小麦（Triticum aestivum L.）是世界上种植最广泛的粮食作物之一，由Blumeria graminis f. sp. Tritici (Bgt)侵染引起的小麦白粉病是小麦最主要的病害之一，严重威胁小麦生产。本实验室前期克隆了小麦广谱抗白粉病基因Pm21（NLR1-V），并克隆了NLR1-V基因的上游启动子序列，对其应答白粉菌的模式研究发现该启动子属于典型的病原菌诱导型启动子。因此，研究NLR1-V基因的转录调控机制对解析其广谱抗性机制具有十分重要的意义。本研究利用酵母单杂交技术筛选NLR1-V启动子互作蛋白，并对互作蛋白进行了调控功能的分析，从转录精准调控的角度解析NLR1-V介导的小麦白粉病抗性分子机制。主要结果如下：

1、NLR1-V启动子互作蛋白的筛选与验证

（1）利用酵母单杂交技术筛选NLR1-V启动子互作蛋白：前期研究表明NLR1-V启动子ATG上游-889bp~-335bp区段可以响应白粉菌诱导，为了确保筛选范围的全面性，针对NLR1-V启动子-1019bp ~ -317bp区段，构建涵盖不同片段的诱饵载体；将诱饵质粒分别转化酵母细胞，对诱饵菌株进行自激活实验寻找适宜的Aba筛库浓度。筛选小麦酵母文库：利用本实验室前期构建的小麦cDNA文库，针对通过自激活验证的诱饵菌株Y1H[pJM1-AbAi]和Y1H[pJM2-AbAi]分别筛选，共获得303个阳性酵母单克隆；对非冗余后测序序列进行注释，挑选部分候选互作蛋白基因进行克隆，进一步在酵母系统中进行反式激活验证，发现Ta-FBOXL和Ta-ZINC是经过验证的互作蛋白。筛选水稻酵母文库：利用水稻转录因子酵母文库，针对通过自激活验证的诱饵菌株Y1H[pJM5-AbAi]进行筛库实验，初步筛选获得5个阳性单克隆，其中三个基因注释为转录因子；克隆获得3个转录因子在小麦中的直系同源基因，构建了3个直系同源基因的AD载体并进行反式激活验证，发现Ta-R1-MYB是经过验证的互作蛋白。

（2）利用烟草双荧光素酶系统验证启动子-蛋白的互作关系：构建载体：依据酵母单杂交实验结果，针对NLR1-V启动子的-516bp~-317bp区段和-1019bp~-680bp区段，构建了载体LUC-0800-2、LUC-0800-3；针对候选互作蛋白Ta-FBOXL、Ta-ZINC和Ta-R1-MYB构建了载体pBI121:Ta-ZINC、pBI121:Ta-FBOXL、pBI121:Ta-R1-MYB；将构建成功的5个载体质粒分别转化农杆菌。酶活测定：将候选蛋白基因载体与携带对应区段启动子载体共同转化烟草，利用Tecan酶标仪对烟草叶片进行酶活测定，发现转录因子Ta-R1-MYB、Ta-ZINC与NLR1-V启动子存在相互作用，而候选互作蛋白Ta-FBOXL与NLR1-V启动子不存在互作。

2、Ta-R1-MYB和Ta-ZINC的生物信息学分析

Ta-R1-MYB和Ta-ZINC两个基因与NLR1-V启动子的互作经过了酵母和双荧光素酶系统验证，因此，本研究针对这两个基因进行了进一步生物信息学分析。

（1）Ta-R1-MYB的生物信息学分析：在小麦南农9918中克隆Ta-R1-MYB全长序列，其CDS长1932bp、编码蛋白643aa，与中国春参考基因的TraesCS3B02G448900.1序列相似度99.74%，命名为Ta-R1-MYB。该基因编码蛋白包含一个telomere repeat binding factor-like DNA-binding domain结构域，该结构域属于SANT/MYB-like家族，具有结合DNA的功能。在普通小麦中共鉴定到MYB家族基因740个，所有的MYB蛋白都含有SANT/MYB-like结构域。MYB蛋白共分为1R、2R、3R、4R四种类型，1R型蛋白共277个成员，构建1R型系统进化树被分成3类，Ta-R1-MYB互作蛋白属于1R型的第二类，所在分支的进化方向为调节转录活动和维持染色体结构。

（2）Ta-ZINC的生物信息学分析：在小麦92R137中克隆Ta-ZINC的全长序列，其CDS长465bp、编码蛋白155aa，与中国春参考基因的TraesCS1D02G131000.1序列相似度100%，命名为Ta-ZINC。该基因编码蛋白包含一个AN1-like Zinc finger domain结构域，属于锌指家族，具有结合DNA的功能。在普通小麦中共鉴定到锌指家族基因60个，所有的锌指蛋白都含有锌指结构域，互作蛋白基因可能参与逆境胁迫应答。

3、互作基因对NLR1-V的转录调控及功能分析

（1）互作蛋白对NLR1-V的转录调控：利用小麦南农9918叶片接种白粉菌并在侵染后不同时间段取样，研究Ta-R1-MYB和Ta-ZINC互作蛋白基因对白粉菌的应答模式，发现Ta-R1-MYB基因对白粉菌侵染具有明显的应答效应。

（2）互作蛋白参与NLR1-V抗病途径的功能分析：利用VIGS技术在携带NLR1-V基因的抗白粉病小麦南农9918中沉默Ta-R1-MYB和Ta-ZINC基因。与对照相比，2个基因沉默叶片上单位面积内的发育菌丝数量均显著增加。进一步对Ta-R1-MYB和Ta-ZINC沉默叶片中NLR1-V基因的表达情况进行分析，发现NLR1-V在基因沉默植株中几乎不受白粉菌诱导。以上结果表明，Ta-R1-MYB和Ta-ZINC通过调控NLR1-V的表达，参与调控Pm21介导的广谱抗白粉病通路。

（3）互作蛋白相关的NLR1-V启动子顺式作用元件预测：通过多种生物信息网站预测Ta-R1-MYB和Ta-ZINC转录因子相关的NLR1-V启动子互作区段的顺式作用元件，共预测到5个MYB元件，2个锌指元件。推测Ta-R1-MYB和Ta-ZINC基因可能通过与NLR1-V启动子互作，调控其转录活性，参与其抗性途径。

Wheat (Triticum aestivum L.) is one of the most widely cultivated food crops in the world. Powdery mildew caused by Blumeria graminis f. sp. Tritici (Bgt) is one of the most serious wheat diseases and severely threatens wheat production. In our laboratory, the Pm21 gene (NLR1-V), a wheat broad-spectrum powdery mildew resistance gene, was cloned and the upstream promoter sequence of NLR1-V gene was isolated. Characterization of the NLR1-V promoter showed that it was a typical pathogen-inducible promoter. Therefore, it is of great significance to study the transcriptional regulation mechanism of NLR1-V gene to elucidate its broad-spectrum resistance mechanism. In this study, yeast single hybridization was used to screen NLR1-V promoter interaction proteins, and the regulatory functions of NLR1-V promoter interaction proteins were analyzed. This study can help elucidate the molecular mechanism of NLR1-V mediated powdery mildew resistance in wheat. The main results are as follows:

1. Identification and validation of NLR1-V promoter interacting proteins 

(1) Identification of NLR1-V promoter interaction proteins using yeast single hybridization: Previous studies have shown that the -889bp~-335bp region in the upstream of NLR1-V promoter can respond to Bgt induction. The bait vectors covering different segments were constructed corresponding to different region of the NLR1-V promoter -1019bp ~ -317bp segment. The bait plasmids were transformed into yeast cells respectively, and the bait strains were self-activated to find the appropriate Aba sieve concentration. Screening of interaction proteins using wheat yeast library: Using the wheat cDNA library constructed previously in our laboratory, the bait strains Y1H[pJM1-AbAi] and Y1H[pJM2-AbAi] previously verified by self-activation were used to screen the interacting proteins, and a total of 303 positive yeast monoclones were obtained. The non-redundant sequences were annotated, then some candidate interaction protein genes were selected for cloning. Ta-FBOXL and Ta-ZINC were finally verified to be the real positive interaction proteins by transactivation in yeast system. Screening of interaction proteins using rice yeast library: Using the rice transcription factor yeast library, a screening experiment was performed against the bait strain Y1H [pJM5-AbAi] verified by self-activation, and 5 positive monoclonal were preliminarily screened, among which 3 genes were annotated as transcription factors. Three orthologous genes of rice transcription factors in wheat were cloned, and AD vectors were constructed for the transactivation. Ta-R1-MYB was found to be the verified interaction protein.

(1) Verification of promoter-protein interaction using tobacco luciferase assay system: Construction of recombinant vectors: Based on the results of yeast single hybridization experiments, vectors LUC-0800-2 and LUC-0800-3 were constructed covering the -516bp~-317bp and -1019bp~-680bp regions of NLR1-V promoter. Vectors pBI121:Ta-ZINC, pBI121:Ta-FBOXL, pBI121:Ta-R1-MYB were constructed inserted with the candidate interacting proteins Ta-FBOXL, Ta-ZINC and Ta-R1-MYB driven by 35S promoter. The five vectors were transformed into Agrobacterium tumefaciens respectively. Enzyme activity determination: The vector inserted with candidate protein gene and the vector inserted with the corresponding promoter region were used to transform tobacco. The enzyme activity of tobacco leaves was measured by Tecan microplate analyzer, and it was found that the transcription factors Ta-R1-MYB and Ta-ZINC could interact with the NLR1-V promoter. However, no interaction between Ta-FBOXL and the NLR1-V promoter was observed.

2. Bioinformatics analysis of Ta-R1-MYB and Ta-ZINC gene family

The interaction of Ta-R1-MYB and Ta-ZINC with NLR1-V promoters was validated by yeast and luciferase assay systems, so further bioinformatics analysis of Ta-R1-MYB and Ta-ZINC was performed to analyze the evolution.

(1) Bioinformatics analysis of Ta-R1-MYB: The full-length sequence of Ta-R1-MYB was cloned from wheat Nannong 9918 with the 1932bp-CDS encoding a 643 aa protein. The similarity with the Chinese spring reference gene TraesCS3B02G448900.1 was 99.74%. The protein contains a telomere repeat binding factor-like DNA-binding domain, which belongs to the SANT/MYB-like family and has the ability to bind DNA. A total of 740 MYB family genes were identified in common wheat, and all the MYB proteins contained SANT/MYB-like domains. MYB proteins could be divided into four types, including 1R, 2R, 3R and 4R, and there are totally 277 members in the 1R clade. The phylogenetic tree of members in the 1R clade could be further divided into three groups, and Ta-R1-MYB belongs to the group 2. 

(2) Bioinformatics analysis of Ta-ZINC: The full-length sequence of Ta-ZINC was cloned from wheat 92R137 with the 465bp-CDS encoding a 155 aa protein. The similarity of Ta-ZINC to TraesCS1D02G131000.1 was 100%. The protein contains an AN1-like zinc finger domain, which belongs to the zinc finger family and has the DNA-binding function. A total of 60 zinc finger family genes were identified in common wheat. The phylogenetic three showed that Ta-ZINC belonged to a branch which was mostly involved in the response to stresses.

3. Transcriptional regulation and functional analysis of interacting protein genes on NLR1-V

(1) Response of Ta-R1-MYB and Ta-ZINC to Bgt: In order to study the response pattern of Ta-R1-MYB and Ta-ZINC to Bgt, the leaves of wheat Nannong9918 were inoculated with Bgt and sampled at different time points after infection. It was found that Ta-R1-MYB gene was obviously responsive to Bgt. 

(2) Function of Ta-R1-MYB and Ta-ZINC involved in NLR1-V-mediated resistance pathway: Ta-R1-MYB and Ta-ZINC genes were silenced in the powdery mildew resistance wheat Nannong 9918 by VIGS technique. Compared with the control, the number of developed hyphae per unit area on Ta-R1-MYB or Ta-ZINC silenced leaves increased significantly. Further analysis of NLR1-V gene expression showed that the response of NLR1-V to Bgt in the Ta-R1-MYB or Ta-ZINC silenced leaves was significantly decreased. These results suggested that Ta-R1-MYB and Ta-ZINC are involved in the NLR1-V-mediated broad-spectrum powdery mildew resistance pathway through regulating the its transcription. 

(3) Prediction of the cis-elements of NLR1-V promoter associated with the interacting proteins: Cis-elements of the NLR1-V promoter interaction segment associated with Ta-R1-MYB and Ta-ZINC transcription factors were predicted through various bioinformatic tools. A total of 5 MYB elements and 2 zinc finger elements were predicted. It was speculated that Ta-R1-MYB and Ta-ZINC genes might interact with NLR1-V promoter to regulate their transcriptional activity and take part in the resistance pathway.

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