稻曲病菌侵染水稻颖花的酵母双杂交cDNA文库构建与应用

doi:10.3864/j.issn.0578-1752.2016.05.006

摘要/Abstract

摘要： 【目的】近年来稻曲病日益严重，但目前对稻曲病菌(Ustilaginoidea virens)与水稻相互作用机制仍不清楚。论文旨在利用水稻感病颖花材料构建酵母双杂交文库并筛选稻曲病菌效应因子互作蛋白，为解析稻曲病菌侵染水稻机制提供理论基础。【方法】以感病水稻Chuannong H2S为试验材料，在水稻破口前5—7 d左右，从水稻穗中上部接种PSB摇培7 d的稻曲病菌荧光标记菌株P4，接种13 d后取颖花进行激光共聚焦观察，收集感病颖花。提取感病颖花总RNA，使用含有Oligo(dT)的接头引物反转录cDNA第一链，并PCR扩增ds cDNA，通过琼脂糖凝胶电泳切胶回收ds cDNA片段，与载体pGADT7-Rec进行同源重组，转化酵母菌株Y187构建酵母双杂交cDNA文库。然后用稻曲病菌效应因子Uv_1261基因构建诱饵载体，将诱饵载体转化入Y2HGold酵母菌株，通过酵母双杂交自激活验证后以该诱饵载体筛选酵母双杂交文库，最终在SD/-Ade/-His/-Leu/-Trp/X-α-Gal平板上筛选出生长状况良好的蓝色单菌落，经测序得到候选互作蛋白的序列，通过NCBI在线网站进行Blast分析和Uniprot在线网站进行gene ontology（GO）注释。【结果】经检测，文库滴度为5.7×10⁸cfu/mL，平均插入片段大小为750 bp，表明稻曲病菌侵染水稻颖花cDNA文库质量较高。用Uv_1261构建诱饵载体，转化Y2HGold后，转化菌可在SD/-Trp、SD/-Trp/X-α-Gal平板上生长，不能在SD/-Trp/X-α-Gal/AbA平板上生长，表明Uv_1261无自激活现象。以该诱饵载体筛选文库，对筛选到的候选互作蛋白进行测序验证并Blast分析获得了56个来自稻曲病菌或水稻的候选互作蛋白，其中有28个候选互作蛋白来自稻曲病菌，有16个来自水稻，以及12个未知蛋白。经GO注释显示，来自稻曲病菌中的候选互作蛋白参与了17个生物过程，包括翻译、代谢过程、氧化还原及胞内氨基酸合成等；分子功能包括金属离子结合活性、水解酶活性及ATP结合活性等；细胞组分包括核糖体、细胞质及线粒体等。来自水稻中的候选互作蛋白参与了11个生物过程，包括蛋白质去磷酸化、糖代谢及翻译等；分子功能包括金属离子结合活性、核苷酸结合活性及转移酶活性等；细胞组分包括核糖体、膜及细胞核等。【结论】该cDNA文库质量较好，能成功地用于稻曲病菌效应蛋白的互作蛋白筛选，可为研究稻曲病菌与水稻相互作用的分子机制提供重要资源。

关键词: 稻曲病菌, 水稻, 效应蛋白, cDNA文库, 酵母双杂交

Abstract: 【Objective】The occurrence of rice false smut disease is increasing in rice production worldwide, but the underlying mechanism of Ustilaginoidea virens-rice interaction is still unclear. The objective of this study is to construct a yeast two-hybrid library from rice spikelets infected with U. virens, and apply it for screening interacting proteins of a U. virens effector Uv_1261, so as to provide valuable resource for studying the mechanism of U. virens-rice interaction. 【Method】A susceptible indica rice Chuannong H2S was challenged with the inoculum of a GFP-tagged U. virens strain P4. Infected rice spikelets were collected after confirmation by a confocal laser scanning miscroscope at 13 days post inoculation (dpi), when pathogen hyphae extended into inner floral organs of rice spikelets. The total RNA was isolated from U. virens-infected spikelets, and used for synthesizing the first-strand cDNA by Oligo (dT) primer. ds cDNA was amplified by PCR and purified by agarose gel electrophoresis before mixed with vector pGADT7-Rec. The yeast two-hybrid cDNA library was constructed by homologous recombination technology in yeast line Y187. Then Uv_1261 bait vector was constructed and subjected to self-activation verification. Then the library was screened, and the blue colonies that grew well in medium containing SD/-Ade/-His/-Leu/-Trp/X-α-Gal were selected as candidate interacting proteins of Uv_1261. After sequencing confirmation, these candidates were annotated and classified by Blast and gene ontology (GO) analyses. 【Result】The cDNA library titer was 5.7×10⁸cfu/mL and the average length of the insertions was 750 bp, indicating that this library is of high quality. Yeast transformation analysis showed that Uv_1261 had no self-activation. Screening for the library with Uv_1261 revealed 56 candidate interacting proteins including 28 proteins in U.virens, 16 protiens in rice and 12 unknown proteins. The candidate interacting proteins from U. virens were involved in 17 biological processes including translation, metabolic process, oxidation-reduction process, cellular amino acid biosynthetic process, etc; or possessed molecular functions such as metal ion binding, hydrolase activity, ATP binding, etc; or were associated with cell components like ribosome, cytoplasm, mitochondrion, etc. Uv_1261-interacting proteins from rice were involved in 11 biological processes including protein dephosphorylation, carbohydrate metabolic process, translation, etc; or possessed molecular functions such as metal ion binding, nucleotide binding, transferase activity; or were associated with cell components like ribosome, membrane, nucleus, etc. 【Conclusion】The cDNA library has good quality, and is suitable for identifying interacting proteins of U. virens effectors. This work can provide a valuable resource for studying molecular interaction between U. virens and rice.

Key words: Ustilaginoidea virens, rice, effector, cDNA library, yeast two-hybrid

王宇秋，李国邦，杨娟，黎良，赵志学，樊晶，王文明. 稻曲病菌侵染水稻颖花的酵母双杂交cDNA文库构建与应用[J]. 中国农业科学, 2016, 49(5): 865-873.

WANG Yu-qiu, LI Guo-bang, YANG Juan, LI Liang, ZHAO Zhi-xue, FAN Jing, WANG Wen-ming. Construction and Application of a Yeast Two-Hybrid cDNA Library from Rice Spikelets Infected with Ustilaginoidea virens[J]. Scientia Agricultura Sinica, 2016, 49(5): 865-873.

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