受青枯菌诱导的花生根酵母双杂交文库构建和AhRRS5互作蛋白的筛选

Construction of yeast two-hybrid cDNA library induced by Ralstonia solana-cearum and interaction protein screening for AhRRS5 in peanut

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1.44×10^(7) 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。

The overexpression of peanut AhRRS5 gene can significantly improve tobacco resistance to bacterial wilt in previous studies.To further explore signaling pathway of the NBS-LRR resistance protein AhRRS5 responding to Ralstonia solanacearum infection in peanut,the interaction proteins of AhRRS5 were screened by yeast two hybrid technology based on the construction of peanut root normalized three-frame libraries induced by Ralstonia solanacearum.Total RNA was extracted from peanut roots at different time points after Ralstonia solanacearum infection.The mRNA was isolated and purified,then double stranded cDNA was synthesized and normalized.The primary and secondary libraries were constructed by homologous recombination method.The titer of the secondary library was 1.44×10^(7) cfu mL^(-1),the recombination rate was 100%,and the average length encoded by the inserted cDNA was more than 1000 bp.The bait vector pGBKT7-AhRRS5 was constructed by enzyme digestion and ligation method.Result showed that there was no toxicity and auto-activation in the yeast cells.The AD library plasmid and bait vector pGBKT7-AhRRS5 were co-transformed into yeast Y2H gold strain.After several screening and rotation verification,12 candidate proteins were obtained,which were involved in plant growth and development,energy metabolism,hormone signal transduction,stress response and so on.The interaction of AhRRS5 with AhSBT1.6 was further confirmed by bimolecular fluorescence com-plementation assays(BiFC)in vivo.The relative expression levels of AhSBT1.6 genes revealed that there were significant differ-ences in different tissues based on transcriptome profiling,indicating the potential involvement of AhSBT1.6 in regulating bacte-rial with resistance in peanut.This study lays a foundation for further study on the mechanism of NBS-LRR resistance protein AhRRS5 and its interaction protein in bacterial wilt resistance defense of peanut.