CN110183525B - 小麦抗条锈病相关的txr蛋白及其编码基因与应用 - Google Patents
小麦抗条锈病相关的txr蛋白及其编码基因与应用 Download PDFInfo
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- CN110183525B CN110183525B CN201910514241.0A CN201910514241A CN110183525B CN 110183525 B CN110183525 B CN 110183525B CN 201910514241 A CN201910514241 A CN 201910514241A CN 110183525 B CN110183525 B CN 110183525B
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Abstract
本发明涉及小麦抗病领域，具体的涉及一种培育条锈病抗性降低的转基因植物的方法，包括将抑制TXR核苷酸转录的DNA片段导入受体植物中，得到转基因植物的步骤；所述转基因植物的条锈病抗性低于所述受体植物。通过病毒诱导的基因沉默和CRISPR/Cas9基因编辑技术，降低TXR基因在小麦叶片中的表达，使抗条锈病的小麦品种水原11对无毒条锈菌小种CYR17的侵染，反应型由0‑1级变为3‑4级。由此证明小麦的TXR基因参与了小麦对条锈菌的抗性调控。
Description
技术领域
本发明属于生物技术领域，具体涉及一种与小麦抗条锈病相关的TXR蛋白及其编码基因与应用。
背景技术
小麦条锈病是由小麦条锈菌侵染引起的一种世界性真菌病害。小麦条锈菌属于柄锈菌属的条形柄锈菌小麦专化型(Pucciniastriiformis f.sp.tritici)(Zhao et al.,2016)，是一种活体寄生真菌。
条锈病是危害中国及世界小麦生产的严重病害。尽管化学药剂可以有效控制小麦病害造成的经济损失，但出于对环境安全和人类健康的考虑，培育并推广抗病品种依然被认为是最根本、最经济和最安全的途径(Line and Chen,1995；Xu et al.,2013)。抗病基因的克隆与功能研究对于小麦抗病分子机制的研究和进行分子抗病育种具有十分重要的意义。
发明内容
本发明的目的是提供一种小麦条锈病抗性相关的基因及其编码蛋白。
一种培育条锈病抗性降低的转基因植物的方法，包括将抑制TXR基因转录的物质导入受体植物中，得到转基因植物的步骤；所述转基因植物的条锈病抗性低于所述受体植物。
其中，所述TXR蛋白具有如序列7或序列8或序列9所述的氨基酸序列。
其中，所述TXR蛋白的编码基因具有如序列1或序列2或序列3所述的DNA序列。
其中，所述抑制TXR基因转录的物质为：BSMV病毒载体α、BSMV病毒载体β和γ-TXR,所述γ-TXR为γ-TXR-1或γ-TXR-2，所述γ-TXR-1为将序列表中的序列2自5’末端起第109-314位核苷酸的序列按基因表达的相反方向插入BSMV-VIGS病毒载体γ链的NheI酶切位点处，且保持BSMV-VIGS病毒载体γ链的其它序列不变的得到的TXR基因沉默载体,所述γ-TXR-1为将序列表中的序列2自5’末端起第1623-1795位核苷酸的序列按基因表达的相反方向插入BSMV-VIGS病毒载体γ链的NheI酶切位点处，且保持BSMV-VIGS病毒载体γ链的其它序列不变的得到的TXR基因沉默载体。。
其中，利用CRISPR/Cas9基因编辑系统实现抑制受体植物中所述的抑制TXR基因转录，从而实现抑制受体植物中所述TXR蛋白质的含量和/或活性。
培育条锈病抗性提高的转基因植物的方法，包括提高受体植物中TXR蛋白的表达量和/或活性，得到转基因植物的步骤；所述转基因植物的条锈病抗性高于所述受体植物。
其中，所述TXR蛋白具有如序列7或序列8或序9所述的氨基酸序列。
其中，所述TXR蛋白的编码基因具有如序列1或序列2或序列3所述的DNA序列。
其中，所述转基因植物的条锈病抗性高于所述受体植物体现在：转基因植物的条锈病菌孢子形成或扩展低于受体植物。
其中，所述受体植物为单子叶植物，所述单子叶植物具体为小麦。
本发明所提供的小麦条锈病抗性相关的蛋白，命名为TXR，来源于普通小麦(Triticumaestivum L.)品种水原11，其核苷酸序列与氨基酸序列示于序列表中序列1-3和序列7-9。该基因位于小麦的第三部分同源群染色体上，并分别将来自3A、3B和3D染色体上的TXR基因序列暂命名为TXR-3A-Suwon11、TXR-3B-Suwon11和TXR-3D-Suwon11。通过病毒诱导的基因沉默和CRISPR/Cas9基因编辑技术，降低TXR基因在小麦叶片中的表达，使抗条锈病的小麦品种水原11对无毒条锈菌小种CYR17的侵染，反应型由0-1级变为3-4级。由此证明小麦的TXR基因参与了小麦对条锈菌的抗性调控。
附图说明
图1为从小麦品种水原11基因组中克隆获得到的3个TXR基因拷贝的KASP标记分型对3个基因拷贝的染色体定位分析。A、B和C分别代表162、643和1245bp位置处的KASP标记分型结果。TXR1、TXR2和TXR3分别代表以以水原11基因组DNA为模板，扩增得到的TXR基因的三个拷贝序列，CS为中国春。红色的三角形表示VIC荧光基团，位于X轴，蓝色的圆形表示FAM荧光基团，位于Y轴，绿色的正方形表示杂合型。×表示分型不明确。
图2为BSMV-VIGS下调TXR基因表达后抗条锈病普通小麦品种水原11叶片中TXR基因的相对表达量。图示沉默片段TXR-1和TXR-2均可极显著下调TXR基因的表达(“**”代表P≤0.01)。Mock：模拟接种的空白对照植株；BSMV:GFP：转GFP基因的负对照植株；BSMV:TXR-1和BSMV:TXR-2分别代表利用沉默片段TXR-1和沉默片段TXR-2下调TXR基因表达的植株。
图3为下调TXR基因表达的水原11植株失去对条锈菌小种CYR17的抗性，沦为高感(反应型为3～4)。Mock：模拟接种的空白对照植株；BSMV:GFP：转GFP基因的负对照植株；BSMV:TXR-1和BSMV:TXR-2分别代表利用沉默片段TXR-1和沉默片段TXR-2下调TXR基因表达的植株。
图4为BSMV-VIGS下调TXR表达后水原11叶片产生H2O2的细胞数目减少A：条锈菌小种CYR17侵染的水原11叶片，箭头示积累活性氧的气孔保卫细胞。B：BSMV-VIGS降低TXR在水原11中的表达后，CYR17侵染时积累活性氧的气孔保卫细胞数量显著减少。C：BSMV-VIGS降低TXR在水原11中的表达后，CYR17侵染时产生活性氧的气孔保卫细胞数量统计结果，与对照Mock和BSMV:GFP相比显著减少。
图5为CRISPR/Cas9系统中针对TXR基因的sgRNA活性检测。b1表示靶标TXR基因的sgRNA。-T7E1表示分别以pTaU6-b1载体质粒(b1)转化的原生质体DNA为模板和野生型(WT)原生质体DNA为模板进行PCR扩增得到的TXR基因片段，经变性和复性后未加T7E1酶处理。+T7E1表示分别以pTaU6-b1载体质粒(b1)转化的原生质体DNA为模板和野生型(WT)原生质体DNA为模板进行PCR扩增得到的TXR基因片段，经变性和复性后加T7E1酶处理。红色箭头示突变条带。
图6为TXR基因编辑植株的抗条锈病鉴定及其突变类型的测序检测结果。A：TXR基因通过CRISPR/Cas9系统被编辑后，在T1代植株叶片中表达量显著降低。B：TXR基因被编辑突变后，水原11植株失去对条锈菌生理小种CYR17的抗性，沦为高感。C：T1代植株突变类型的测序检测结果。CK：对照植株。38-1和38-4分别表示编号为38的T0代植株自交获得的两个不同的T1代单株。38-1-3A：TXR-3A-Suwon11在38-1株系中的突变类型。38-1-3B：TXR-3B-Suwon11在38-1株系中的突变类型。38-1-3D：TXR-3D-Suwon11在38-1株系中的突变类型。38-4-3A：TXR-3A-Suwon11在38-4株系中的突变类型。38-4-3B：TXR-3B-Suwon11在38-4株系中的突变类型。38-4-3D：TXR-3D-Suwon11在38-4株系中的突变类型。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的定量实验，均设置3次重复，结果取平均值。
下述实施例中的BSMV-VIGS病毒载体(包括α、β和γ质粒)和GFP(BSMV:GFP)质粒在文献“Holzberg S.,Brosio P.,Gross C.,Pogue G.Barley stripe mosaic virus-induced gene silencing in a monocot plant.Plant J.,2002,30:315-327.”中公开过，公众可从中国科学院遗传与发育生物学研究所获得。
下述实施例中的条锈菌(Blumeriagraminis f.sp.tritici)生理小种CYR17在文献“Huanbin Zhou,Shaofang Li,Zhiyong Deng,Xianping Wang,Tao Chen,JinsongZhang,Shouyi Chen,Hongqing Ling,Aimin Zhang,Daowen Wang and XiangqiZhang.Molecular analysis of three new receptor-like kinase genes fromhexaploid wheat and evidence for their participation in the wheathypersensitive response to stripe rust fungus infection.The Plant Journal，2007，52:420-434”中公开过，公众可从中国科学院遗传与发育生物学研究所获得。
下述实施例中的小麦品种水原11(Triticumaestivum var.Suwon 11)在文献“杨作民。小麦对条锈病抗性遗传的研究。作物学报，1981，7(2)：81-90。”中公开过。公众可从中国科学院遗传与发育生物学研究所获得。
下述实施例中的质粒pTaU6和pJIT163-2NLSCas9在文献“Qiwei Shan,YanpengWang,Jun Li,Caixia Gao Genome editing in rice and wheat using the CRISPR/Cassystem.Nature Protocols,2014,9(10):2395-2410.”中公开过。小麦幼胚转化由转化平台完成。公众可从中国科学院遗传与发育生物学研究所获得。
下述实施例中的GKP Buffer含有50mM甘氨酸(glycine)、30mM K2HPO4(pH9.2)、1％(质量百分比)膨润土(bentonite)和1％(质量百分比)硅藻土(celite)。
实施例一、TXR基因cDNA和DNA序列的克隆
1、小麦cDNA和基因组序列的获得
1)小麦基因组DNA和总RNA的提取
取0.2g新鲜小麦叶片，放入装有钢珠的2mL离心管中，用液氮速冻后，在组织研磨仪中将材料研磨成粉末。加入0.8mL裂解液(1％SLS,417mMTris/HCl pH8.0,417mMNaCl,83mM EDTA)，剧烈震荡，涡旋充分混匀，冰上静置10min，使细胞充分裂解。向离心管中加入与裂解液等体积的提取液(Tris饱和酚/氯仿/异戊醇＝25:24:1)，缓慢摇匀至乳浊液，冰上(或室温)静置10min，12000rpm离心10min，将上清液移至新的离心管中。重复此操作一次。吸取上清液至另一个1.5mL离心管，加入上清液0.6倍体积的预冷异丙醇，充分混匀，于-20℃沉淀30min。4℃，10000rpm离心10min后，弃上清，加入1mL 75％乙醇洗涤沉淀两次，每次4℃，10000rpm离心5min后弃上清。然后用无水乙醇洗涤，离心后弃上清，倒置离心管，室温干燥DNA。向干燥的DNA中加入100μL的TE缓冲液，待DNA充分溶解后按1/1000的比例加入RNaseA，消解RNA。用NanoDrop微量核酸蛋白分析仪测定DNA样品的浓度与纯度，用1％的琼脂糖凝胶电泳鉴定DNA的完整性。
采用RNA提取试剂盒提取普通小麦品种水原11的总RNA。RNA提取试剂盒购自PEXBIO公司(货号：A010400)，具体的提取操作步骤和条件要求按照产品说明书进行。
2)RNA的反转录
利用Takara公司的反转录试剂盒(货号：RR047A)对上述步骤1)中获得的小麦叶片总RNA进行反转录，获得cDNA。具体的操作步骤和条件要求按照产品说明书进行。
2、TXR基因DNA和cDNA序列的PCR扩增与测序
以上述步骤1中获得的DNA为模板，利用引物对QCTXRF:5'-TTATGGTRATGAAGATGGAG-3'和QCTXRR2:5'-CACCATGTTCATGGCACGA-3'，以水原11(抗条锈菌生理小种CYR17)基因组DNA为模板进行PCR扩增。
PCR反应体系：DNA模板0.5μL(<0.5μg)，dNTP(2.5mM)4μl，QCTXRF(10μM)1μl，QCTXRR2(10μM)1μl，5×TransStartFastPfu Buffer 10μl，TransStartFastPfu DNAPolymerase 1μl(2.5U)，最后用水补足至50μl。PCR反应程序：95℃预变性2min；然后95℃变性20s，58℃退火20s，72℃延伸40s，35个循环；最后72℃延伸5min。
将上述PCR扩增产物经1％琼脂糖凝胶分离纯化后连接到
-Blunt ZeroCloning Vector(Transgen CB501)载体上，得到重组质粒pEASY-T-TXR，转化大肠杆菌感受态细胞DH5α，并用通用引物M13F对细菌单克隆进行一代测序。
大量测序结果表明，PCR扩增得到的长度为1857bp的DNA序列即为TXR基因全长序列，位于小麦的三个不同染色体上的三个不同的基因拷贝分别命名为TXR-3A-Suwon11、TXR-3B-Suwon11和TXR-3D-Suwon11。TXR基因三个拷贝的基因组DNA全长序列分别为序列表中的序列1、序列2和序列3。
以上述步骤1中获得的cDNA为模板，利用引物对QCTXRF:5'-TTATGGTRATGAAGATGGAG-3'和QCTXRR2:5'-CACCATGTTCATGGCACGA-3'，以水原11(抗条锈菌生理小种CYR17)cDNA为模板进行PCR扩增。
PCR反应体系：cDNA模板0.5μL，dNTP(2.5mM)4μl，QCTXRF(10μM)1μl，QCTXRR2(10μM)1μl，5×TransStartFastPfu Buffer 10μl，TransStartFastPfuDNA Polymerase 1μl(2.5U)，最后用水补足至50μl。PCR反应程序：95℃预变性2min；然后95℃变性20s，58℃退火20s，72℃延伸40s，35个循环；最后72℃延伸5min。
将上述PCR扩增产物经1％琼脂糖凝胶分离纯化后连接到
-Blunt ZeroCloning Vector(Transgen CB501)载体上，得到重组质粒pEASY-T-TXR，转化大肠杆菌感受态细胞DH5α，并用通用引物M13F对细菌克隆进行一代测序。
大量测序结果表明，PCR扩增得到的长度为1857bp的cDNA序列即为TXR基因的编码区全长序列，并将其对应的位于三个不同染色体的三个不同的基因拷贝分别命名为TXR-3A-Suwon11、TXR-3B-Suwon11和TXR-3D-Suwon11。TXR基因三个拷贝的基因编码区全长序列分别为序列表中的序列4、序列5和序列6。相应地将TXR基因编码的蛋白分别命名为TXR-3A-Suwon11、TXR-3B-Suwon11和TXR-3D-Suwon11蛋白。TXR蛋白由618个氨基酸残基组成，其氨基酸序列即为序列表中的序列7-9。
实施例二、利用KASP标记对TXR基因进行染色体定位
1、KASP分子标记引物设计与合成
1)将实施例一中从小麦基因组DNA中扩增获得的3个TXR基因拷贝，进行同源比对，分别选择162(A/G)、643(G/A)和1245(C/T)bp位置的SNP位点进行引物设计。
2)将选定SNP位点及其上游19nt分别设计为两条正向引物，再在两条正向引物的5'端加FAM或VIC荧光报告基团标签序列，其中FAM标签序列为5'-GAAGGTGACCAAGTTCATGCT-3'和VIC标签序列为5'-GAAGGTCGGAGTCAACGGATT-3'。再选定SNP位点下游合适位置选取20nt左右的核苷酸序列作为反向引物，使最终PCR扩增获得的片段长度为50～80bp。反向引物不加标签。三个SNP位点的KASP标记引物分别为：
SNP162F1:5'-gaaggtgaccaagttcatgctCTGAGGGTCTTGTTGCTGCA-3'、
SNP162F2:5'-gaaggtcggagtcaacggattCTGAGGGTCTTGTTGCTGCG-3'、
SNP162R:5'-GCAGTTTATTTTGCAAGTCCACA-3'、
SNP643F1:5'-gaaggtgaccaagttcatgctCACCAGTCTCAAATGGCACTAG-3'、
SNP643F2:5'-gaaggtcggagtcaacggattCACCAGTCTCAAATGGCACTAA-3'、
SNP643R:5'-CTTGCAAGCACAGCAGGATT-3'、
SNP1245F1:5'-gaaggtgaccaagttcatgctACCAGTTCCAAGTCTGGTGTC-3'、
SNP1245F2:5'-gaaggtcggagtcaacggattACCAGTTCCAAGTCTGGTGTT-3'和
SNP1245R:5'-TCCATCAAGAACAGCTGCTCT-3'。
3)上述所有KASP标记引物均由北京华大基因股份有限公司合成。
2、模板DNA准备
按照实施例一中基因组DNA提取方法，分别提取中国春缺体-四体系N3AT3B、N3BT3D和N3DT3A的基因组DNA。用天根公司生产的质粒小提试剂盒(货号：DP103-03)分别提取实施例一中包含TXR基因序列全长的3个大肠杆菌单克隆的质粒DNATXR1、TXR2和TXR3。
3、KASP标记分型及染色体定位
1)上述3个SNP位点的KASP标记引物分别加ddH2O溶解，使浓度为100μM，然后按两个上游引物分别加12μL，下游引物加30μL，最后再加46μL ddH2O，混合均匀，配制成引物mix。
2)10μL PCR反应体系中加入5μL2×KASP Master mix，0.14μL引物mix，0.1μg DNA模板，最后加ddH2O至10μL，混合均匀。PCR扩增程序为94℃预变性15min；94℃变性20s，61-55℃退火/延伸60s，每循环一次降0.6℃，共10个循环；94℃变性20s，55℃退火/延伸60s，共26个循环。
3)PCR反应结束后，于ABI7500荧光定量PCR仪中，25-30℃，10min，一个循环，读取荧光数据，进行基因分型。
4)上述3个SNP位点的基因分型结果分别为附图中图1的A、B和C图。根据A图可以判断TXR2定位于3B染色体，根据B图可以判断TXR1定位于3A染色体，根据C图可以判断TXR3定位于3D染色体。
实施例三、利用BSMV-VIGS实验验证TXR基因的抗条锈病功能
1、沉默TXR基因小麦的获得
1)诱导TXR基因下调BSMV-VIGS载体系统的构建
(1)根据实施例1中TXR-3B基因序列，在basic-Helix-Loop-Helix结构域以外的非保守区选取了两个沉默片段，一个片段位于编码区的靠近N-端处，具体位置为+109bp～+314bp，长206bp，命名为沉默序列TXR-1，利用引物对V-TXR-F1：5'-ATTGCTAGCTTTGCATACTTGACAAAAG-3'和V-TXR-R1：5'-TAAGCTAGCGGTTCACGGCAAGAGC-3'进行PCR扩增获得。该片段与TXR-3A-Suwon11在+161bp位置存在一个SNP差异，与TXR-3D-Suwon11在+161bp和+203bp位置各存在一个SNP差异。另一个片段靠近C-端，起始于+1623bp，到+1795bp结束，长173bp，命名为沉默序列TXR-2，利用引物对V-TXR-F2：5'-ATTGCTAGCCATCCAGGTGGTGCAAGA-3'和V-TXR-R2：5'-TAAGCTAGCAGCCAGGGCACTTGAT-3'进行PCR扩增获得。该片段与TXR-3A-Suwon11在+1775bp位置存在一个SNP差异，与TXR-3D-Suwon11的序列完全相同。
上述引物对V-TXR-F1和V-TXR-R1的核苷酸序列以及引物对V-TXR-F2和V-TXR-R2的核苷酸序列中的下划线序列即为限制性内切酶NheI的酶切识别位点。
(2)载体的构建：具体操作步骤如下所述。
将上述步骤1获得的沉默序列TXR-1和TXR-2分别反向插入BSMV-VIGS病毒载体γ的NheI酶切位点处，且保持其它序列不变，得到重组载体γ-TXR-1和γ-TXR-2。
以引物对V-TXR-F1和γ-strain-p:5'-CAACTGCCAATCGTGAGTAGG-3'对重组载体γ-TXR-1进行PCR扩增和测序鉴定，阳性克隆即为将TXR基因编码区cDNA序列(序列表中的序列2)自5’末端起第109-314位核苷酸的序列按基因表达的相反方向插入BSMV-VIGS病毒载体γ链的NheI酶切位点处，且保持其它序列不变的TXR基因沉默载体γ-TXR-1。
以引物对V-TXR-F2和γ-strain-p对重组载体γ-TXR-2进行PCR扩增和测序鉴定，阳性克隆即为将TXR基因编码区cDNA序列(序列表中的序列2)自5’末端起第1623-1795位核苷酸的序列按基因表达的相反方向插入BSMV-VIGS病毒载体γ链的NheI酶切位点处，且保持其它序列不变的TXR基因沉默载体γ-TXR-2。
BSMV-VIGS病毒载体α、β和γ-GFP载体共同构成病毒载体系统BSMV:GFP。
BSMV-VIGS病毒载体α、β和重组载体γ-TXR-1共同构成可沉默TXR基因的病毒沉默载体系统BSMV:TXR-1。
BSMV-VIGS病毒载体α、β和重组载体γ-TXR-2共同构成可沉默TXR基因的病毒沉默载体系统BSMV:TXR-2。
2)BSMV体外转录
(1)用MluI酶切BSMV病毒载体α链、γ-GFP载体、重组载体γ-TXR-1和重组载体γ-TXR-2，用SpeI酶切BSMV病毒载体β链，分别得到线性化质粒。
(2)以上述步骤(1)获得的线性化质粒为模板进行体外转录，分别得到体外转录的BSMV病毒载体α、β、γ-GFP、γ-TXR-1和γ-TXR-2。体外转录反应按照RiboMAXTMLarge ScaleRNA Production System-T7(Promega公司产品，货号：P1300)说明书进行操作。转录反应体系和条件分别为：反应总体积20.0μl，包括线性化质粒(1.0μg)和无RNase水共6.5μl，5×Transcription Buffer 4.0μl，m7G(5')ppp(5')G 1.5μl(Promega公司产品，货号：P1718)，rNTPPreMix 6.0μl，Enzyme Mix 2.0μl，37℃反应4h，转录产物置-70℃保存备用。
3)BSMV接种
将水原11播种于营养土中，待生长至二叶期，分别取8μl BSMV:TXR-1重组病毒载体溶液和BSMV:TXR-2重组病毒载体溶液涂抹接种于水原11平展的第二叶上，10min后用灭菌超纯水喷施叶面，覆保鲜膜保湿24h，之后转为22℃正常条件培养，分别得到转BSMV:TXR-1植株和BSMV:TXR-2植株。同时，部分植株接种BSMV:GFP重组病毒载体溶液，得到转BSMV:GFP植株，部分植株涂抹1×GKP Buffer，得到模拟接种植株。
上述BSMV:TXR-1重组病毒载体溶液是将体外转录的BSMV-VIGS载体α、β和γ-TXR-1经无RNase水稀释3倍后等量混合，再加入等体积的2×GKP Buffer得到的溶液。
上述BSMV:TXR-2重组病毒载体溶液是将体外转录的BSMV-VIGS载体α、β和γ-TXR-2用无RNase水稀释3倍后等体积混合，再加入等体积的2×GKP Buffer得到的溶液。
上述转BSMV:TXR-1和BSMV:TXR-2的植株即为沉默TXR基因的小麦植株，转BSMV:GFP的植株即为阴性对照植株，涂抹1植GKP Buffer的模拟接种植株即为空白对照植株。
4)沉默TXR基因小麦的RT-PCR验证
BSMV-VIGS系统沉默TXR基因效果的具体检测方法如下：将上述步骤3)获得的转BSMV:TXR-1植株和BSMV:TXR-2植株、转BSMV:GFP植株及模拟接种植株(MOCK)在正常条件下培养10天后，取第三叶提取总RNA，反转录后通过定量PCR检测TXR基因的相对表达量，设置TaEF-1α为内参基因，相对表达量通过ΔΔCT法计算得到，由ABI7500型荧光定量PCR仪自带计算公式计算。
检测TXR相对表达量的定量PCR引物对序列为
qTXR-F：5'-GTGCCATTTGAGACTGGTGTGC-3'和qTXR-R：5'–AGACCTGGTGACCTCTCAATGTG-3'，检测内参基因TaEF-1α的定量PCR引物对序列即为TaEF-1αF：5'-TGGTGTCATCAAGCCTGGTATGGT-3'和TaEF-1αR：5'-ACTCATGGTGCATCTCAACGGACT-3'。
TXR基因的相对表达量的检测结果如图2所示。从图2可以看出，与转BSMV植株以及MOCK植株相比，转BSMV:TXR-1和转BSMV:TXR-2植株中TXR基因的相对表达量较阴性对照植株(BSMV:GFP)和空白对照植株(MOCK)都极显著地降低了，说明本实验所选择的两个沉默片段TXR-1和TXR-2都是有效的。
5)TXR基因沉默植株的条锈病抗性分析
将有效沉默了TXR基因表达的植株(BSMV:TXR-1和BSMV:TXR-2)、阴性对照植株(BSMV:GFP)，以及空白对照植株(MOCK)在第三叶展开后(约16天龄)接种条锈病菌生理小种CYR17，接种14天后观察第四叶发病情况。
结果如图3所示。模拟接种的空白对照植株(MOCK)的叶片没有任何症状，反应型为0级(免疫)。转BSMV:GFP的阴性对照植株叶片可见明显的病毒过敏反应(HR)斑，但没有孢子产生，反应型为0-0；级。而TXR基因沉默效果明显的转BSMV:TXR-1植株和转BSMV:TXR-2植株没有明显的HR反应，出现大量的条锈病菌孢子堆，反应型为3-4级，呈高感表型。
以上结果表明TXR基因表达量的降低使得水原11对条锈病菌生理小种CYR17的抗性完全丧失，沦为高感。由此证明TXR是一个参与小麦抗条锈病反应过程的重要基因。
实施例四、通过活性氧检测实验验证TXR的功能
按照实施例二的方法，获得降低TXR基因表达的水原11植株。将有效沉默了TXR基因表达的植株(BSMV:TXR-1和BSMV:TXR-2)、阴性对照植株(BSMV:GFP)，以及空白对照植株(MOCK)在第三叶展开后(约16天龄)接种条锈病菌生理小种CYR17。接种48小时后剪取叶片中部4cm左右的叶段进行DAB染色：(1)将叶片放入50mL离心管中，加入DAB染色液(1mg/mL，调节pH至5.8。现用现配，避光保存)没过叶片，28℃染色12h。(2)弃去染色液，以无水乙醇对叶片进行脱色漂白。清水漂洗2-3次，50％甘油固定，显微镜观察并统计产生活性氧的细胞数目。
结果如图4A所示，水原11接种CYR17后，孢子萌发长出芽管，芽管识别并通过气孔入侵叶肉细胞。对照中，TXR基因没有被沉默，此时被入侵的气孔两侧保卫细胞会有过氧化物的积累(图4A)，而TXR被沉默后，产生活性氧的保卫细胞数目显著减少(图4C)。
以上结果表明TXR可以作为正向调控因子参与小麦对条锈病的抗性反应。
实施例五、利用CRISPR/Cas9基因编辑验证TXR基因的抗条锈病功能
1、TXR基因编辑植株的获得
1)TXR基因编辑引导序列引物设计
为保证基因功能沉默的有效性，避开靠近C端的区域，针对TXR N端900bp以内基因序列搜索NGG(或CCN)PAM序列。再根据pTaU6载体的Bbs I酶切位点序列，连同PAM及其上游(或下游)19-20nt的碱基序列，一起设计为sgRNA(single guide RNA)引物b1F：5'-CAATGATGATGCCAAACAGCGG-3'和b1R：5'-CCGCTGTTTGGCATCATCATTG-3'。为进行后续的酶切检测，设计跨越sgRNA部分的PCR扩增引物Testb1F和Testb1R，用于检测sgRNA引导活性及突变效果。引物Testb1F的序列为：5'-ATGGTAATGAAGATGGAGGTTGAG-3'和Testb1R的序列为：5'-CGAAGTACATAGATGCTAGGAAGAA-3'。
2)载体构建
将引物b1F和b1R用ddH2O稀释成10μM，然后分别取9μL，并加入2μLddH2O，配成20μL反应体系，于普通PCR仪上，按照程序94℃，5min，90-10℃，每1min降10℃，对寡核苷酸序列进行退火，退火产物于冰上放置备用。pTaU6载体用Bbs I限制性内切酶，按照以下方法进行酶切、检测和载体构建：5μL
buffer，2μg pTaU6载体质粒，1μLBbs I(20U/μL)，加ddH2O至50μL，混合均匀，于37℃反应30min，加入10μL 6×Gel loading dye终止反应；用1％琼脂糖凝胶电泳检测并切胶纯化；用T4DNA连接酶将退火后的sgRNA b1与酶切过的pTaU6载体连接，构建成基因编辑载体pTaU6-b1。
3)sgRNA引导活性检测
将pTaU6-b1载体质粒与pJIT163-2NLSCas9(Shan et al.,2014)质粒用PEG法共转化普通小麦品种科农199的原生质体，并用单转pJIT163-GFP(Shan et al.,2014)质粒的小麦原生质体作为阳性对照，23℃培养48h。在荧光显微镜下观察原生质体状态并通过统计有荧光信号的原生质体数目来计算转化效率。原生质体饱满且呈圆形，转化效率在70％以上，即可以进行后续检测实验。
离心收集培养48h后的原生质体，用植物基因组DNA提取试剂盒(TIANGEN公司产品，货号：DP305)提取基因组DNA。然后用检测引物Test1F和Test1R扩增包含突变目标区域的基因片段。取5μL PCR反应产物，分别加入1.1μL10×T7E1 buffer和4.4μLddH2O，混合均匀后于PCR仪上进行变性与退火反应(95℃，5min，95-15℃，每分钟降10℃)，以形成杂合双链DNA片段。然后在反应产物中加入0.5μL(2.5units)的T7E1核酸内切酶，37℃消解1h。消解产物用2％的琼脂糖凝胶电泳检测。以pTaU6-b1载体质粒转化的原生质体DNA为模板进行PCR扩增得到的TXR基因片段，经变性、复性和T7E1酶切后，电泳图谱中出现了3条带，即除了一条与野生型对照相同的一条带以外，还有两条较短的酶切带(图5)。说明在pTaU6-b1载体质粒转化的小麦原生质体中TXR基因发生了突变，即这说明b1确实具有引导活性(图5)。
4)TXR基因编辑植株的获得
用步骤3)构建的基因编辑载体质粒pTaU6-b1与pJIT163-2NLSCas9共转化水原11的幼胚，经愈伤组织诱导和分化培养，获得水原11再生植株。转化过程由中国科学院遗传与发育生物学研究所遗传转化平台完成，公众可商业化联系该平台完成转化。按照步骤3)中的检测方法，用引物对Testb1F和Testb1R对全部再生植株进行目标基因突变检测，将获得的T0代突变株系，于4℃冷室春化4周，然后移栽到营养土中进行常规培养。T0代植株经自交获得T1代TXR基因编辑植株的种子。
2、TXR基因编辑植株的抗条锈病鉴定
1)荧光定量PCR检测TXR基因编辑植株中TXR基因的表达
将上述步骤1中获得的T1代种子，分别播种于10cm×10cm分别播种于方形花盆中，对生长7天左右的T1代基因编辑株系及对照株系，剪取叶片，按照实施例一和实施例二中的方法，进行RNA提取、RNA反转录和荧光定量PCR，检测TXR基因的相对表达量。定量PCR检测结果如图6A所示，与空白对照相比，TXR基因的表达量在T1代基因编辑植株中显著降低。
2)TXR基因编辑植株条锈病抗性检测
对生长10天左右的水原11TXR基因编辑植株及其空白对照株系进行条锈菌菌系CYR17的大量接菌，14天后观察叶片表型。大量接菌鉴定结果如图6B所示。对照植株叶片上几乎没有条锈菌孢子的出现，TXR基因编辑T1代株系叶片上则有大量的条锈菌孢子堆出现。
3)TXR基因编辑植株中TXR基因编辑类型的检测
取上述步骤2)中生长21天左右的T1代植株和对照植株的叶片，按照实施例一中的方法，提取基因组DNA，并以此为模板，用引物对Testb1F和Testb1R(序列表中序列19)进行PCR扩增。PCR反应体系：DNA模板0.5μL(<0.5μg)，dNTP(2.5mM)4μl，QCTXRF(10μM)1μl，QCTXRR2(10μM)1μl，5×TransStartFastPfu Buffer10μl，TransStartFastPfu DNAPolymerase 1μl(2.5U)，最后用水补足至50μl。PCR反应程序：95℃预变性2min；然后95℃变性20s，58℃退火20s，72℃延伸40s，35个循环；最后72℃延伸5min。
将上述PCR扩增产物经1％琼脂糖凝胶分离纯化后连接到
-Blunt ZeroCloning Vector(Transgen CB501)载体上，得到重组质粒pEASY-T-Testb1，转化大肠杆菌感受态细胞Trans-T1(全式金生物技术有限公司，货号：CD501-01)，将菌液平铺在含有100μg/ml氨苄青霉素的LB固体培养基上。12h后挑取单克隆，各选取30个菌液PCR鉴定为阳性的单克隆用通用引物M13F对其进行一代测序。测序结果如图6C所示。对照植株中TXR基因没有发生突变，在T1代基因编辑植株中，所有的TXR-3A-Suwon11和TXR-3D-Suwon11均有不同形式的突变，TXR-3B-Suwon11则表现为部分突变。
以上结果表明，水原11基因组中4个或4个以上TXR基因alleles的突变，可以使TXR基因的表达显著下调，使水原11有抗条锈生理小种CYR17转为感条锈菌生理小种CYR17。由此证明小麦TXR基因参与小麦对条锈病的抗性过程，对小麦抗性育种具有利用价值。
序列表
<110> 中国科学院遗传与发育生物学研究所
<120> 小麦抗条锈病相关的TXR蛋白及其编码基因与应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1857
<212> DNA
<213> 小麦(Triticum aestivum L.)
<400> 1
atggtaatga agatggaggt tgaggaggac ggtgccaatg gaggaaatgg tggggcatgg 60
actgaggagg accgagacct cagcaccact gtgctaggaa gagatgcatt tgcatacttg 120
acaaaagggg gcggtaccat atctgagggt cttgttgctg catcgtcacc tgtggacttg 180
caaaataaac tgcaggagct tatcgaatca gagcatcctg gtgctggttg gaactacgcc 240
atcttctggc agctttcacg cacaaagtct ggtgatcttg tccttgggtg gggtgatggc 300
tcttgccgtg aacccaatga tgctgagttg gcagctgctg cttctgcagg caatgatgat 360
gccaaacagc ggatgtggaa gcgtgtactg cagcggctgc acaaagcatt tggtggtgct 420
gatgaggagg attatgctcc cactattggt caggtgacag atacagaaat gttcttccta 480
gcatctatgt acttcgcgtt tccgcgtcgt gccggtgctc ctggtcaagt ttttgcagct 540
ggcctccctc tctgggttcc caattctgag cgcaatgtat tcccagctaa ttactgttac 600
cggggatacc ttgcaagcac agcaggattt agaactatct tgctagtgcc atttgagact 660
ggtgtgcttg agctgggttc gatgcagcag gtggctgaga gttctgacac tctccagacc 720
ataaagtcag tctttgcggg gacaggtggc aataaggata taatcccgag tcgtgaagga 780
aatggtcaca ttgagaggtc accaggtctg gcaaagattt ttggcaagga tttgaacctt 840
ggtcggtctt cagcagggcc agtgattggg gtatcgaaag tagatgaaag gccgtgggaa 900
cagaggactg ctggtggagg gagctcattg cttcccaatg tccagaaagg attgcagagt 960
ttcacttgga gtcaggcccg gggcctgaat tctcaccagc agaagtttgg caatggtata 1020
ctgatagtga gtaatgaagc tacacacggc aacaatagaa ccgcggacag ctccactaca 1080
acacagtttc agcttcagaa agcacctcag ctccagaaac taccacttct tcagaaacca 1140
ccacagctag tggagccgct gcagatggtc aaccagcaac agctgcagcc acaggcgcct 1200
aggcaaatag attttagtgc agggaccagt tccaagtctg gtgtcctggt tacaagagca 1260
gctgttcttg atggagatag ttcggaggtg aatggcttgt gtaaagagga agggacaaca 1320
cctgtcatag aggaccgacg gccaaggaag aggggaagaa agcctgcgaa tgggagagag 1380
gagccgctga atcatgttga ggctgagcgt caaaggaggg agaagctcaa ccagcggttc 1440
tatgcgttga gagccgttgt gcccaacatc tcgaaaatgg acaaggcttc cctgctgggt 1500
gatgcaatag catacatcac tgaccttcag aagaagctca aagatatgga gacggagaga 1560
gaacgatttc ttgagtctgg tatgggggat ccaagggagc gagcccctag accagaggtt 1620
gacatccagg tggtgcaaga cgaggttctg gttcgagtta tgtctccatt ggagaaccat 1680
ccggcaagga aggtctttga agcgtttgaa gaggcggacg tccgggtagg ggagtcgaaa 1740
ctcacaggca acaatggaac ggtagtgcat tcctttatca tcaagtgccc tggctccgaa 1800
cagcaaacaa gggagaaagt gattgctgca atgtctcgcg ccatgagctc agtgtga 1857
<210> 2
<211> 1857
<212> DNA
<213> 小麦(Triticum aestivum L.)
<400> 2
atggtaatga agatggaggt tgaggaggac ggtgccaatg gaggaaatgg tggggcatgg 60
actgaggagg accgagacct cagcaccact gtgctaggaa gagatgcatt tgcatacttg 120
acaaaagggg gcggtaccat atctgagggt cttgttgctg cgtcgtcacc tgtggacttg 180
caaaataaac tgcaggagct tatcgaatca gagcatcctg gtgctggttg gaactacgcc 240
atcttctggc agctttcacg cacaaagtct ggtgatcttg tccttgggtg gggtgatggc 300
tcttgccgtg aacccaatgg tgctgagttg gcagctgctg cttctgcagg caatgatgat 360
gccaaacagc ggatgcggaa gcgtgtactg cagcggttgc acaaagcatt tggtggtgct 420
gatgaggagg attatgctcc cactattggt caggtgacag atacagaaat gttcttccta 480
gcatctatgt acttcgcgtt tccacgtcgt gccggtgctc ctggtcaagt ttttgcagct 540
ggcctccctc tctgggttcc caattctgag cgcaatgtat tctcagccaa ttactgttac 600
cggggatacc ttgcaagcac agcaggattt agaactatct tgttagtgcc atttgagact 660
ggtgtgcttg agctgggttc gatgcagcag gtggctgaga gttctgacac tctccagacc 720
ataaagtctg tctttgcggg gacaggtggc aataaggata taattccgag ccgggaagga 780
aatggtcaca ttgagaggtc accaggtctg gcaaagattt ttggcaagga tttgaacctt 840
ggtcggtctt cagcagggcc agtgattggg gcatcgaaag tagatgaaag gccatgggaa 900
cagaggactg ctggtggagg gagctcattg cttcccaatg tccagaaagg attgcagagt 960
ttcacttgga gtcaggcccg gggcctgaat tctcaccagc agaagtttgg caatggtata 1020
ctgatagtga gtaatgaagc tacacacggc aacaatagag ccgcggacag ctccactaca 1080
acacagtttc agcttcagaa agcacctcag ctccagaaac taccacttct tcagaaacca 1140
ccacagctag tgaagccact gcagatggtc aaccagcaac agctgcagcc acaggcgcct 1200
aggcaaatag attttagtgc agggaccagt tccaagtctg gtgtcctggt tacaagagca 1260
gctgttcttg atggagatag ttcagaggtg aatggcttgt gtaaagagga agggacaaca 1320
cctgtcatag aggaccgacg gccaaggaag aggggaagaa agcccgcaaa tgggagggag 1380
gagccgctga atcatgttga ggctgagcgt caaaggaggg agaggctcaa ccaacggttc 1440
tatgcgttga gagccgttgt gcctaacatc tcgaaaatgg acaaggcctc cctattgggc 1500
gatgcaatag cttacatcac tgaccttcag aagaagctca aagatatgga gacggagaga 1560
gaacgatttc ttgagtctgg tatggtggat ccaagggagc gagcccctag accagaggtt 1620
gacatccagg tggtgcaaga cgaggttctg gttcgagtta tgtctccatt ggagaaccat 1680
ccggtaaaga aggtctttga agcgtttgaa gaggcggacg tccgggtagg ggagtcgaaa 1740
ctcacaggca acaatggaac ggtagtgcat tccttcatca tcaagtgccc tggctctgaa 1800
cagaaaacga gggagaaagt gattgctgca atgtctcgcg ccatgagctc agtgtga 1857
<210> 3
<211> 1857
<212> DNA
<213> 小麦(Triticum aestivum L.)
<400> 3
atggtaatga agatggaggt tgaggaggtc ggtgccaatg gaggaaatgg tggagcatgg 60
actgaggagg accgagacct cagcaccact gtgctaggta gagatgcatt tgcatacttg 120
acaaaagggg gcggtaccat atctgagggt cttgttgctg catcgtcacc tgtggacttg 180
cagaataaac tgcaggagct tattgaatca gagcatcctg gtgctggttg gaactacgcc 240
atctcctggc agcttccacg cacaaagtct ggtgatcttg tccttgggtg gggtgatggc 300
tcttgccgtg aacccaatga tgctgagttg gcagctgctg cttctgcagg caatgatgat 360
gccaaacagc ggatgcggaa gcgtgtactg caacggttgc acagagcatt tggtgctgct 420
gatgaggagg attatgctcc cactattggt caggtgacag atacagaaat gttcttccta 480
gcatctatgt acttcgcgtt tccgcgtcgt gccggtgctc ctggtcaagt ttttgcagct 540
ggcctccctc tctgggttcc caattctgag cgcaatgtat tcccagccaa ttactgttac 600
cggggatacc ttgcaagcac agcagggttt agaactatcc tgttagtgcc atttgagact 660
ggtgtgcttg agctgggttc gatgcagcag gtggctgaga gttctgacac tctccagacc 720
ataaagtctg tctttgcggg gacaggtggc aataaggata taattccgag tcgtgaagga 780
aatggtcaca ttgagaggtc accaggtctg gcaaagattt ttggcaagga tttgaacctt 840
ggtcggtctt cagcagggcc agtggttggg gtatcgaaag tagatgaaag gtcatgggaa 900
cagaggactg ctggtggagg gagctcagtg cttcccaatg tccagaaagg attgcagagt 960
ttcacttgga gtcaggcccg gggcctgaat tctcaccagc agaagtttgg caatggtata 1020
ctgatagtga gtaatgaagc tacacacggc aacaatagag ccacagacag ctccactaca 1080
acacagtttc agctacagaa agcacctcag ctccagaaac taccacttct tcagaaacca 1140
ccacagctag tgaagccact gcagatggcc aaccagcaac agctgcagcc acaggcgcct 1200
aggcaaatag attttagtgc agggaccagt tccaagtctg gtgttctggt tacaagagca 1260
gctgttcttg atggagatag ttcaggggtg aacggcttgt gtaaagagga agggacaaca 1320
cctgtcatag aggaccgacg gccaaggaag aggggaagaa agcctgcgaa tgggagagag 1380
gagccgctga atcatgttga ggctgagcgt caaaggaggg agaagctcaa ccagcggttc 1440
tatgctttga gagccgttgt gcccaacatc tcgaaaatgg acaaggcctc tctgttgggc 1500
gatgcaatag catacatcac tgaccttcaa aagaagctca aagatatgga gacggagaga 1560
gaacgatttc ttgagtctgg tatggttgat cctagggagc gagcccctag accagaggtt 1620
gccatccagg tggtgcaaga cgaggttctg gttcgagtta tgtctccatt ggagaaccat 1680
ccggtaaaga aggcctttga agcgtttgaa gaggcggacg tccgggtagg ggagtcgaaa 1740
ctcacaggca acaatggaac ggtagtgcat tccttcatca tcaagtgccc tggctccgaa 1800
cagcaagcga gagaaaaagt gatcgctgca atgtctcgcg ccatgagctc agtgtga 1857
<210> 4
<211> 1857
<212> DNA
<213> 小麦(Triticum aestivum L.)
<400> 4
atggtaatga agatggaggt tgaggaggac ggtgccaatg gaggaaatgg tggggcatgg 60
actgaggagg accgagacct cagcaccact gtgctaggaa gagatgcatt tgcatacttg 120
acaaaagggg gcggtaccat atctgagggt cttgttgctg catcgtcacc tgtggacttg 180
caaaataaac tgcaggagct tatcgaatca gagcatcctg gtgctggttg gaactacgcc 240
atcttctggc agctttcacg cacaaagtct ggtgatcttg tccttgggtg gggtgatggc 300
tcttgccgtg aacccaatga tgctgagttg gcagctgctg cttctgcagg caatgatgat 360
gccaaacagc ggatgtggaa gcgtgtactg cagcggctgc acaaagcatt tggtggtgct 420
gatgaggagg attatgctcc cactattggt caggtgacag atacagaaat gttcttccta 480
gcatctatgt acttcgcgtt tccgcgtcgt gccggtgctc ctggtcaagt ttttgcagct 540
ggcctccctc tctgggttcc caattctgag cgcaatgtat tcccagctaa ttactgttac 600
cggggatacc ttgcaagcac agcaggattt agaactatct tgctagtgcc atttgagact 660
ggtgtgcttg agctgggttc gatgcagcag gtggctgaga gttctgacac tctccagacc 720
ataaagtcag tctttgcggg gacaggtggc aataaggata taatcccgag tcgtgaagga 780
aatggtcaca ttgagaggtc accaggtctg gcaaagattt ttggcaagga tttgaacctt 840
ggtcggtctt cagcagggcc agtgattggg gtatcgaaag tagatgaaag gccgtgggaa 900
cagaggactg ctggtggagg gagctcattg cttcccaatg tccagaaagg attgcagagt 960
ttcacttgga gtcaggcccg gggcctgaat tctcaccagc agaagtttgg caatggtata 1020
ctgatagtga gtaatgaagc tacacacggc aacaatagaa ccgcggacag ctccactaca 1080
acacagtttc agcttcagaa agcacctcag ctccagaaac taccacttct tcagaaacca 1140
ccacagctag tggagccgct gcagatggtc aaccagcaac agctgcagcc acaggcgcct 1200
aggcaaatag attttagtgc agggaccagt tccaagtctg gtgtcctggt tacaagagca 1260
gctgttcttg atggagatag ttcggaggtg aatggcttgt gtaaagagga agggacaaca 1320
cctgtcatag aggaccgacg gccaaggaag aggggaagaa agcctgcgaa tgggagagag 1380
gagccgctga atcatgttga ggctgagcgt caaaggaggg agaagctcaa ccagcggttc 1440
tatgcgttga gagccgttgt gcccaacatc tcgaaaatgg acaaggcttc cctgctgggt 1500
gatgcaatag catacatcac tgaccttcag aagaagctca aagatatgga gacggagaga 1560
gaacgatttc ttgagtctgg tatgggggat ccaagggagc gagcccctag accagaggtt 1620
gacatccagg tggtgcaaga cgaggttctg gttcgagtta tgtctccatt ggagaaccat 1680
ccggcaagga aggtctttga agcgtttgaa gaggcggacg tccgggtagg ggagtcgaaa 1740
ctcacaggca acaatggaac ggtagtgcat tcctttatca tcaagtgccc tggctccgaa 1800
cagcaaacaa gggagaaagt gattgctgca atgtctcgcg ccatgagctc agtgtga 1857
<210> 5
<211> 1857
<212> DNA
<213> 小麦(Triticum aestivum L.)
<400> 5
atggtaatga agatggaggt tgaggaggac ggtgccaatg gaggaaatgg tggggcatgg 60
actgaggagg accgagacct cagcaccact gtgctaggaa gagatgcatt tgcatacttg 120
acaaaagggg gcggtaccat atctgagggt cttgttgctg cgtcgtcacc tgtggacttg 180
caaaataaac tgcaggagct tatcgaatca gagcatcctg gtgctggttg gaactacgcc 240
atcttctggc agctttcacg cacaaagtct ggtgatcttg tccttgggtg gggtgatggc 300
tcttgccgtg aacccaatgg tgctgagttg gcagctgctg cttctgcagg caatgatgat 360
gccaaacagc ggatgcggaa gcgtgtactg cagcggttgc acaaagcatt tggtggtgct 420
gatgaggagg attatgctcc cactattggt caggtgacag atacagaaat gttcttccta 480
gcatctatgt acttcgcgtt tccacgtcgt gccggtgctc ctggtcaagt ttttgcagct 540
ggcctccctc tctgggttcc caattctgag cgcaatgtat tctcagccaa ttactgttac 600
cggggatacc ttgcaagcac agcaggattt agaactatct tgttagtgcc atttgagact 660
ggtgtgcttg agctgggttc gatgcagcag gtggctgaga gttctgacac tctccagacc 720
ataaagtctg tctttgcggg gacaggtggc aataaggata taattccgag ccgggaagga 780
aatggtcaca ttgagaggtc accaggtctg gcaaagattt ttggcaagga tttgaacctt 840
ggtcggtctt cagcagggcc agtgattggg gcatcgaaag tagatgaaag gccatgggaa 900
cagaggactg ctggtggagg gagctcattg cttcccaatg tccagaaagg attgcagagt 960
ttcacttgga gtcaggcccg gggcctgaat tctcaccagc agaagtttgg caatggtata 1020
ctgatagtga gtaatgaagc tacacacggc aacaatagag ccgcggacag ctccactaca 1080
acacagtttc agcttcagaa agcacctcag ctccagaaac taccacttct tcagaaacca 1140
ccacagctag tgaagccact gcagatggtc aaccagcaac agctgcagcc acaggcgcct 1200
aggcaaatag attttagtgc agggaccagt tccaagtctg gtgtcctggt tacaagagca 1260
gctgttcttg atggagatag ttcagaggtg aatggcttgt gtaaagagga agggacaaca 1320
cctgtcatag aggaccgacg gccaaggaag aggggaagaa agcccgcaaa tgggagggag 1380
gagccgctga atcatgttga ggctgagcgt caaaggaggg agaggctcaa ccaacggttc 1440
tatgcgttga gagccgttgt gcctaacatc tcgaaaatgg acaaggcctc cctattgggc 1500
gatgcaatag cttacatcac tgaccttcag aagaagctca aagatatgga gacggagaga 1560
gaacgatttc ttgagtctgg tatggtggat ccaagggagc gagcccctag accagaggtt 1620
gacatccagg tggtgcaaga cgaggttctg gttcgagtta tgtctccatt ggagaaccat 1680
ccggtaaaga aggtctttga agcgtttgaa gaggcggacg tccgggtagg ggagtcgaaa 1740
ctcacaggca acaatggaac ggtagtgcat tccttcatca tcaagtgccc tggctctgaa 1800
cagaaaacga gggagaaagt gattgctgca atgtctcgcg ccatgagctc agtgtga 1857
<210> 6
<211> 1857
<212> DNA
<213> 小麦(Triticum aestivum L.)
<400> 6
atggtaatga agatggaggt tgaggaggtc ggtgccaatg gaggaaatgg tggagcatgg 60
actgaggagg accgagacct cagcaccact gtgctaggta gagatgcatt tgcatacttg 120
acaaaagggg gcggtaccat atctgagggt cttgttgctg catcgtcacc tgtggacttg 180
cagaataaac tgcaggagct tattgaatca gagcatcctg gtgctggttg gaactacgcc 240
atctcctggc agcttccacg cacaaagtct ggtgatcttg tccttgggtg gggtgatggc 300
tcttgccgtg aacccaatga tgctgagttg gcagctgctg cttctgcagg caatgatgat 360
gccaaacagc ggatgcggaa gcgtgtactg caacggttgc acagagcatt tggtgctgct 420
gatgaggagg attatgctcc cactattggt caggtgacag atacagaaat gttcttccta 480
gcatctatgt acttcgcgtt tccgcgtcgt gccggtgctc ctggtcaagt ttttgcagct 540
ggcctccctc tctgggttcc caattctgag cgcaatgtat tcccagccaa ttactgttac 600
cggggatacc ttgcaagcac agcagggttt agaactatcc tgttagtgcc atttgagact 660
ggtgtgcttg agctgggttc gatgcagcag gtggctgaga gttctgacac tctccagacc 720
ataaagtctg tctttgcggg gacaggtggc aataaggata taattccgag tcgtgaagga 780
aatggtcaca ttgagaggtc accaggtctg gcaaagattt ttggcaagga tttgaacctt 840
ggtcggtctt cagcagggcc agtggttggg gtatcgaaag tagatgaaag gtcatgggaa 900
cagaggactg ctggtggagg gagctcagtg cttcccaatg tccagaaagg attgcagagt 960
ttcacttgga gtcaggcccg gggcctgaat tctcaccagc agaagtttgg caatggtata 1020
ctgatagtga gtaatgaagc tacacacggc aacaatagag ccacagacag ctccactaca 1080
acacagtttc agctacagaa agcacctcag ctccagaaac taccacttct tcagaaacca 1140
ccacagctag tgaagccact gcagatggcc aaccagcaac agctgcagcc acaggcgcct 1200
aggcaaatag attttagtgc agggaccagt tccaagtctg gtgttctggt tacaagagca 1260
gctgttcttg atggagatag ttcaggggtg aacggcttgt gtaaagagga agggacaaca 1320
cctgtcatag aggaccgacg gccaaggaag aggggaagaa agcctgcgaa tgggagagag 1380
gagccgctga atcatgttga ggctgagcgt caaaggaggg agaagctcaa ccagcggttc 1440
tatgctttga gagccgttgt gcccaacatc tcgaaaatgg acaaggcctc tctgttgggc 1500
gatgcaatag catacatcac tgaccttcaa aagaagctca aagatatgga gacggagaga 1560
gaacgatttc ttgagtctgg tatggttgat cctagggagc gagcccctag accagaggtt 1620
gccatccagg tggtgcaaga cgaggttctg gttcgagtta tgtctccatt ggagaaccat 1680
ccggtaaaga aggcctttga agcgtttgaa gaggcggacg tccgggtagg ggagtcgaaa 1740
ctcacaggca acaatggaac ggtagtgcat tccttcatca tcaagtgccc tggctccgaa 1800
cagcaagcga gagaaaaagt gatcgctgca atgtctcgcg ccatgagctc agtgtga 1857
<210> 7
<211> 618
<212> PRT
<213> 小麦(Triticum aestivum L.)
<400> 7
Met Val Met Lys Met Glu Val Glu Glu Asp Gly Ala Asn Gly Gly Asn
1 5 10 15
Gly Gly Ala Trp Thr Glu Glu Asp Arg Asp Leu Ser Thr Thr Val Leu
20 25 30
Gly Arg Asp Ala Phe Ala Tyr Leu Thr Lys Gly Gly Gly Thr Ile Ser
35 40 45
Glu Gly Leu Val Ala Ala Ser Ser Pro Val Asp Leu Gln Asn Lys Leu
50 55 60
Gln Glu Leu Ile Glu Ser Glu His Pro Gly Ala Gly Trp Asn Tyr Ala
65 70 75 80
Ile Phe Trp Gln Leu Ser Arg Thr Lys Ser Gly Asp Leu Val Leu Gly
85 90 95
Trp Gly Asp Gly Ser Cys Arg Glu Pro Asn Asp Ala Glu Leu Ala Ala
100 105 110
Ala Ala Ser Ala Gly Asn Asp Asp Ala Lys Gln Arg Met Trp Lys Arg
115 120 125
Val Leu Gln Arg Leu His Lys Ala Phe Gly Gly Ala Asp Glu Glu Asp
130 135 140
Tyr Ala Pro Thr Ile Gly Gln Val Thr Asp Thr Glu Met Phe Phe Leu
145 150 155 160
Ala Ser Met Tyr Phe Ala Phe Pro Arg Arg Ala Gly Ala Pro Gly Gln
165 170 175
Val Phe Ala Ala Gly Leu Pro Leu Trp Val Pro Asn Ser Glu Arg Asn
180 185 190
Val Phe Pro Ala Asn Tyr Cys Tyr Arg Gly Tyr Leu Ala Ser Thr Ala
195 200 205
Gly Phe Arg Thr Ile Leu Leu Val Pro Phe Glu Thr Gly Val Leu Glu
210 215 220
Leu Gly Ser Met Gln Gln Val Ala Glu Ser Ser Asp Thr Leu Gln Thr
225 230 235 240
Ile Lys Ser Val Phe Ala Gly Thr Gly Gly Asn Lys Asp Ile Ile Pro
245 250 255
Ser Arg Glu Gly Asn Gly His Ile Glu Arg Ser Pro Gly Leu Ala Lys
260 265 270
Ile Phe Gly Lys Asp Leu Asn Leu Gly Arg Ser Ser Ala Gly Pro Val
275 280 285
Ile Gly Val Ser Lys Val Asp Glu Arg Pro Trp Glu Gln Arg Thr Ala
290 295 300
Gly Gly Gly Ser Ser Leu Leu Pro Asn Val Gln Lys Gly Leu Gln Ser
305 310 315 320
Phe Thr Trp Ser Gln Ala Arg Gly Leu Asn Ser His Gln Gln Lys Phe
325 330 335
Gly Asn Gly Ile Leu Ile Val Ser Asn Glu Ala Thr His Gly Asn Asn
340 345 350
Arg Thr Ala Asp Ser Ser Thr Thr Thr Gln Phe Gln Leu Gln Lys Ala
355 360 365
Pro Gln Leu Gln Lys Leu Pro Leu Leu Gln Lys Pro Pro Gln Leu Val
370 375 380
Glu Pro Leu Gln Met Val Asn Gln Gln Gln Leu Gln Pro Gln Ala Pro
385 390 395 400
Arg Gln Ile Asp Phe Ser Ala Gly Thr Ser Ser Lys Ser Gly Val Leu
405 410 415
Val Thr Arg Ala Ala Val Leu Asp Gly Asp Ser Ser Glu Val Asn Gly
420 425 430
Leu Cys Lys Glu Glu Gly Thr Thr Pro Val Ile Glu Asp Arg Arg Pro
435 440 445
Arg Lys Arg Gly Arg Lys Pro Ala Asn Gly Arg Glu Glu Pro Leu Asn
450 455 460
His Val Glu Ala Glu Arg Gln Arg Arg Glu Lys Leu Asn Gln Arg Phe
465 470 475 480
Tyr Ala Leu Arg Ala Val Val Pro Asn Ile Ser Lys Met Asp Lys Ala
485 490 495
Ser Leu Leu Gly Asp Ala Ile Ala Tyr Ile Thr Asp Leu Gln Lys Lys
500 505 510
Leu Lys Asp Met Glu Thr Glu Arg Glu Arg Phe Leu Glu Ser Gly Met
515 520 525
Gly Asp Pro Arg Glu Arg Ala Pro Arg Pro Glu Val Asp Ile Gln Val
530 535 540
Val Gln Asp Glu Val Leu Val Arg Val Met Ser Pro Leu Glu Asn His
545 550 555 560
Pro Ala Arg Lys Val Phe Glu Ala Phe Glu Glu Ala Asp Val Arg Val
565 570 575
Gly Glu Ser Lys Leu Thr Gly Asn Asn Gly Thr Val Val His Ser Phe
580 585 590
Ile Ile Lys Cys Pro Gly Ser Glu Gln Gln Thr Arg Glu Lys Val Ile
595 600 605
Ala Ala Met Ser Arg Ala Met Ser Ser Val
610 615
<210> 8
<211> 618
<212> PRT
<213> 小麦(Triticum aestivum L.)
<400> 8
Met Val Met Lys Met Glu Val Glu Glu Asp Gly Ala Asn Gly Gly Asn
1 5 10 15
Gly Gly Ala Trp Thr Glu Glu Asp Arg Asp Leu Ser Thr Thr Val Leu
20 25 30
Gly Arg Asp Ala Phe Ala Tyr Leu Thr Lys Gly Gly Gly Thr Ile Ser
35 40 45
Glu Gly Leu Val Ala Ala Ser Ser Pro Val Asp Leu Gln Asn Lys Leu
50 55 60
Gln Glu Leu Ile Glu Ser Glu His Pro Gly Ala Gly Trp Asn Tyr Ala
65 70 75 80
Ile Phe Trp Gln Leu Ser Arg Thr Lys Ser Gly Asp Leu Val Leu Gly
85 90 95
Trp Gly Asp Gly Ser Cys Arg Glu Pro Asn Gly Ala Glu Leu Ala Ala
100 105 110
Ala Ala Ser Ala Gly Asn Asp Asp Ala Lys Gln Arg Met Arg Lys Arg
115 120 125
Val Leu Gln Arg Leu His Lys Ala Phe Gly Gly Ala Asp Glu Glu Asp
130 135 140
Tyr Ala Pro Thr Ile Gly Gln Val Thr Asp Thr Glu Met Phe Phe Leu
145 150 155 160
Ala Ser Met Tyr Phe Ala Phe Pro Arg Arg Ala Gly Ala Pro Gly Gln
165 170 175
Val Phe Ala Ala Gly Leu Pro Leu Trp Val Pro Asn Ser Glu Arg Asn
180 185 190
Val Phe Ser Ala Asn Tyr Cys Tyr Arg Gly Tyr Leu Ala Ser Thr Ala
195 200 205
Gly Phe Arg Thr Ile Leu Leu Val Pro Phe Glu Thr Gly Val Leu Glu
210 215 220
Leu Gly Ser Met Gln Gln Val Ala Glu Ser Ser Asp Thr Leu Gln Thr
225 230 235 240
Ile Lys Ser Val Phe Ala Gly Thr Gly Gly Asn Lys Asp Ile Ile Pro
245 250 255
Ser Arg Glu Gly Asn Gly His Ile Glu Arg Ser Pro Gly Leu Ala Lys
260 265 270
Ile Phe Gly Lys Asp Leu Asn Leu Gly Arg Ser Ser Ala Gly Pro Val
275 280 285
Ile Gly Ala Ser Lys Val Asp Glu Arg Pro Trp Glu Gln Arg Thr Ala
290 295 300
Gly Gly Gly Ser Ser Leu Leu Pro Asn Val Gln Lys Gly Leu Gln Ser
305 310 315 320
Phe Thr Trp Ser Gln Ala Arg Gly Leu Asn Ser His Gln Gln Lys Phe
325 330 335
Gly Asn Gly Ile Leu Ile Val Ser Asn Glu Ala Thr His Gly Asn Asn
340 345 350
Arg Ala Ala Asp Ser Ser Thr Thr Thr Gln Phe Gln Leu Gln Lys Ala
355 360 365
Pro Gln Leu Gln Lys Leu Pro Leu Leu Gln Lys Pro Pro Gln Leu Val
370 375 380
Lys Pro Leu Gln Met Val Asn Gln Gln Gln Leu Gln Pro Gln Ala Pro
385 390 395 400
Arg Gln Ile Asp Phe Ser Ala Gly Thr Ser Ser Lys Ser Gly Val Leu
405 410 415
Val Thr Arg Ala Ala Val Leu Asp Gly Asp Ser Ser Glu Val Asn Gly
420 425 430
Leu Cys Lys Glu Glu Gly Thr Thr Pro Val Ile Glu Asp Arg Arg Pro
435 440 445
Arg Lys Arg Gly Arg Lys Pro Ala Asn Gly Arg Glu Glu Pro Leu Asn
450 455 460
His Val Glu Ala Glu Arg Gln Arg Arg Glu Arg Leu Asn Gln Arg Phe
465 470 475 480
Tyr Ala Leu Arg Ala Val Val Pro Asn Ile Ser Lys Met Asp Lys Ala
485 490 495
Ser Leu Leu Gly Asp Ala Ile Ala Tyr Ile Thr Asp Leu Gln Lys Lys
500 505 510
Leu Lys Asp Met Glu Thr Glu Arg Glu Arg Phe Leu Glu Ser Gly Met
515 520 525
Val Asp Pro Arg Glu Arg Ala Pro Arg Pro Glu Val Asp Ile Gln Val
530 535 540
Val Gln Asp Glu Val Leu Val Arg Val Met Ser Pro Leu Glu Asn His
545 550 555 560
Pro Val Lys Lys Val Phe Glu Ala Phe Glu Glu Ala Asp Val Arg Val
565 570 575
Gly Glu Ser Lys Leu Thr Gly Asn Asn Gly Thr Val Val His Ser Phe
580 585 590
Ile Ile Lys Cys Pro Gly Ser Glu Gln Lys Thr Arg Glu Lys Val Ile
595 600 605
Ala Ala Met Ser Arg Ala Met Ser Ser Val
610 615
<210> 9
<211> 618
<212> PRT
<213> 小麦(Triticum aestivum L.)
<400> 9
Met Val Met Lys Met Glu Val Glu Glu Val Gly Ala Asn Gly Gly Asn
1 5 10 15
Gly Gly Ala Trp Thr Glu Glu Asp Arg Asp Leu Ser Thr Thr Val Leu
20 25 30
Gly Arg Asp Ala Phe Ala Tyr Leu Thr Lys Gly Gly Gly Thr Ile Ser
35 40 45
Glu Gly Leu Val Ala Ala Ser Ser Pro Val Asp Leu Gln Asn Lys Leu
50 55 60
Gln Glu Leu Ile Glu Ser Glu His Pro Gly Ala Gly Trp Asn Tyr Ala
65 70 75 80
Ile Ser Trp Gln Leu Pro Arg Thr Lys Ser Gly Asp Leu Val Leu Gly
85 90 95
Trp Gly Asp Gly Ser Cys Arg Glu Pro Asn Asp Ala Glu Leu Ala Ala
100 105 110
Ala Ala Ser Ala Gly Asn Asp Asp Ala Lys Gln Arg Met Arg Lys Arg
115 120 125
Val Leu Gln Arg Leu His Arg Ala Phe Gly Ala Ala Asp Glu Glu Asp
130 135 140
Tyr Ala Pro Thr Ile Gly Gln Val Thr Asp Thr Glu Met Phe Phe Leu
145 150 155 160
Ala Ser Met Tyr Phe Ala Phe Pro Arg Arg Ala Gly Ala Pro Gly Gln
165 170 175
Val Phe Ala Ala Gly Leu Pro Leu Trp Val Pro Asn Ser Glu Arg Asn
180 185 190
Val Phe Pro Ala Asn Tyr Cys Tyr Arg Gly Tyr Leu Ala Ser Thr Ala
195 200 205
Gly Phe Arg Thr Ile Leu Leu Val Pro Phe Glu Thr Gly Val Leu Glu
210 215 220
Leu Gly Ser Met Gln Gln Val Ala Glu Ser Ser Asp Thr Leu Gln Thr
225 230 235 240
Ile Lys Ser Val Phe Ala Gly Thr Gly Gly Asn Lys Asp Ile Ile Pro
245 250 255
Ser Arg Glu Gly Asn Gly His Ile Glu Arg Ser Pro Gly Leu Ala Lys
260 265 270
Ile Phe Gly Lys Asp Leu Asn Leu Gly Arg Ser Ser Ala Gly Pro Val
275 280 285
Val Gly Val Ser Lys Val Asp Glu Arg Ser Trp Glu Gln Arg Thr Ala
290 295 300
Gly Gly Gly Ser Ser Val Leu Pro Asn Val Gln Lys Gly Leu Gln Ser
305 310 315 320
Phe Thr Trp Ser Gln Ala Arg Gly Leu Asn Ser His Gln Gln Lys Phe
325 330 335
Gly Asn Gly Ile Leu Ile Val Ser Asn Glu Ala Thr His Gly Asn Asn
340 345 350
Arg Ala Thr Asp Ser Ser Thr Thr Thr Gln Phe Gln Leu Gln Lys Ala
355 360 365
Pro Gln Leu Gln Lys Leu Pro Leu Leu Gln Lys Pro Pro Gln Leu Val
370 375 380
Lys Pro Leu Gln Met Ala Asn Gln Gln Gln Leu Gln Pro Gln Ala Pro
385 390 395 400
Arg Gln Ile Asp Phe Ser Ala Gly Thr Ser Ser Lys Ser Gly Val Leu
405 410 415
Val Thr Arg Ala Ala Val Leu Asp Gly Asp Ser Ser Gly Val Asn Gly
420 425 430
Leu Cys Lys Glu Glu Gly Thr Thr Pro Val Ile Glu Asp Arg Arg Pro
435 440 445
Arg Lys Arg Gly Arg Lys Pro Ala Asn Gly Arg Glu Glu Pro Leu Asn
450 455 460
His Val Glu Ala Glu Arg Gln Arg Arg Glu Lys Leu Asn Gln Arg Phe
465 470 475 480
Tyr Ala Leu Arg Ala Val Val Pro Asn Ile Ser Lys Met Asp Lys Ala
485 490 495
Ser Leu Leu Gly Asp Ala Ile Ala Tyr Ile Thr Asp Leu Gln Lys Lys
500 505 510
Leu Lys Asp Met Glu Thr Glu Arg Glu Arg Phe Leu Glu Ser Gly Met
515 520 525
Val Asp Pro Arg Glu Arg Ala Pro Arg Pro Glu Val Ala Ile Gln Val
530 535 540
Val Gln Asp Glu Val Leu Val Arg Val Met Ser Pro Leu Glu Asn His
545 550 555 560
Pro Val Lys Lys Ala Phe Glu Ala Phe Glu Glu Ala Asp Val Arg Val
565 570 575
Gly Glu Ser Lys Leu Thr Gly Asn Asn Gly Thr Val Val His Ser Phe
580 585 590
Ile Ile Lys Cys Pro Gly Ser Glu Gln Gln Ala Arg Glu Lys Val Ile
595 600 605
Ala Ala Met Ser Arg Ala Met Ser Ser Val
610 615
Claims (8)
1.一种培育条锈病抗性降低的转基因植物的方法，其特征在于，包括抑制受体植物中TXR蛋白的含量和/或活性，得到转基因植物的步骤；所述转基因植物的条锈病抗性弱于所述受体植物，其中所述TXR蛋白为如序列7或序列8或序列9所示的氨基酸序列，所述植物为小麦。
2.根据权利要求1所述的方法，其特征在于：所述TXR蛋白的编码基因为如序列1或序列2或序列3所示的DNA序列。
3.根据权利要求1-2任一所述的方法，其特征在于：所述抑制受体植物中TXR蛋白的含量和/或活性的物质为:BSMV病毒载体α、BSMV病毒载体β和γ-TXR,所述γ-TXR为γ-TXR-1或γ-TXR-2，所述γ-TXR-1为将序列表中的序列2自5’末端起第109-314位核苷酸的序列按基因表达的相反方向插入BSMV-VIGS病毒载体γ链的NheI酶切位点处，且保持BSMV-VIGS病毒载体γ链的其它序列不变的得到的TXR基因沉默载体,所述γ-TXR-2为将序列表中的序列2自5’末端起第1623-1795位核苷酸的序列按基因表达的相反方向插入BSMV-VIGS病毒载体γ链的NheI酶切位点处，且保持BSMV-VIGS病毒载体γ链的其它序列不变的得到的TXR基因沉默载体。
4.一种培育条锈病抗性提高的转基因植物的方法，包括提高受体植物中TXR蛋白的表达量和/或活性，得到转基因植物的步骤；所述转基因植物的条锈病抗性高于所述受体植物，其中所述TXR蛋白为如序列7或序列8或序列9所示的氨基酸序列，所述植物为小麦。
5.根据权利要求4所述的方法，其特征在于：所述TXR蛋白的编码基因为如序列1或序列2或序列3所示的DNA序列。
6.根据权利要求4所述的方法，其特征在于：所述转基因植物的条锈病抗性高于所述受体植物体现在：转基因植物的条锈病菌孢子数低于受体植物。
7.一种权利要求1-3所述的方法或权利要求4-6所述的方法在小麦育种中的应用。
8.根据所述权利要求7所述的应用，其特征在于，选取能够提高受体植物中TXR蛋白的表达量和/或活性种子。
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