中国农业科学 ›› 2020, Vol. 53 ›› Issue (12): 2371-2384.doi: 10.3864/j.issn.0578-1752.2020.12.006
齐悦1,吕峻元1,张悦1,韦杰1,张娜1,杨文香1(),刘大群2(
)
收稿日期:
2019-12-26
出版日期:
2020-06-16
发布日期:
2020-06-25
通讯作者:
杨文香,刘大群
作者简介:
齐悦,E-mail:908952718@qq.com。
基金资助:
QI Yue1,LÜ JunYuan1,ZHANG Yue1,WEI Jie1,ZHANG Na1,YANG WenXiang1(),LIU DaQun2(
)
Received:
2019-12-26
Online:
2020-06-16
Published:
2020-06-25
Contact:
WenXiang YANG,DaQun LIU
摘要:
【目的】由小麦叶锈菌(Puccinia triticina)引起的小麦叶锈病是影响小麦生产的主要病害之一,在小麦与叶锈菌互作的过程中病菌向寄主细胞分泌效应蛋白,以调控寄主防御反应、发挥毒性功能。开展对小麦叶锈菌效应蛋白的研究,探索小麦叶锈菌的致病机制,为病害的持续防控提供依据。【方法】以小麦叶锈菌13-5-72与感病品种Thatcher互作的cDNA为模板扩增效应蛋白Pt18906,通过SignalP 4.1、TargetP 1.1、TMHMM 2.0和EffectorP 2.0软件对Pt18906进行序列特征分析,利用在线软件Swiss-Model预测Pt18906的三级结构,利用在线软件SOPMA预测Pt18906的二级结构。采用实时荧光定量PCR对Pt18906的表达模式进行分析,借助于烟草的异源表达系统对Pt18906进行抑制Bax和INF1诱导的细胞程序性死亡(programmed cell death, PCD)能力验证,利用酵母系统验证Pt18906的信号肽是否具有分泌功能,采用氨基酸逐步缺失的方法缺失突变Pt18906,从而确定其功能毒性motif;通过在烟草中瞬时表达Pt18906-GFP融合蛋白,结合质壁分离技术分析Pt18906的亚细胞定位,得出效应蛋白的作用位点;利用瞬时表达技术在以Thatcher为背景的不含抗病基因和含有不同抗病基因的全套近等基因系上开展Pt18906无毒性功能分析;采用细菌三型分泌系统(Type Ⅲ secretion system)介导的瞬时转化分析Pt18906对寄主防御反应的调控。【结果】从小麦叶锈菌13-5-72与感病品种Thatcher互作6 d的转录组文库中获得一个在接种24 h后显著高表达的、基因全长序列672 bp、编码223个氨基酸的候选效应蛋白Pt18906,该效应蛋白缺乏已知的功能结构域和保守基序,工作环境偏碱性,在烟草细胞中瞬时表达Pt18906,Pt18906能够抑制Bax和INF1诱导的细胞程序性死亡,表明该效应蛋白具有毒性功能,并且通过构建缺失突变体明确其28—47位氨基酸对其毒性功能具有重要作用,该效应蛋白定位于细胞核和细胞质,表明其作用于细胞内。Pt18906在单基因系抗病品种TcLr27+31和TcLr42上能够引起过敏性坏死反应,表明该效应蛋白的无毒性,Pt18906能够引起TcLr27+31中胼胝质的积累和活性氧的迸发,胼胝质随注射时间的增加而逐渐积累,活性氧在注射后的10 min达到最高。【结论】位于28—47位的氨基酸决定Pt18906的毒性主要功能,Pt18906能激发小麦TcLr27+31双层防御反应。
齐悦,吕峻元,张悦,韦杰,张娜,杨文香,刘大群. 小麦叶锈菌效应蛋白Pt18906激发TcLr27+31的双层防御反应[J]. 中国农业科学, 2020, 53(12): 2371-2384.
QI Yue,LÜ JunYuan,ZHANG Yue,WEI Jie,ZHANG Na,YANG WenXiang,LIU DaQun. Puccinia triticina Effector Protein Pt18906 Triggered Two-Layer Defense Reaction in TcLr27+31[J]. Scientia Agricultura Sinica, 2020, 53(12): 2371-2384.
表1
本试验所用引物"
引物类型 Primer type | 引物名称 Primer name | 引物序列 Primer sequence (5′-3′) | 序列长度 Sequence length (bp) |
---|---|---|---|
Pt18906的ORF引物 Primer for Pt18906 ORF 信号肽分泌功能验证 Function verification of signal peptide secretion 异源瞬时表达 Heterogeneous transient expression 亚细胞定位 Subcellular localization 毒性结构域验证 Verification of virulent domain 细菌三型分泌系统 Bacterial type Ⅲ secretion system | ORFPt18906-F ORFPt18906-R SPPt18906-F SPPt18906-R Pt18906-F ΔSPPt18906-F Pt18906-R LBA LBB DPt18906-F DPt18906-R qPt18906-F1 qPt18906-R1 qPt18906-R2 qPt18906-R3 qPt18906-R4 PPt18906-F PPt18906-R | ATGTTTTCAGCAAGTTCAAT CTACTTACCCTTCTCCTTAG CCGGAATTCATGTTTTCAGCAAGTTCAAT CCGCTCGAGAAGCTCAACGGGTGGTAAGC TCCCCCGGGATGTTTTCAGCAAGTTCAAT TCCCCCGGGGCCGAAGTCCAACGACACGC GCGTCGACCTACTTACCCTTCTCCTTAG CAATCACAGTGTTGGCTTGC GACCCTATGGGCTGTGTTG CCCATCGATGCCGAAGTCCAACGACACGC TCCCCCGGGCTTACCCTTCTCCTTAGGAT CCCATCGATGCTCCACTCAAAAACGGTGA TCCCCCGGGTTGGTTGTTAACGTCCTCAA TCCCCCGGGGACCGCCATGTATCCGGCTA TCCCCCGGGGGGGTCTATCTCTGTAGGAT TCCCCCGGGTATGCTCCCGTATTTGCATG CACCATGTTTTCAGCAAGTTCAAT CTACTTACCCTTCTCCTTAG | 20 20 29 29 29 29 28 21 20 29 29 29 29 29 29 29 24 20 |
图7
在小麦中过表达Pt18906激发坏死反应 A:在TcLr27+31过表达Pt18906激发坏死Overexpression of Pt18906 in TcLr27+31 stimulates HR;B:在TcLr42过表达Pt18906激发坏死Overexpression of Pt18906 in TcLr42 stimulates HR 1:注射的表型结果Phenotypic results of injection;2:4倍荧光显微镜下的H2O2 The H2O2 under fluorescence microscope (4×);3:20倍荧光显微镜下的H2O2 The H2O2 under fluorescence microscope (20×)"
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