作物学报 ›› 2021, Vol. 47 ›› Issue (11): 2134-2146.doi: 10.3724/SP.J.1006.2021.04254
陈玉婷1,2(), 刘露1,2, 楚盼盼1,2, 魏嘉贤1,2, 钱慧娜1,2, 陈华1,2,3, 蔡铁城1,2,3, 庄伟建1,2,3,*(), 张冲1,2,3,*()
CHEN Yu-Ting1,2(), LIU Lu1,2, CHU Pan-Pan1,2, WEI Jia-Xian1,2, QIAN Hui-Na1,2, CHEN Hua1,2,3, CAI Tie-Cheng1,2,3, ZHUANG Wei-Jian1,2,3,*(), ZHANG Chong1,2,3,*()
摘要:
前期研究报道超表达花生AhRRS5基因能够显著提高烟草抗青枯病水平, 为进一步探究NBS-LRR类抗病蛋白AhRRS5在花生应答青枯菌胁迫的信号通路, 本研究在构建花生受青枯菌诱导的根部组织均一化三框文库的基础上, 通过酵母双杂交技术筛选AhRRS5的互作蛋白。通过改良的CTAB法提取青枯菌诱导后不同时间点的花生根部组织样品总RNA, 分离纯化mRNA并合成双链cDNA, 并基于同源重组方法分别构建酵母双杂交初级和次级文库。构建的酵母双杂交次级cDNA文库库容为1.44×107 cfu mL-1, 重组率为100%, 插入片段大小在1000 bp以上。通过酶切连接法构建pGBKT7-AhRRS5诱饵载体, 在酵母细胞中无自激活和毒性活性, 与酵母双杂交文库共转酵母Y2H Gold菌株后, 经多次筛库和回转验证, 最终获得12个候选互作蛋白, 这些蛋白涉及到植物生长发育、能量代谢、激素信号转导、胁迫响应等多个方面。通过双分子荧光互补(bimolecular fluorescence complementation, BiFC)验证了AhSBT1.6和AhRRS5的体内互作。转录组数据显示, 花生AhSBT1.6基因在不同组织中表达差异显著; 实时荧光定量PCR显示, 该基因在抗青枯病花生品种中受青枯菌诱导上调表达, 推测AhSBT1.6可能参与调控花生青枯病抗性。研究结果为进一步研究NBS-LRR类抗病蛋白AhRRS5和互作蛋白在花生青枯病抗性防御的作用机制奠定了基础。
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