌的菌丝生长 ...

1995

9.737

0.0

... 2 转基因植株的PCR检测采用改良的CTAB法^[9], 从T₂至T₃代植株叶片中提取基因组DNA ...

2011

0.0

1.8267

Acta Agron Sin. 2011, 37(10):1897 - 1903 DOI:10.3724/SP.J.1006.2011.01897

Utilization of Tissue Specific Expressing Promoter RSS1P inTiERF1 Transgenic Wheat

组织特异表达启动子RSS1P在转TiERF1基因小麦中的应用

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 / 农业部作物遗传育种重点实验室，北京 100081; 2山东农业大学农学院，山东泰安 2701082; 3江苏省农业科学院，江苏南京 210014

李钊^1,2,庄洪涛¹，杜丽璞¹，周淼平³，蔡士宾³，徐惠君¹，李斯深^2,*，张增艳^1,*

1 National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Crop Genetic and Breeding, Ministry of Agriculture / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 College of Agronomy, Shandong Agricultural University, Tai’an 271018, China; 3 Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

A sucrose synthase-1 promoter (RSS1P) was cloned from rice leaf, and subcloned in-fused with 5′-terminate of the gene of ethylene responsive factor of Thinopyrum intermedium (TiERF1) to form theRSS1P::TiERF1expressing cassette. The expressing cassette replaced theUbi::bargene expression cassette of pAHC20 to produce the phloem-specific expressing vector pA20-RSS1P::TiERF1. The selective markbarwas deleted in the pA20-RSS1P::TiERF1vector, which is safe for the ecosystem. Using the method of bombardment, the vector DNAs of pA20-RSS1P::TiERF1 andpAHC20 withbargene were co-transformed to young embryo callus of wheat cultivar Yangmai 12. TheRSS1P::TiERF1 transgenic plants in T₀ and T₁ generations were subjected to PCR, PCR-Southern, RT-PCR, and Q-RT-PCR analyses, andRhizoctonia cerealis resistance tests. The molecular detection results showed that the transgeneRSS1P::TiERF1 was introduced into Yangmai 12 and was inheritable. In theRSS1P::TiERF1 transgenic wheat plants, expression ofTiERF1was detected in root, stem, and leaf except seed. The highest expression level was observed in root. Compared to the untransformated Yangmai 12, resistance toR. cerealis in the RSS1P::TiERF1 transgenic wheat plants was obviously improved, which was similar to that in the Ubi::TiERF1 transgenic wheat plants. The agronomic traits ofRSS1P::TiERF1 transgenic plants had no obvious changes compared to untransformated Yangmai 12. These results suggest thatRSS1P promoter is feasible to be used for developing new transgenic wheat germplasm.

从水稻叶片中克隆了一个韧皮部组织特异表达的水稻蔗糖合酶启动子(RSS1P)，将RSS1P与中间偃麦草乙烯反应因子基因TiERF1相融合构成组织特异表达的TiERF1基因表达盒，取代pAHC20中Ubi::bar基因表达盒，构建成无选择标记的韧皮部组织特异表达的pA20-RSS1P::TiERF1载体。利用基因枪将pA20-RSS1P::TiERF1与pAHC20载体混合、共轰击小麦品种扬麦12的幼胚愈伤组织，获得转RSS1P::TiERF1基因小麦。对该转基因小麦T₀和T₁代植株进行PCR、PCR-Southern、半定量RT-PCR和荧光定量PCR分析，证实外源RSS1P::TiERF1基因已转入受体，并且具有可遗传性；转入的RSS1P::TiERF1基因仅在根、茎、叶中表达，以根部表达量最高，在种子内不表达。纹枯病抗性鉴定和主要农艺性状考察结果表明，与受体扬麦12相比，转RSS1P::TiERF1基因小麦对纹枯病的抗性有明显提高，与转Ubi::TiERF1基因小麦的抗病性相当，而且转RSS1P::TiERF1基因小麦的农艺性状没有明显改变，说明可以利用RSS1P启动子创造更实用的转基因小麦新种质。

... RSS1P-1038U (5′-CATCCCCAACCAGTCCTTTT-3′)位于RSS1P启动子序列上, TIET-2088L (5′-CTCGGTAGTGC TTCCCCCT-3′)位于TiERF1 序列上^[10] ...

1985

2.179

0.0

... 1 苗期人工接种鉴定利用PDA培养基在25℃下培养小麦全蚀病菌12~15 d, 待菌丝布满培养基后切成直径 1 cm的圆形菌饼, 按照Penrose^[11]菌饼法接种 ...

2004

0.0

0.7181

Journal of Yunnan Agricultural University. 2004, (4):384 - 386

Resistance Assessments of High Yield Wheat Cultivars to Take-all Disease

超高产小麦品种(系)对全蚀病的抗性鉴定

高小宁;刘起丽;黄丽丽;魏国荣;康振生

在人工接种全蚀病菌的条件下,以小偃6号小麦品种为对照,评价了38个超高产小麦品种(系)在温室和田间对全蚀病的抗病性.结果表明,品种(系)间发病严重度及病情指数明显不同,存在比较稳定的抗病性差异.连续3年的室内盆栽和田间试验结果表明,西农1376,132,西农918,(97)148,R859等品种(系)对小麦全蚀病表现出明显和稳定的抗病性.

... 参照高小宁等^[12]的方法制备接种体, 将谷粒与水按照1 ...

2000

0.0

... 接种120 h感病受体的中柱层已有菌丝出现(图3-E), 说明此时菌丝已经进入中柱, 这与刘常宏和商鸿生^[13]的研究结果一致, 而10ZGY-92的中柱中并未发现菌丝(图3-F) ...

2003

0.0

1.1098

... 因此, 本研究依据温室接种后小麦材料的黑根面积、总根面积、严重度(黑根面积与总根面积的比值)^[14]、黑茎面积、黑茎率等指标鉴定苗期的抗病性, 并结合病圃评价小麦苗期、成株期抗性, 同时对导入转TiERF1-RC7 基因小麦进行分子检测及利用扫描电镜观察病原菌丝情况, 进一步探讨了转TiERF1-RC7 基因小麦抗全蚀病机制 ...

1997

0.0

... 扫描电镜(SEM)表明, 全蚀病菌的侵入部位大多集中在根毛区, 少数从茎侵入, 这与王裕中等^[15]及郝祥之等^[16]的报道结果相同 ...

1982

0.0

... 扫描电镜(SEM)表明, 全蚀病菌的侵入部位大多集中在根毛区, 少数从茎侵入, 这与王裕中等^[15]及郝祥之等^[16]的报道结果相同 ...