摘要
【目的】揭示β1-微管蛋白基因(β1-tub)和β2-微管蛋白基因(β2-tub)在赤霉病菌(Gibberella zeae)对多菌灵的抗性过程中所起的作用。【方法】采用PCR克隆测序法测定Js449(EC50=7.911μg·m L-1)、Js462(EC50=6.515μg·m L-1)、Js484(EC50=5.031μg·m L-1)、Js506(EC50=8.455μg·m L-1)和Js519(EC50=6.280μg·m L-1)等5个多菌灵抗性菌株的α-、β1-、β2-、γ-tub序列,并与敏感菌株HG-1(EC50=0.552μg·m L-1)进行比对。采用实时荧光定量PCR(q PCR)测定β1-tub和β2-tub在多菌灵胁迫下的抗性菌株Js506中的相对表达量。构建β1-tub和β2-tub的超量表达载体,分别在HG-1中表达。运用split PCR获得含有潮霉素磷酸转移酶基因和目标基因的融合片段,并对Js506进行原生质体转化,通过同源重组获得β2-tub的敲除体和互补体。对菌株Js506、HG-1及其突变体分别进行多菌灵敏感性测定、菌落生长观察和致病力测定。【结果】基因比对结果表明,5个抗性菌株的α-、β1-、γ-tub基因序列与敏感菌株的一致。对β2-tub序列比对结果表明,Js449、Js462和Js506菌株的第167位氨基酸由苯丙氨酸(Phe)变为酪氨酸(Tyr)。Js484菌株的第200位氨基酸由苯丙氨酸(Phe)变为酪氨酸(Tyr)。Js519菌株的第198位氨基酸由谷氨酸(Glu)变为谷氨酰胺(Gln)。5μg·m L-1多菌灵能诱导Js506菌株的β1-tub表达量显著上调(P<0.05)。10μg·m L-1的多菌灵对Js506菌株的β2-tub表达量影响不显著。β1-tub的超量表达使HG-1突变体的EC50增加至2.839μg·m L-1,抗药性显著增强(P<0.05)。β2-tub超量表达突变体的抗性水平与野生型菌株无显著差异。对Js506菌株的β2-tub进行敲除试验,分别获得了2个转化体(△β2tub-Js506-1、△β2tubJs506-2),经潮霉素抗性筛选、PCR和Southern杂交验证,确认2个转化体均不含有β2-tub。与野生型菌株Js506相比,敲除体△β2tub-Js506-1(EC50=0.078μg·m L-1)和△β2tub-Js506-2(EC50=0.072μg·m L-1)对多菌灵均表现为超级敏感,且菌落生长变慢,致病力显著下降(P<0.05)。对△β2tub-Js506-1进行互补转化获得了2个互补体,β2tub-Js506-C1(EC50=7.521μg·m L-1)和β2tub-Js506-C2(EC50=7.243μg·m L-1),2个互补转化体均使敲除体△β2tubJs506-1基本恢复了抗性、菌落生长速率和致病力。【结论】5个赤霉病菌菌株对多菌灵的抗性与β2-tub的第167、198、200位密码子突变有关,与α-、β1-和γ-tub序列的突变无关。β1-tub在多菌灵胁迫下诱导表达,且β1-tub的超量表达能增强赤霉病菌对多菌灵的抗性。β2-tub是小麦赤霉病菌对多菌灵抗性所必需的,β1-tub和β2tub均能影响赤霉病菌对多菌灵的抗性。
【Objective】The objective of this study is to reveal the function of β1-tub and β2-tub in resistance of Gibberella zeae to carbendazim.【Method】α-,β1-,β2- and γ-tub of 5 carbendazim resistant strains,Js449（EC50=7.911 μg·m L-1）,Js462（EC50=6.515 μg·m L-1）,Js484（EC50=5.031 μg·m L-1）,Js506（EC50=8.455 μg·m L-1） and Js519（EC50=6.280 μg·m L-1）,were cloned and sequenced,and alignment were carried out among these sequences with those of HG-1（EC50=0.552 μg·m L-1）,a carbendazim sensitive strain,respectively.Relative expression levels of β1-tub and β2-tub in Js506,a resistant strain,in response to carbendazim were detected using real-time quantitative PCR（q PCR）.The overexpression vectors harboring β1-tub and β2-tub were constructed and transferred into HG-1.Hygromycin phosphotransferase gene and flanking sequences of target gene were fused by split PCR.The entire β2-tub locus was deleted from Js506 and complementation was also performed by protoplast transformation and homologous recombination.Sensitivity to carbendazim,colony growth and pathogenicity of Js506,HG-1 and their mutants were tested.【Result】 No mutation was detected in α-,β1- and γ-tub of 5 resistant strains based on DNA sequence alignment with corresponding sequences of HG-1.The multiple sequence alignment for β2-tub revealed a mutation（Phe 167 Tyr） at the codon 167 in Js449,Js462 and Js506 and at the codon 200 in Js484.A change at the codon 198 from Glu to Gln was also detected in Js519.Expression of β1-tub was induced in Js506 by treatment of 5 μg·m L-1 carbendazim at a significant level of 0.05.Carbendazim at 10 μg·m L-1 did not exert an influence on expression level of β2-tub in Js506 significantly.Overexpression of β1-tub in HG-1 resulted in an increase of the value of EC50 up to 2.839 μg·m L-1 and enhanced resistance to carbendazim（P〈0.05）.No significant difference was presented in resistance level between the mutant with overexpressed β2-tub and the wild strain.Two transformants,Δβ2tub-Js506-1 and Δβ2tub-Js506-2,were generated by knocking out assay and validation was carried out by hygromycin screening,PCR amplification and Southern blotting.Compared with the parent strain,both two deletion mutants of β2-tub locus displayed supersensitivity to carbendazim,slower growth of colony,and reduced pathogenicity（P〈0.05）.Two complementation mutants of △β2tub-Js506-1,β2tub-Js506-C1（EC50=7.521 μg·m L-1） and β2tub-Js506-C2（EC50=7.243 μg·m L-1）,were identified and these biological characteristics were almost restored in the two transformants by genetic complementation.【Conclusion】Resistance of 5 strains was correlated with point mutations at codons 167,198 and 200 in the β2-tub but not with mutations in the β1-tub sequence.Treatment of culture with carbendazim induced β1-tub transcript levels and overexpression of β1-tub improved its resistance to carbendazim.β2-tub is necessary for resistance of G.zeae to the fungicide,and both two tubulin genes are able to affect the resistance.
出处
《中国农业科学》
CAS
CSCD
北大核心
2015年第4期695-704,共10页
Scientia Agricultura Sinica
基金
国家科技支撑计划(2012BAD19B04)
国家公益性行业(农业)科研专项(201303016)
关键词
小麦赤霉病菌
微管蛋白基因
多菌灵
超量表达
Gibberella zeae（Schwein.） Petch
tubulin gene
carbendazim
overexpression
