禾谷镰孢菌（Fusarium graminearum）是引起赤霉病最主要的病原菌之一。赤霉病不仅严重影响小麦的产量，而且降低小麦的品质，禾谷镰孢菌可产生多种衍生真菌毒素及次生代谢物质，如脱氧雪腐镰刀菌烯醇（Deoxynivalenol，DON）和雪腐镰刀菌烯醇（Nivalenol，NIV）等，可引起人类和哺乳动物中毒，严重威胁着人和动物的健康。灰葡萄孢菌（Botrytis cinerea）引起的灰霉病是我国许多果树、蔬菜、草莓、花卉等植物上的重要病害，尤其在保护地生产的蔬菜及草莓上引起果实腐烂, 损失比较严重。 自1970年沈阳化工研究院张少铭等实现多菌灵的工业化生产以来，多菌灵等苯并咪唑类杀菌剂用于赤霉病和灰霉病的防治至今已有40多年的历史。苯并咪唑类药剂可与植物病原真菌细胞的β-微管蛋白结合，阻止纺锤丝的形成，从而抑制细胞有丝分裂，植物病原真菌在生物进化过程中保持了微管蛋白基因的随机突变性质，以适应不良的生存环境，因此植物病原真菌对苯并咪唑类杀菌剂的抗药性大多是病原真菌细胞内编码β-微管蛋白的某些特定氨基酸发生突变，使苯并咪唑类杀菌剂与作用靶标的亲和性下降或丧失而表现抗药性，并且大多数植物病原真菌田间抗性菌株的氨基酸突变一般位于β-微管蛋白的198位或200位两个编码位点。灰葡萄孢菌（Botrytis cinerea）β-微管蛋白198位的谷氨酸（Glu-E）突变为丙氨酸（Ala-A）、赖氨酸（Lys-K）或缬氨酸（Val-V）都导致其对多菌灵产生高水平抗性，或200位的苯丙氨酸（Phe-F）突变为酪氨酸（Tyr-Y）导致其对多菌灵产生中等水平抗性产生。禾谷镰孢菌（Fusarium graminearum）中存在β1和β2两个微管蛋白基因，并且禾谷镰孢菌对多菌灵不同敏感型菌株的β1-微管蛋白基因相同，没有发生突变，而β2-微管蛋白73、167、198或200位氨基酸突变能够导致禾谷镰孢菌对多菌灵产生不同水平的抗药性，73位谷氨酰胺（Gln-Q）突变为精氨酸（Arg-R）或198位谷氨酸（Glu-E）突变为亮氨酸（Leu-L）导致禾谷镰孢菌对多菌灵产生高水平抗药性，167或200位苯丙氨酸（Phe-F）突变为酪氨酸（Tyr-Y）导致禾谷镰孢菌对多菌灵产生中等水平抗药性。说明禾谷镰孢菌对多菌灵的抗性机制不同于灰葡萄孢菌。 本文采用Double-joint PCR方法体外构建含抗药性编码的灰葡萄孢菌β-微管蛋白基因载体，通过PEG介导的原生质体转化法同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因，并进一步敲除获得的转化子菌株中另外的β2-微管蛋白基因或β1-微管蛋白基因，获得抗多菌灵β-微管蛋白基因同源置换禾谷镰孢菌β1-微管蛋白基因转化子菌株及其β2-微管蛋白基因敲除突变体菌株、同源置换禾谷镰孢菌β2-微管蛋白基因转化子菌株及其β1-微管蛋白基因敲除突变体菌株。通过半定量RT-PCR测定抗多菌灵β-微管蛋白基因同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因后在禾谷镰孢菌中的表达情况，测定禾谷镰孢菌菌株和各转化子菌株对多菌灵的敏感性、菌丝生长速率、产分生孢子能力、产子囊壳能力和致病力等生物学特性，以探明不同植物病原真菌间微管蛋白基因能否替代以及抗多菌灵灰葡萄孢菌的β-微管蛋白基因能否在禾谷镰孢菌中表达，抗多菌灵β-微管蛋白基因、禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因的相互作用关系，为进一步揭示禾谷镰孢菌对多菌灵的抗性机制，以及多菌灵与β-微管蛋白的作用机制提供参考依据。本文的主要研究结果如下所述。 本文采用Double-joint PCR方法体外构建含抗药性编码的β-微管蛋白基因载体，通过PEG介导的原生质体转化法同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因，成功获得了同源置换后的转化子菌株，表明不同植物病原真菌间β-微管蛋白基因可以替代。 通过测定抗多菌灵β-微管蛋白基因同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因获得的转化子菌株对多菌灵的敏感性结果表明，抗多菌灵β-微管蛋白基因同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因后禾谷镰孢菌对多菌灵敏感性降低，但不表现抗药性。 通过半定量RT-PCR测定了抗多菌灵β-微管蛋白基因同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因后在禾谷镰孢菌中的表达水平，结果表明β-微管蛋白基因在禾谷镰孢菌中在mRNA水平能够表达，同源置换β1-微管蛋白基因后的表达水平和β1-微管蛋白基因相比没有显著差异，同源置换β2-微管蛋白基因后的表达水平和β2-微管蛋白基因相比显著降低。 通过测定抗多菌灵β-微管蛋白基因同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因转化子菌株对应的β2-微管蛋白基因和β1-微管蛋白基因敲除突变体菌株对多菌灵的敏感性、菌丝生长速率等生物学特性，结果表明禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因对抗多菌灵β-微管蛋白基因在禾谷镰孢菌中的功能表达没有抑制作用，突变体对多菌灵仍然敏感，不表现抗药性。 通过测定禾谷镰孢菌野生型菌株、禾谷镰孢菌野生型菌株的β1-微管蛋白基因和β2-微管蛋白基因敲除突变体菌株、抗多菌灵β-微管蛋白基因同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因的转化子菌株及其敲除相应微管蛋白基因的突变体菌株对多菌灵的敏感性、菌落生长速率、产分生孢子能力、产子囊壳能力和致病力等生物学特性，结果表明β2-微管蛋白基因对禾谷镰孢菌是必需的而β1-微管蛋白基因是非必需的，但是两者在功能上是互补的，抗多菌灵β-微管蛋白基因同源置换禾谷镰孢菌β1-微管蛋白基因和β2-微管蛋白基因能够恢复禾谷镰孢菌有性生殖和无性生殖能力以及致病力。

Fusarium graminearum, which is the overriding pathogen of Fusarium head blight (FHB) on wheat, is a leading cause of economic loss in these crops. In addition to reducing seed mass and quality, the fungus contaminates grain with toxic metabolites such as DON and NIV that are a threat to human and other mammals’ health. Grey mold (Botrytis cinerea) is an important plant disease on several economic crops in China, especially results in severe loss to vegetables and strawberrys in protected fields, inducing fruit rot. Carbendazim and other benzimidazole fungicides have been used to control FHB and grey mold for more than 40 years since Shenyang Research Institute of Chemical Industry industrialized manufacture in 1970 led by Zhang Shaoming. Benzimidazole fungicides combine with β-tubulin in plant pathogenic fungi’s cell, preventing the formation of spindle fiber, thus inhibiting mitosis. Plant pathogenic fungi retain the tubulin gene’s random mutation in evolutionary process to adapt to infaust medium. Therefore, most resistance of plant pathogenic fungi to benzimidazole fungicides owes to some definite amino acid mutation of β-tubulin, which causes decreased affinity between benzimidazole fungicides and the target. Amino acid mutation is located in β-tubulin site 198 or 200 generally in most field resistant strains. The site-mutation of β-tubulin at codon198 (Glu to Ala or Lys or Val ) may lead to high resistance while mutation at codon167(Phe to Tyr) and codon200(Phe to Tyr) cause medium resistance to MBC in Botrytis cinerea. Fusarium graminearum contains β1-tubulin and β2-tubulin. Besides, β1-tubulin in strains of dirrenernt sensitivity patterns is identical, without any mutation. However, site mutation of β2-tubulin at codon 73, 167, 198 or 200 can result in distinct resistance, Q73R or E198L to high level while F167Y or F200Y to medium level. Data above shows that the mechanism of resistance to MBC in Fusarium graminearum to Botrytis cinerea. In this article we adopted Double-joint PCR to construct the vector containing MBC resistant β-tubulin gene in vitro, using PEG-mediated protoplast transformation to homologous replace β1-tubulin gene and β2-tubulin gene in Fusarium graminearum. Furthermore, we deleted the original β1-tubulin gene and β2-tubulin gene of mutants we got above. Accordingly, we obtained mutant of Fusarium graminearum that β1/β2-tubulin gene knock-out mutants, MBC-resistant β-tubulin gene replacing β1/β2-tubulin gene transformants respectively and corresponding knock-outs. We assayed the sensitivity to MBC, mycelial growth rate, conidia productivity, perithecium productivity, pathogenicity and other biological characteristics of different Fusarium graminearum transformants by testing the expression of MBC-resistant β-tubulin gene via homologous replacement with β1-tubulin gene and β2-tubulin gene in Fusarium graminearum using semiquantitative RT-PCR to study whether β-tubulin gene of different plant pathogenic fungi can be substituted and if the MBC-resistant β-tubulin gene of Botrytis cinerea can express in Fusarium graminearum which provided reference to further research the resistance mechanism of Fusarium graminearum to MBC and the mechanism of action between MBC and β-tubulin. Below are our main findings. We adopted Double-joint PCR to construct the vector containing MBC resistant β-tubulin gene in vitro, using PEG-mediated protoplast transformation to homologous replace β1-tubulin gene and β2-tubulin gene in Fusarium graminearum, and successfully got the transformants which proved that β-tubulin gene of different plant pathogenic fungi can be substituted. The test of sensitivity to MBC of mutant strains containing β-tubulin gene substituted for β1-tubulin gene and β2-tubulin gene respectively showed that the mutant strains had lower sensitivity to MBC, however, without resistance. The test of the expression of MBC-resistant β-tubulin gene via homologous replacement with β1-tubulin gene and β2-tubulin gene in Fusarium graminearum using semiquantitative RT-PCR indicated that β-tubulin gene can express on mRNA level in Fusarium graminearum, and there were no significant deviation between the expression of β-tubulin gene after homologous replacement with β1-tubulin gene and β1-tubulin gene while notable reduction after β-tubulin gene replaced β2-tubulin gene than β2-tubulin gene. The test of the sensitivity to MBC, mycelial growth rate, conidia productivity, perithecium productivity, pathogenicity and other biological characteristics of Fusarium graminearum transformants of MBC-resistant β-tubulin gene replacing β1-tubulin gene and β2-tubulin gene separately manifested that β1-tubulin gene and β2-tubulin gene of Fusarium graminearum had no inhibition to the expression of MBC-resistant β-tubulin gene of Botrytis cinerea in Fusarium graminearum and the mutant remained sensitive to MBC. The test of the sensitivity to MBC, mycelial growth rate, conidia productivity, perithecium productivity, pathogenicity and other biological characteristics of Fusarium graminearum β1/β2-tubulin gene knockout mutant strains, MBC-resistant β-tubulin gene replacing β1-tubulin gene and β2-tubulin gene separately and their corresponding knockouts proclaimed that β2-tubulin gene was essential for Fusarium graminearum while β1-tubulin gene was not, nevertheless, two were complementary, and MBC-resistant β-tubulin gene replacing β1-tubulin gene and β2-tubulin gene separately can both repair the sexual and asexual reproduction capability and pathogenicity of Fusarium graminearum.