| 张露凡，宋普文，毋柳柳，胡海燕，李成伟.小麦果糖激酶基因TaFRK与TaPFT的互作分析[J].麦类作物学报,2021,(11):1339 |
| 小麦果糖激酶基因TaFRK与TaPFT的互作分析 |
| Interaction Analysis of Fructokinase TaFRK and TaPFT Genes in Wheat |
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| DOI：10.7606/j.issn.1009-1041.2021.11.03 |
| 中文关键词: 小麦 赤霉病 酵母双杂交 双分子荧光互补 TaPFT RT-qPCR TaFRK |
| 英文关键词:Wheat Fusarium head blight Yeast-two-hybrid Two-molecule fluorescence complementation TaPFT RT-qPCR TaFRK |
| 基金项目:国家自然科学基金项目（31872129）；河南省科技攻关计划项目（192102110131，192102110030） |
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| 中文摘要: |
| 小麦赤霉病是全球性病害，严重威胁着中国的小麦粮食安全。目前已经明确的抗赤霉病位点有7个（ Fhb1~ Fhb7）。 其中 Fhb1位点是3BS染色体上稳定性和效应最大的抗扩展位点。 TaPFT是 Fhb1位点上的抗性基因之一，但其抗性机理并不清楚。本研究以TaFRK为诱饵蛋白通过酵母双杂交技术筛选小麦酵母双杂交文库，从而得到互作基因 TaFRK，其编码蛋白为果糖激酶。进一步通过构建酵母双杂交载体和双分子荧光互补载体分析 TaFRK与 TaPFT之间的互作关系，并通过qPCR分析 TaFRK受到禾谷镰刀菌孢子诱导后的表达模式。通过酵母双杂交试验发现，共转pGBKT7 - TaPFT和pGADT7 - TaFRK 的酵母菌株Y2HGlod在含有X-α-Gal和金担子素A（Aureobasidin A,AbA）的四缺培养基SD/-His-Leu-Trp-Ade上长出蓝色菌落，证明 TaFRK与 TaPFT在酵母中互作。通过双分子荧光互补试验发现，在共转YN- TaPFT和YC- TaFRK表达载体的烟草叶片中可观察到强烈的黄色荧光信号，表明 TaFRK与 TaPFT在植物细胞中能够发生相互作用。实时荧光定量PCR分析发现，禾谷镰刀菌孢子侵染抗性小麦后， TaFRK基因表达量升高，说明 TaFRK基因的表达受禾谷镰刀菌侵染诱导。本研究结果对进一步研究 TaPFT的赤霉病抗性机制具有一定的参考 价值。 |
| 英文摘要: |
| Fusarium head blight (FHB) is a global disease,and also a serious threat to China's wheat food security. At present,there are seven FHB resistance genes( Fhb1 -Fhb7 ). Fhb1 locus is the most stable and effective anti-extension gene on 3BS chromosome. Wheat PFT(pore-forming toxin-like) (TaPFT ) is one of the genes that confer resistance to Fhb1 ,but its resistance mechanism is not clear. In this experiment,we used PFT as bait to screen wheat yeast-two-hybrid library to obtain the interaction gene TaFRK . The protein encoded by TaFRK is fructokinase. In this study,the interaction between TaFRK and TaPFT was analyzed by constructing yeast two hybrid vector and bimolecular fluorescent complementary vector. The expression pattern of TaFRK induced by Fusarium graminearum spores was analyzed by real-time PCR. A yeast two hybrid experiment was conducted,the yeast strain Y2HGlod transformed with pGBKT7 - TaPFT and pGADT7 - TaFRK showed normal growth and blue color on SD/-His-Leu-Trp-Ade medium containing Aureobasidin A(AbA）and X-α-Gal,indicating that TaFRK interacts with TaPFT in yeast cells. In the bimolecular fluorescence complementation experiment,strong yellow fluorescence signal was observed in the cytoplasm of Nicotiana benthamiana cells that were co-transformed with YN-TaPFT and YC-TaFRK vectors,which further indicated that TaFRK interacts with TaPFT in plant cells. Real-time PCR analysis found that the expression of TaFRK gene was increased after inoculation with Fusarium graminearum spores,indicating that the expression of TaFRK gene was induced by Fusarium graminearum infection. The results have certain reference value for further research on the resistance mechanism of TaPFT against to FHB. |
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