中国农业科学 ›› 2015, Vol. 48 ›› Issue (4): 695-704.doi: 10.3864/j.issn.0578-1752.2015.04.07

• 植物保护 • 上一篇    下一篇

β1-和β2-微管蛋白基因在赤霉病菌抗多菌灵中的作用

曾凡松,尹合兴,史文琦,汪华,杨立军,龚双军,张学江,向礼波,喻大昭   

  1. 湖北省农业科学院植保土肥研究所/农业部华中作物有害生物综合治理重点实验室,武汉430064
  • 收稿日期:2014-09-02 出版日期:2015-02-16 发布日期:2015-02-16
  • 通讯作者: 喻大昭,Tel:027-87380089;E-mail:dazhaoyu@china.com
  • 作者简介:曾凡松,Tel:027-87380681;E-mail:zengfansong2005@126.com。尹合兴,Tel:027-87380681;E-mail:yhxmy123456@163.com 。曾凡松与尹合兴为同等贡献作者。
  • 基金资助:
    国家科技支撑计划(2012BAD19B04)、国家公益性行业(农业)科研专项(201303016)

Function Analysis of β1-tub and β2-tub in Resistance of Gibberella zeae to Carbendazim

ZENG Fan-song, YIN He-xing, SHI Wen-qi, WANG Hua, YANG Li-jun, GONG Shuang-jun, ZHANG Xue-jiang, XIANG Li-bo, YU Da-zhao   

  1. Institute for Plant Protection and Soil Fertilizer, Hubei Academy of Agricultural Sciences/Laboratory of Integrated Pest Management on Crop in Central China, Ministry of Agriculture, Wuhan 430064
  • Received:2014-09-02 Online:2015-02-16 Published:2015-02-16

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=7.911 μg·mL-1)、Js462(EC50=6.515 μg·mL-1)、Js484(EC50=5.031 μg·mL-1)、Js506(EC50=8.455 μg·mL-1)和Js519(EC50=6.280 μg·mL-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=0.552 μg·mL-1)进行比对。采用实时荧光定量PCR(qPCR)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)。5 μg·mL-1多菌灵能诱导Js506菌株的β1-tub表达量显著上调(P<0.05)。10 μg·mL-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2.839 μg·mL-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、△β2tub- Js506-2),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=0.078 μg·mL-1)和△β2tub-Js506-2(EC50=0.072 μg·mL-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=7.521 μg·mL-1)和β2tub-Js506-C2(EC50=7.243 μg·mL-1),2个互补转化体均使敲除体△β2tub- Js506-1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β2-tub均能影响赤霉病菌对多菌灵的抗性。

关键词: 小麦赤霉病菌, 微管蛋白基因, 多菌灵, 超量表达

Abstract: 【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·mL-1), Js462 (EC50=6.515 μg·mL-1), Js484 (EC50=5.031 μg·mL-1), Js506 (EC50=8.455 μg·mL-1) and Js519 (EC50=6.280 μg·mL-1), were cloned and sequenced, and alignment were carried out among these sequences with those of HG-1 (EC50=0.552 μg·mL-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 (qPCR). 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·mL-1 carbendazim at a significant level of 0.05. Carbendazim at 10 μg·mL-1 did not exert an influence on expression level of b2-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·mL-1 and enhanced resistance to carbendazim (P<0.05). No significant difference was presented in resistance level between the mutant with overexpressed b2-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 b2-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·mL-1) and β2tub-Js506-C2 (EC50=7.243 μg·mL-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.

Key words: Gibberella zeae (Schwein.) Petch, tubulin gene, carbendazim, overexpression