引用本文
单天雷, 洪彦涛, 杜丽璞, 徐惠君, 魏学宁, 张增艳. 抗根腐病的 TaMYB86过表达转基因小麦的创制与分子功能鉴定 . 作物学报, 2016, 42(10): 1429-1436
SHAN Tian-Lei, HONG Yan-Tao, DU Li-Pu, XU Hui-Jun, WEI Xue-Ning, ZHANG Zeng-Yan. Development and Characterization of TaMYB86-Overexpressing Transgenic Wheat Lines with Resistance to Common Root Rot . ACTA AGRONOMICA SINICA, 2016, 42(10): 1429-1436  
Permissions
Copyright©2016, 作物学报编辑部
作物学报编辑部
抗根腐病的 TaMYB86过表达转基因小麦的创制与分子功能鉴定
单天雷, 洪彦涛, 杜丽璞, 徐惠君, 魏学宁, 张增艳*
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
*通讯作者(Corresponding author): 张增艳, E-mail: zhangzengyan@caas.cn, Tel: 010-82108781

第一作者联系方式: E-mail: tlshan8023@163.com; Tel: 15600642564

收稿日期:2016-03-10 接受日期:2016-07-11 网络出版日期:2016-08-01
基金:本研究由国家转基因生物新品种培育重大专项(2016ZX08002-001-004)和国家自然科学基金项目(31471494)资助
摘要

小麦根腐病是一种难以防治的小麦土传病害。 TaMYB86是一个小麦中受根腐病菌诱导表达的MYB编码基因。本文构建了 TaMYB86的过表达转基因载体pUbi:MYC-TaMYB86, 利用基因枪介导法将其转入推广小麦品种扬麦16。对转 TaMYB86基因小麦T0-T3代植株进行分子特征分析和抗病鉴定。PCR检测结果表明, 外源 TaMYB86已转入3个转基因小麦株系中; qRT-PCR结果显示, TaMYB86在3个转基因小麦株系中的表达量显著高于在未转基因扬麦16中的表达量, 约为未转基因扬麦16中的5~6倍, 表明 TaMYB86可在转基因小麦中过量转录; Western杂交结果表明, 引入的 TaMYB86可在上述3个转基因小麦株系中翻译表达。对转 TaMYB86基因小麦与未转基因扬麦16进行根腐病菌接种与抗病鉴定表明, 3个转 TaMYB86基因小麦株系在T1~T3代的根腐病病情指数分别为31.75、50.00、45.00; 37.75、37.50、38.50; 41.75、31.25、37.50; 在3次鉴定中未转基因扬麦16的根腐病病情指数分别为75.04、54.17、65.38, 转 TaMYB86基因小麦T1~T3代的根腐病抗性均显著高于未转基因扬麦16 ( P< 0.01)。与未转基因扬麦16相比, 转 TaMYB86基因小麦中3个下游防卫基因( PR10、 PR17c和 Chit1)的转录水平也显著上调。以上结果说明, TaMYB86过表达可显著增强转基因小麦的根腐病抗性, 在小麦防御根腐病过程中起正向调控作用。

关键词: 小麦; MYB转录因子; 防御反应; 小麦根腐病; 转基因
Development and Characterization of TaMYB86-Overexpressing Transgenic Wheat Lines with Resistance to Common Root Rot
SHAN Tian-Lei, HONG Yan-Tao, DU Li-Pu, XU Hui-Jun, WEI Xue-Ning, ZHANG Zeng-Yan*
National Key Facility for 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
Fund:The study was supported by the National Major Project for Developing New GM Crops (2016ZX08002-001-004) and the National Natural Science Foundation of China (31471494)
Abstract

Wheat common root rot, mainly caused by Bipolaris sorokiniana, is a difficultly prevent soil-borne disease of wheat ( Triticum aestivum L.) worldwide. TaMYB86, a B. sorokiniana-induced wheat MYB gene ,encodes a MYB transcription factor. We constructed the TaMYB86 overexpression vector pUbi:MYC-TaMYB86 and introduced TaMYB86 into Yangmai 16 via the particle bombardment. The TaMYB86 transgenic wheat lines on generations of T0-T3 were underwent by molecular characteristics analysis and disease resistance evaluation. The PCR and quantitative RT-PCR results showed that the alien TaMYB86was introduced into three transgenic wheat lines, and the relative transcriptional level of TaMYB86was apparently higher in transgenic wheat lines than in non-transformed Yangmai 16. As Western blot results presented, the introduced MYC-TaMYB86 gene was translated into the MYC-TaMYB86 protein in the three overexpressing transgenic lines, but not in non-transformed Yangmai 16. The infection types and disease indexes of three TaMYB86 transgenic wheat lines were significantly lower than those of non-transformation Yangmai 16 ( t-test, P< 0.01). The transcript levels of 3 wheat defense genes ( PR10, PR17c, and Chit1) were significantly elevated in three transgenic wheat lines than in the non-transformed Yangmai 16. These results indicate that overexpression of TaMYB86 enhances significantly resistance to B. sorokiniana in transgenic wheat lines and TaMYB86 plays a positive role in defense response to B. sorokiniana.

Keyword: Triticum aestivum; MYB transcription factor; Defense response; Common root rot; Transgene

小麦根腐病是一种世界性的、难以防治的小麦土传病害[1], 其病原菌为平脐蠕孢菌Bipolaris sorokiniana (有性态为禾旋孢腔菌Cochliobolus sativus)。据2015年全国农技推广中心报道, 我国华北、黄淮和东北麦区均发现小麦根腐病, 在黄淮麦区和华北南部麦区危害呈上升态势。小麦根腐病是全生育期典型的多阶段性病害, 从苗期到抽穗结实期都能发生, 小麦种子、幼芽、幼苗、成株根系、叶片、茎和穗都可受害, 一般可造成产量损失10%~30%, 严重地块减产超过50% [2, 3]。目前, 生产上大面积推广品种和育种材料对根腐病的抗性普遍较差, 常规抗病育种进展缓慢, 主要原因是抗性鉴定和单株选择困难, 缺乏高抗根腐病材料, 加之抗性遗传机制复杂[3]。因此, 发掘、克隆抗根腐病基因对丰富小麦抗病资源有重要的意义, 同时开展相关基因的功能研究, 开发可用于辅助育种的分子标记, 对抗根腐病小麦育种具有指导作用。

植物MYB转录因子是一个大家族。MYB转录因子含有高度保守的MYB结构域, 具有结合靶标基因启动子的作用。一个MYB结构域含有50~52个氨基酸残基, 能形成3个α 螺旋, 并且最终能形成螺旋-转角-螺旋(helix-turn-helix, HTH)的高级结构。每个MYB结构域重复的第2和第3个α 螺旋能直接与目标DNA结合, 被称为“ 识别螺旋” [4]。根据结构域重复单位的数目和位置可将MYB分为1R-MYB (MYB- related)、2R-MYB (R2R3-MYB)、3R-MYB和4R-MYB共4个亚族, 以R2R3-MYB的数量最多[4]。MYB转录因子参与许多植物生长发育的过程[5, 6, 7, 8]及非生物逆境防御反应[9, 10, 11, 12]。一些MYB转录因子也参与植物抗病反应, 例如拟南芥R2R3-MYB转录因子BOTRYTIS SUSCEPTIBLE1 (BOS1)参与寄主对灰葡萄孢菌(Botrytis cinerea)和甘蓝链格孢菌(Alternaria brassicicola)等病原菌的防御反应, 该基因突变体bos1对这些病原菌的敏感性增加[13]。过表达拟南芥R2R3-MYB转录因子基因AtMYB96能增强寄主的耐旱能力[14]和对假单胞杆菌(Pseudomonas syringae)的抗性[15]。大麦MYB转录因子基因HvMYB6参与大麦抗白粉病反应, 该基因沉默后使大麦对白粉病菌(Blumeria graminis)的敏感性增加, 而该基因过表达则增强转基因大麦的抗病性[16]。本实验室克隆了中间偃麦草(Thinopyrum intermedium)的R2R3-MYB基因TiMYB2R-1, 该基因超强表达后显著提高了转基因小麦对全蚀病菌(Gaeumannomyces graminisvar.tritici)的抗性[17]。Al-Attala等[18]通过沉默TaMYB4基因(小麦MYB转录因子基因), 降低了小麦植株对条锈病的抗性, 证实TaMYB4是抗条锈病反应的正向调控因子。Zhang等[19]通过VIGS (virus-induced gene silencing)沉默川农19中的TaLHY基因, 使该小麦品种对条锈病的抗性降低, 而且还无法完成抽穗, 表明TaLHY不仅控制小麦抽穗, 而且参与对条锈病的防御反应。

本实验室通过基因芯片分析, 发现一个响应纹枯病菌和根腐病菌侵染的MYB基因TaMYB86(GenBank登录号为KM066946)。为解析TaMYB86的抗病功能, 我们构建了TaMYB86过表达的转基因载体pUbi:MYC-TaMYB86, 用基因枪介导法将其转入小麦商业品种扬麦16中, 创制了TaMYB86过表达的转基因小麦, 并对其T1至T3代进行分子检测与抗病性鉴定, 获得了抗根腐病的转基因小麦新种质。

1 材料与方法
1.1 材料

转基因受体品种扬麦16由江苏里下河农业科学院研究所程顺和课题组提供, 小麦根腐病菌ACC30209由中国农业科学院作物科学研究所李洪杰研究员提供, 含有c-MYC标签的转基因载体质粒pAHC25-MYC由本实验室对转基因载体质粒pAHC25改造和保存[20, 21]。

1.2 pUbi:MYC-TaMYB86转基因表达载体的构建

根据TaMYB86 (GenBank登录号为KM066946)的ORF序列, 设计1对特异引物(TaMYB86-P25-F: (5′ - ATACTAGTATGGGACGTCCGTCGTCC-3′ , 下画线标示SpeI识别位点; TaMYB86-P25-R: 5′ -TCAGAA GTATGGTTCCAATT-3′ ), 利用PrimeSTAR HS DNA Polymerase和PCR扩增法, 在ORF完整序列上游引入SpeI 酶切位点, 然后用Spe I酶切扩增产物, 回收目的片段; 再用SpeI、EcoI CRI (与SacI识别酶切序列一致, 为同裂酶, 但EcoI CRI酶切后产生平末端)酶切单子叶植物表达载体pAHC25-MYC, 回收载体骨架, 并连接, 构建成TaMYB86过表达转化载体pUbi:MYC-TaMYB86 (图1)。通过测序分析确定构建的基因表达载体的正确性。在转化载体中, TaMYB86基因上游与6个MYC标签序列相连, 转录由玉米泛素(ubiquitin, Ubi)启动子驱动, 被农杆菌胭脂碱合酶终止子(Agrobacterium tumefaciensnopaline synthase, Tnos)终止, 该载体还含有1个Bar基因表达盒, 可为后续选择利用Bialaphos筛选转化再生植株提供抗性筛选标记。

图1 表达载体pUbi:MYC-TaMYB86的构建Fig. 1 Construction of expression vector pUbi:MYC-TaMYB86

1.3 转TaMYB86基因小麦植株的获得

采用徐惠君等[22]报道的基因枪介导法, 将构建好的pUbi:MYC-TaMYB86载体DNA与适量金粉混合, 转化小麦扬麦16幼胚愈伤组织, 经过分化、Bialaphos筛选、再生、移栽, 获得转基因小麦T0代植株。收获成活株中经PCR检测为阳性植株的种子, 单株播种, 获得T1代植株, 单株播种T1代转基因植株, 依此类推。

1.4 转基因小麦的PCR检测

在小麦四叶期, 从每个成活植株取1片叶, 采用CTAB法[23]提取基因组DNA。将该基因组DNA作为模板, 利用TaMYB86基因ORF的2条特异序列(TaMYB86-ZJF1: 5′ -TCCGAGAACCTGGGCTAC-3′ ; TaMYB86-ZJF2: 5′ -TTTTGATTTCAACTTGGAATT GG-3′ )分别作为上游引物, 以表达载体Tnos特异序列(Tnos-R: 5′ -AAAACCCATCTCATAAATAACG-3′ )作为下游引物, 对转TaMYB86基因T0~T3代植株进行巢式PCR检测。以转基因质粒pUbi:MYC-TaMYB86为阳性对照, 以未转基因扬麦16的基因组DNA为阴性对照, 预期扩增产物片段为281 bp。第1轮扩增体系含2× EcoTaq PCR SuperMix (全式金公司) 12.5 µ L、上游引物TaMYB86-ZJF1 (10 µ mol L-1)、下游引物Tnos-R (10 µ mol L-1)各1 µ L、模板DNA 100 ng、

补ddH2O至25 µ L。扩增程序为94℃ 5 min; 30× (94℃ 45 s, 61℃ 30 s, 72℃ 30 s), 72℃ 5 min, 16℃保存。然后, 利用引物对TaMYB86-ZJF2/Tnos-R进行第2轮扩增, 反应体系成分和浓度与第1轮扩增基本相同, 仅将上游引物换成TaMYB86-ZJF2, 模板变为第1轮PCR产物的50倍稀释液1.0 μ L。扩增程序为94℃ 5 min; 30× (94℃ 45 s, 48℃ 30 s, 72℃ 30 s), 72℃ 5 min, 16℃保存。扩增产物用1.5%琼脂糖凝胶电泳进行检测, 紫外照相, 记录结果。

1.5 转基因小麦株系中TaMYB86及防卫基因的转录水平

用TRIZOL试剂盒(Invitrogen)提取转TaMYB86基因小麦植株接种基部茎部总RNA。用DNase I (大连宝生物)去除基因组DNA。利用FastQuant cDNA第一链合成试剂盒(天根生化)合成第1链cDNA。

以合成的cDNA为模板, 用SuperReal 荧光定量预混试剂增强版(天根生化)在ABI PRISMR 7500实时荧光定量PCR仪(ABI, 美国)上进行qRT-PCR分析。反应体系含2× SuperReal PreMix Plus 12.5 µ L、上游引物(10 µ mol L-1)、下游引物(10 µ mol L-1)各0.75 µ L、cDNA模板5.0 µ L、50× ROX Reference Dye 0.50 µ L, 补RNase-free ddH2O至25 µ L。扩增条件为95℃预变性15 min; 95℃变性10 s, 56℃退火20 s, 72℃延伸32 s, 40个循环。以小麦肌动蛋白基因TaActin (TaAct-A: 5′ -CACTGGAATGGTCAAGGCTG-3′ ; TaAct- B: 5′ -CTCCATGTCATCCCAGTTG-3′ )为内参基因。TaMYB86定量扩增的上游引物为TaMYB86-QF (5′ -TCCGAGAACCTGGGCTACG-3′ ), 下游引物为TaMYB86-QR (5′ -CGAGGAGGCTCTGTTCTTGG-3′ )。用2-Δ Δ Ct法[24]计算目标基因的相对表达量, Δ CT = CTTaMYB86 - CTTaActin, Δ Δ CT = Δ CT试验样品 - Δ CT基准样品。每个反应均有3次独立的重复实验。

转录因子TaMYB86的3个下游防卫基因为PR10(CA613496)、PR17c (TA65181)和Chit1 (CA665185), 其上、下游引物对依次是PR10-Q-F (5′ -CGTGGAG GTAAACGATGAG-3′ ) / PR10-Q-R (5′ -GCTAAGTG TCCGGGGTAAT-3′ )、PR17c-Q-F (5′ -ACGACATCAC GGCGAGGT-3′ ) / PR17c-Q-R (5′ -CACGGGGAAAG AGAGGATGA-3′ )和Chit1-Q-F (5′ -ATGCTCTGGGA CCGATACTT-3′ ) / Chit1-Q-R (5′ -AGCCTCACTTTG TTCTCGTTTG-3′ )。利用上述qRT-PCR方法, 分析TaMYB86过表达转基因小麦株系中这3个防卫基因的转录水平。

1.6 转TaMYB86基因株系的Western杂交分析

以3个转基因株系中抗根腐病的T3代植株接种基部茎秆为材料, 提取总蛋白, 在液氮中充分研磨, 加入蛋白质提取液混匀。提取液含62.5 mmol L-1 Tris-HCl (pH 7.4)、10%甘油、0.1% SDS、2 mmol L-1 Na2EDTA、1 mmol L-1 PMSF (phenyl methane sulfonyl fluoride)和5% β -巯基乙醇。将上述混合液置于冰上10 min后, 4℃下13 400 × g离心20 min, 取含总蛋白的上清液进行SDS-PAGE, 经湿转法转至PVDF膜上, 与稀释900倍的anti-c-MYC 抗体(一抗, 北京全式金生物技术有限公司)杂交, 用TBST将杂交膜洗干净后, 与稀释1000倍的ProteinFind Goat Anti-Mouse IgG(H+L)抗体(二抗, 北京全式金生物技术有限公司)第二次杂交, 化学发光后曝光显影, 检测c-MYC-TaMYB86融合蛋白的表达。

1.7 小麦抗根腐病鉴定

在煮熟的麦粒上培养平脐蠕孢菌, 麦粒表面长满菌丝后待用。在小麦分蘖盛期, 用消毒镊子夹取长满菌丝的麦粒, 放入麦苗根基部, 每株放4~5粒, 保湿3~5 d[25]。

收获时按单株鉴定根腐病严重程度, 按5级标准[25]划分小麦根腐病的病级(infection type, IT), 其中, 0级为全株无病; 1级为叶鞘有少量病斑, 叶鞘病斑面积小于总面积的1/4; 2级为病菌侵入茎秆, 茎杆病斑面积介于1/4~1/2之间; 3级为茎杆病斑面积介于1/2~ 3/4之间; 4级为茎杆病斑面积大于3/4, 茎秆已软腐。

式中, DI为病情指数(disease index), Xi为第i病级的株数。

2 结果与分析
2.1 转TaMYB86基因小麦的获得与PCR检测

由图1和测序结果可知, 载体pUbi:MYC- TaMYB86构建成功, TaMYB86的插入位置和方向均正确。

用基因枪介导法将构建好的pUbi:MYC- TaMYB86载体DNA与适量金粉混合轰击小麦扬麦16的幼胚愈伤组织1200块, 经过分化、Bialaphos筛选、再生、移栽, 获得转基因小麦28株T0代植株。利用特异引物的PCR检测表明, 有5株阳性植株, 转化率0.42%。以种子数量足够多的3个抗根腐病的转基因小麦株系(MO86-7、MO86-14和MO86-28) T0~T3代植株叶片基因组DNA作为模板, 利用转基因特异的引物对转基因小麦T0至T3代植株PCR检测表明, 3个转基因株系(MO86-7、MO86-14和MO86-28)中均能检测到外源TaMYB86 (图2), 说明导入的TaMYB86在这3个转基因小麦株系中能稳定遗传。

图2 转基因小麦T0~T3植株外源TaMYB86的PCR检测
P: 转基因表达载体pUbi:MYC-TaMYB86质粒; WT: 未转基因扬麦16; MO86-7, MO86-14, MO86-28: TaMYB86过表达株系。Fig. 2 PCR patterns of TaMYB86 in T0-T3 plants of TaMYB86transgenic wheat lines
P: pUbi:MYC-TaMYB86 plasmid as the positive control; WT: non-transformed wheat (recipient) Yangmai 16; MO86-7, MO86-14, MO86-28: 3 TaMYB86transgenic wheat lines.

2.2 转基因小麦中TaMYB86的转录水平分析

qRT-PCR分析结果表明, TaMYB86在3个抗根腐病的转基因小麦株系中的表达量均显著高于未转基因扬麦16, 约为未转基因扬麦16的5~6倍(图3), 说明这3个抗根腐病的转基因小麦株系中TaMYB86基因能够超量表达。

图3 过表达株系中TaMYB86的转录水平分析
WT: 未转基因扬麦16; MO86-7、MO86-14、MO86-28: TaMYB86过表达株系。每个样本进行3次重复实验, 采用t检验分析差异显著性; * * P < 0.01概率水平极显著。Fig. 3 qRT-PCR analysis of TaMYB86transcriptional levels in transgenic wheat lines
WT: wild type, non-transformed Yangmai 16; MO86-7, MO86-14, MO86-28: TaMYB86transgenic wheat lines. Three biological
replicates for each line were averaged and statistically treated
(t-test; * * P < 0.01).

2.3 TaMYB86受根腐菌的诱导表达分析

利用qRT-PCR方法, 分析接种根腐菌后不同时间点未转基因扬麦16与转基因株系中TaMYB86基因的表达水平。结果表明, 在未转基因扬麦16和过表达转基因小麦中, TaMYB86的转录水平均受根腐菌侵染的诱导; 与未转基因扬麦16相比, 转基因小麦中TaMYB86的转录水平更高, 并且更快达到峰值, 说明过表达的TaMYB86能更快、更强地响应根腐菌的侵染, 从而增强小麦对根腐菌的防御(图4)。

图4 根腐病菌接种后TaMYB86的表达模式
Y16: 未转基因扬麦16; MO86: TaMYB86过表达株系MO86-7、MO86-14、MO86-28的混合样品。每个样本进行3次重复实验。Fig. 4 Transcriptional patterns of TaMYB86 in B. sorokiniana-inoculated wheat
Y16: non-transformed Yangmai 16; MO86: pooled samples of transgenic wheat lines MO86-7, MO86-14, and MO86-28.

2.4 转TaMYB86基因小麦中c-MYC-TaMYB86融合蛋白的Western杂交分析

在转基因载体pUbi:MYC-TaMYB86中TaMYB86上游与c-MYC标签序列相连, 便于对转基因植株中翻译表达的c-MYC-TaMYB86融合蛋白进行Western杂交分析。结果显示, 3个转TaMYB86基因小麦株系中蛋白可与anti-c-MYC抗体产生杂交条带, 而未转基因扬麦16 (WT)不能杂交出条带, 表明c-MYC- TaMYB86基因可在3个转基因阳性株系(MO86-7、MO86-14、MO86-28)中翻译表达(图5)。

2.5 TaMYB86的过表达增强了转基因小麦对根腐病的抗性

根腐病抗性鉴定结果(表1)显示, 与未转基因扬麦16 (WT)相比, 3个过表达TaMYB86的转基因小麦株系(MO86-7、MO86-14和MO86-28)的病级极显著降低(t-test; P < 0.01)。T1代过表达株系的平均病级分别为1.27、2.00和1.80, 而未转基因扬麦16平均病级为3.00; T2代过表达株系的平均病级为1.51、1.50和1.54, 而未转基因扬麦16病级为2.17; T3代3个过表达株系的平均病级分别为1.67、1.25和1.50, 而未转基因扬麦16病级为2.62。转TaMYB86基因的过表达株系各代间的病级有所不同, 但每代转基因植株的抗性均极显著高于未转基因扬麦16 (WT), 说明TaMYB86过表达显著增强了转基因小麦对根腐病的抗性。

2.6 TaMYB86正向调控小麦下游防卫基因的转录水平

为了解析TaMYB86在小麦防御根腐病菌侵染的机制, 用qRT-PCR方法分析了接种根腐病菌47 d的TaMYB86过表达株系中3个下游防卫基因(PR10、PR17c和Chit1)的转录水平。这3个基因是PR10、PR17c和Chit1。正如图6所示, 与未转基因扬麦16相比, 3个下游防卫基因在TaMYB86过表达株系中的转录水平显著升高, 且下游基因的表达模式TaMYB86相同。以上结果证明, TaMYB86可以正向调控小麦中下游防卫基因的表达。

图5 转TaMYB86基因过表达株系和未转基因扬麦16的Western杂交分析
WT: 未转基因扬麦16; MO86-7, MO86-14, MO86-28: TaMYB86过表达株系。箭头指示MYC-TaMYB86融合蛋白的位置。Fig. 5 Western blot pattern of TaMYB86-overexpressed transgenic lines and non-transformed Yangmai 16 using an anti-MYC antibody
WT: wild type, non-transformed Yangmai 16; MO86-7, MO86-14, MO86-28: TaMYB86transgenic wheat lines. The arrow shows the position of MYC-TaMYB86 fusion protein.

表1 转TaMYB86基因过表达株系与未转基因扬麦16的根腐病抗性鉴定 Table 1 Common root rot response of TaMYB86-overexpressed transgenic lines and non-transformed wheat Yangmai 16
3 讨论

植物MYB转录因子在发育及抵抗生物或非生物逆境方面发挥重要作用, 一些植物MYB转录因子受病原物诱导表达, 从而激活下游基因来参与抗病反应。拟南芥AtMYB30受黄单胞菌(Xanthomonas campestrispv. campestris)和假单胞杆菌(Pseudomonas syringae pv.tomato)诱导表达[26], 并参与过敏性反应(hypersensitive response, HR), 过表达AtMYB30增强了寄主的HR表型及对假单胞杆菌的抗性[27]。拟南芥BOS1 (AtMYB108)受灰葡萄孢菌和甘蓝链格孢菌诱导表达, bos1突变体对这些病原菌和渗透胁迫的敏感性增加[13]。本研究中TaMYB86是受根腐病菌诱导表达的R2R3-MYB转录因子基因。

为了解析TaMYB86的防御功能, 本研究构建了该基因的过表达载体pUbi:MYC-TaMYB86, 并将其成功转入小麦商业品种扬麦16中, 获得5个转基因小麦株系。选取其中种子数量多的3个株系进行分子检测和抗病鉴定。结果说明, TaMYB86基因成功转入3个转基因小麦株系中, 并可稳定转录、过量表达, TaMYB86过表达的转基因小麦植株对根腐病抗性显著提高, 说明TaMYB86在小麦防御根腐病过程中起正向调控作用。但是TaMYB86是否是小麦防御根腐病反应所必需的基因, 以及是否有基因冗余现象, 还有待进一步验证。在植物中, 转录因子通过调控下游防卫基因表达来调节抗病防御反应[28, 29, 30, 31, 32]。一些防卫基因过表达可以显著提高转基因植物的抗病性。例如, 过表达大麦几丁质酶基因、萝卜防御素基因RsAFP2或者小麦脂转运蛋白均可增强转基因植物对病原菌的防御[33, 34, 35]。本研究对防卫基因表达的初步分析表明, 与未转基因扬麦16 (受体)相比, 抗根腐病的TaMYB86过表达转基因小麦中3个防卫基因的表达量显著提高, 说明TaMYB86可能通过调控其下游防卫基因的表达来增强根腐病抗性, 但TaMYB86作用的深入机制还有待进一步阐明。

图6 TaMYB86过表达株系中TaMYB86和下游防卫基因的qRT-PCR分析
接种根腐病菌47 d后TaMYB86过表达株系和未转基因扬麦16 (WT)植株中TaMYB86和下游防卫基因的转录表达。防卫基因包括
PR10 (CA613496)、PR17c (TA65181)和Chit1 (CA665185)。采用t检验分析差异显著性; * * P < 0.01极显著。Fig. 6 qRT-PCR analysis of the relative transcript level of TaMYB86 and three defense genes in TaMYB86-overexpressing transgenic lines
The expression of TaMYB86 and three defense genes in TaMYB86-overexpressing transgenic and non-transformed wheat Yangmai 16 (WT) inoculated with B. sorokiniana for 47 d. Defense genes include PR10 (CA613496), PR17c (TA65181) and Chit1 (CA665185).
Three biological replicates of each line were averaged and statistically treated (t-test; * * P < 0.01).

4 结论

通过基因枪转化、分子检测和抗根腐病鉴定, 创制、筛选出抗根腐病的TaMYB86过表达转基因小麦新种质3份, 证明TaMYB86在小麦防御根腐病过程中起正向调控作用。

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
[1] Kumar J, Schäfer P, Heckelhoven R, Langen G, Baltruschat H, Stein E, Nagarajan S, Kogel K H. Bipolaris sorokiniana, a cereal pathogen of global concern: cytological and molecular approaches towards better control. Mol Plant Pathol, 2002, 3(4): 185-195 [本文引用:1]
[2] 贾廷祥, 吴桂本, 刘传德. 我国小麦根腐性病害研究现状及防治对策. 中国农业科学, 1995, 28: 41-48
Jia Y X, Wu G B, Liu C D. The present research situation and control countermeasure of root rots in wheat. Sci Agric Sin, 1995, 28: 41-48 (in Chinese with English abstract) [本文引用:1]
[3] Li H J, Conner R L, Chen Q, Li H Y, Laroche A, Graf R J, Kuzyk A D. The transfer and characterization of resistance to common root rot from Thinopyrum ponticum to wheat. Genome, 2004, 47: 215-223 [本文引用:2]
[4] Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis. Trends Plant Sci, 2010, 15: 573-581 [本文引用:2]
[5] Haga N, Kato K, Murase M, Araki S, Kubo M, Demura T, Suzuki K, Müller I, Voss U, Jürgens G. Ito M. R1R2R3-Myb proteins positively regulate cytokinesis through activation of KNOLLE transcription in Arabidopsis thaliana. Development, 2007, 134: 1101-1110 [本文引用:1]
[6] Zhou J, Lee C, Zhong R, Ye Z H. MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell, 2009, 21: 248-266 [本文引用:1]
[7] Millar A A, Gubler F. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development. Plant Cell, 2005, 17: 705-721 [本文引用:1]
[8] Muller D, Schmitz G, Theres K. Blind homologous R2R3 Myb genes control the pattern of lateral meristem initiation in Arabidopsis. Plant Cell, 2006, 18: 586-597 [本文引用:1]
[9] Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi- Shinozaki K. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 2003, 15: 63-78 [本文引用:1]
[10] Jung C, Seo J S, Han S W, Koo Y J, Kim C H, Song S I, Nahm B H, Choi Y D, Cheong J J. Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant Physiol, 2008, 146: 623-635 [本文引用:1]
[11] Dai X Y, Xu Y Y, Ma Q B, Xu W Y, Wang T, Xue Y B, Chong K. Overexpression of an R1R2R3 MYB gene, OsMYB3R-2, increases tolerance to freezing, drought, and salt stress in transgenic Arabidopsis. Plant Physiol, 2007, 143: 1739-1751 [本文引用:1]
[12] Su C F, Wang Y C, Hsieh T H, Lu C A, Tseng T H, Yu S M. A novel MYBS3-dependent pathway confers cold tolerance in rice. Plant Physiol, 2010, 153: 145-158 [本文引用:1]
[13] Mengiste T, Chen X, Salmeron J, Dietrich R. The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis. Plant Cell, 2003, 15: 2551-2565 [本文引用:2]
[14] Seo P J, Xiang F, Qiao M, Park J Y, Lee Y N, Kim S G, Lee Y H, Park W J, Park C M. The MYB96 transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis. Plant Physiol, 2009, 151: 275-289 [本文引用:1]
[15] Seo P J, Park C M. MYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis. New Phytol, 2010, 186: 471-483 [本文引用:1]
[16] Chang C, Yu D, Jiao J, Jing S, Schulze-Lefert P, Shen Q H. Barley MLA immune receptors directly interfere with antagonistically acting transcription factors to initiate disease resistance signaling. Plant Cell, 2013, 25: 1158-1173 [本文引用:1]
[17] Liu X, Yang L H, Zhou X Y, Zhou M P, Lu Y, Ma L J, Zhang Z Y. Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease. J Exp Bot, 2013, 64: 2243-2253 [本文引用:1]
[18] Al-Attala M N, Wang X, Abou-Attia M A, Duan X, Kang Z. A novel TaMYB4 transcription factor involved in the defence response against Puccinia striiformis f. sp. tritici and abiotic stresses. Plant Mol Biol, 2014, 84: 589-603 [本文引用:1]
[19] Zhang Z J, Chen J M, Su Y Y, Liu H M, Chen Y E, Luo P G, Du X G, Wang D, Zhang H Y. TaLHY, a 1R-MYB transcription factor, plays an important role in disease resistance against stripe rust fungus and ear heading in wheat. PLoS One, 2015, 10, doi: DOI:10.1371/journal.pone.0127723 [本文引用:1]
[20] Christensen A H, Quail P H. Ubiquitin promoter-based vectors for high-level expression of selectable and /or screenable marker genes in monocotyledonous plants. Transgenic Res, 1996, 5: 213-218 [本文引用:1]
[21] Zhu X L, Yang K, Wei X N, Zhang Q F, Rong W, Du L P, Ye X G, Qi L, Zhang Z Y. The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis. J Exp Bot, 2015, 66: 6591-6603 [本文引用:1]
[22] 徐惠君, 庞俊兰, 叶兴国, 杜丽璞, 李连城, 辛志勇, 马有志, 陈剑平, 陈炯, 程顺和, 吴宏亚. 基因枪介导法向小麦导入黄花叶病毒复制酶基因的研究. 作物学报, 2001, 27: 688-693
Xu H J, Pang J L, Ye X G, Du L P, Li L C, Xin Z Y, Ma Y Z, Chen J P, Chen J, Cheng S H, Wu H Y. Study on the gene transferring of Nib8 into wheat for its resistance to the Yellow mosaic virus by bombardment. Acta Agron Sin, 2001, 27: 688-694 (in Chinese with English abstract) [本文引用:1]
[23] Sharp P J, Kreis M, Shewry P R, Gale M D. Location of β-amylase sequences in wheat and its relatives. Theor Appl Genet, 1988, 75: 286-290 [本文引用:1]
[24] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods, 2001, 25: 402-408 [本文引用:1]
[25] Dong N, Liu X, Lu Y, Du L P, Xu H J, Liu H X, Xin Z Y, Zhang Z Y. Overexpression of TaPIEP1, a pathogen-induced ERF gene of wheat, confers host-enhanced resistance to fungal pathogen Bipolaris sorokiniana. Funct Integr Genomic, 2010, 10: 215-226 [本文引用:2]
[26] Daniel X, Lacomme C, Morel J B, Roby D. A novel myb oncogene homologue in Arabidopsis thaliana related to hypersensitive cell death. Plant J, 1999, 20: 57-66 [本文引用:1]
[27] Vailleau F, Daniel X, Tronchet M, Montillet J L, Triantaphylides C, Roby D. A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Proc Natl Acad Sci USA, 2002, 99: 10179-10184 [本文引用:1]
[28] Zhang H, Zhang D, Chen J, Yang Y, Huang Z, Huang D, Wang X C, Huang R. Tomato stress-responsive factor TSRF1 interacts with ethylene responsive element GCC box and regulates pathogen resistance to Ralstonia solanacearum. Plant Mol Biol, 2004, 55: 825-834 [本文引用:1]
[29] 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]
[30] Zhang Z Y, Liu X, Wang X D, Zhou M P, Zhou X Y, Ye X G, Wei X N. An R2R3 MYB transcription factor in wheat, TaPIMP1, mediates host resistance to Bipolaris sorokiniana and drought stresses through regulation of defense- and stress-related genes. New Phytol, 2012, 196: 1155-1170 [本文引用:1]
[31] Liu X, Yang L H, Zhou X Y, Zhou M P, Lu Y, Ma L J, Ma H X, Zhang Z Y. Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease. J Exp Bot, 2013, 64: 2243-2253 [本文引用:1]
[32] Zhu X L, Qi L, Liu X, Cai S B, Xu H J, Huang R F, Li J R, Wei X N, Zhang Z Y. The wheat ethylene response factor transcription factor pathogen-induced ERF1 mediates host responses to both the necrotrophic pathogen Rhizoctonia cerealis and freezing stresses. Plant Physiol, 2014, 164: 1499-1514 [本文引用:1]
[33] Shin S, Mackintosh C A, Lewis J, Heinen S J, Radmer L, Dill-Macky R, Baldridge G D, Zeyen R J, Muehlbauer G J. Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum. J Exp Bot, 2008, 59: 2371-2378 [本文引用:1]
[34] Li Z, Zhou M P, Zhang Z Y, Ren L J, Du L P, Zhang B Q, Xu H J, Xin Z Y. Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis. Funct Integr Genomics, 2011, 11: 63-70 [本文引用:1]
[35] Zhu X L, Li Z, Xu H J, Zhou M P, Du L P, Zhang Z Y. Overexpression of wheat lipid transfer protein gene TaLTP5 increases resistances to Cochliobolus sativus and Fusarium graminearum in transgenic wheat. Funct Integr Genomics, 2012, 12: 481-488 [本文引用:1]