【作者】 姚红燕；

【导师】 陈利锋；

【作者基本信息】 南京农业大学 ， 植物病理学， 2003， 硕士

【摘要】 本课题着重通过研究小麦赤霉病菌毒素输出泵基因Tri12在小麦穗组织中的表达水平、Tri12对病菌及毒素在寄主组织中的扩散影响及病菌输出的主要毒素种类及其对寄主病程的影响，来明确毒素输出泵基因的表达与其致病性间的关系。 Tri12是镰孢菌Fusarium编码单端孢霉烯族毒素(trichothecenes，以下简称“单族毒素”)输出泵的基因，而产毒基因Tri5编码的单端孢霉二烯合酶催化毒素生物合成中的第一步反应。以禾谷镰孢野生型菌株NRRL 29169为“亲本”，通过基因插入法分别获得Tri12剔除的转化子MT12和Tri5剔除而不产生单族毒素的转化子GZT40。与NRRL 29169相比，MT12在PDA培养基上生长慢，生长量小(P＜0.01)，大型分生孢子较长。致病力测定结果显示，NRRL 29169致病力最强，所致病害可以自接种点向上下扩展至其它小穗；GZT40致病力弱，所致病害不扩展；而MT12的致病力最弱。我们由此推测，Tri12的剔除导致病菌产生的毒素不能被泵出体外毒害寄主，而在病菌体内积累抑制自身生长和毒素的进一步产生，从而降低病菌的致病力。 在研究Tril2剔除对禾谷镰孢菌产毒和耐毒能力的影响试验中，我们发现：MT12在含0-50ppm的脱氧雪腐镰刀菌烯醇(deoxynivalenol，DON)、3-乙酰基脱氧雪腐镰刀菌烯醇(3-acetyldeoxynivalenol，3ADON)和15-乙酰基脱氧雪腐镰刀菌烯醇(15-acetrldeoxynivalenol，15ADON)等单族毒素的PDA培养基上，生长速率无显著性差异。分别在大米粒和小麦粒等固体培养基和葡萄糖-蛋白胨-酵母粉(GYEP)液体培养基上，MT12只产生少量毒素或不产生毒素。因此，我们推测，Tri12基因编码的输出泵蛋白可能是一个双向泵。Tri12的剔除使毒素不能排出而在细胞内积累，抑制病菌生长和进一步产毒；同时，也不能将外界毒素输入细胞内，因而对环境中毒素不敏感。 Tri12与禾谷镰孢单族毒素在寄主组织中的扩散试验表明：MT12接种的穗组织中积累少量的DON、15-AcDON和3-AcDON等3种单族毒素，毒素没有向接种点以外扩散。在DON饲喂试验中，于小麦抽穗后第二天，用DON连续(每天一次，连续10天)等量(每次4μg)饲喂穗中下部的一个小花，然后分部位回收检测。在回收到的DON中，97％来自受喂小穗，其余约3％来自穗的其它部分。其余约3％中有75％来自饲喂点上方的小穗和穗轴，其余来自饲喂点以下的小穗、穗轴和穗颈。在另一小麦赤霉病菌毒素输出泵基因TriIZ的病理作用试验中，只向穗中下部的一个小花一次性饲喂4陀DoN，10天后回收的DoN中，97%仍来自受喂小穗，其余3%分别来自临近饲喂点的上、下各一个小穗(49%)和穗顶端小穗(约39%)以及穗下Icm的穗颈(12%)，其余小穗和穗轴中未检测到DON.这些结果表明，TriIZ影响病菌产生的毒素从产毒细胞中输出并进入寄主组织的过程.毒素如不能顺利输出，便在产毒细胞内积累，抑制病菌自身生长和进一步产毒，也不可能对寄主产生毒害作用;而毒素如能顺利从产毒细胞输出并进入寄主，其中仅有极少量的毒素可从小麦穗的一个小穗扩散到其它小穗.可能正是这些极少量扩散的毒素协助病菌在寄主组织中的扩展. 将Gus编码区呈于TriIZ启动子的控制下，构建了质粒pGusTri12P，使用该质粒转化野生型菌株NRRL 29169，获得GS1212转化子.与NRR工29169相比，GS1212在PDA培养基上生长慢，生长量小(P<0.01)，大型分生抱子较长.通过X一Glu。染色法和定童测定，发现GslZ12的菌丝和抱子中有GUS活性，而NRR工29169中无GUS酶活性或GUS活性很低.接种试验表明:GS 1212菌株的致病力基本丧失;小麦组织中具有较高的GUS背景活性.GslZ12和N班让29169处理后接种小穗中的GUS净比活力有极显著差异(尸<0 .01).同时，接种Gsl 212的穗样中，感病品种宁麦6号的GUS活性高，而且表现出随时间而上升的趋势.抗病品种苏麦3号的GUS活性较低，且呈现随时间下降的趋势.在小麦与赤霉病菌互作过程中，NRR工29169的接种可能促进内源GUS酶的表达或产生类似GUS酶的物质，对应用GUS作为报告基因的试验产生强烈干扰.因此，建议在应用报告基因对赤霉病菌与小麦互作的研究中，应选用除GUS外的其它报告基因.更多还原

【Abstract】 This thesis report the expression in wheat spikelets of Tril2 gene encoding trichothecene efflux pump that is responsible for the transportation of trichothecenes out of fungal cells in Fusarium graminearum, the main toxins of F. graminearum produced and the diffusion of trichotheneces in wheat spike tissuses in relation to Tril2, and the role of trichotheneces to wheat pathogenicity, So we can defmitude the relationship of the expression of trichotheneces efflux pump gene and it’s pathogenicity.Trichothecenes are secondary metabolites, some of which are pathogenicity factors in Fusarium graminearum. With the wild type strain NRRL 29169 as parent strain, we generate a Tril2 knock-out mutant strain (MT12). On potato-dextrose-agar, MT12 grew slower and produced less mycelia than NRRL 29169 (P<0.01), though macroconidia of MT12 were longer than those of NRRL 29169 (P<0.05). Virulence on wheat was determined for NRRL 29169, MT12 and GZT40, a strain with the Tri5 gene disrupted. Tri5 encodes trichothecene synthase that catalyzes the first step of trichothecene biosynthesis. Among the three strains tested, NRRL 29169 was most virulent with disease symptoms spreading from the inoculated spikelets to other spikelets, while the two mutant strains were less virulent with no spreading being observed. From these experiments, we conclude that disruption of Tri12 causes accumulation of trichothecenes in fungal mycelia instead of them being pumped into host tissues. We further believe that trichothecenes are toxic to the fungus itself and that exposure to the toxins inhibits its growth and toxin production. As a result, the pathogenicity of the pathogen may be greatly reduced.In the study of knock-out of Tri12 reduced trichotheneces production sensitivity in F.graminarum, We find that on potato-dextrose-agar containing 0-50 ppm of either deoxynivalenol (DON), 3-AcDON (3-acetyldeoxynivalenol) or 15-AcDON (15-acetyldeoxynivalenol), MT12 showed no significant difference in growth rate, indicating that MT12 is not sensitive to trichothecenes. On wheat grain or rice grain solid meida and glucose-yeast extract-peptone liquid medium, MT12 produced little or no trichothecenes. From these results, we hypothesize that the trichothecene efflux pump encoded by Tri12 is a bidirectional pump. Removal of efflux pump causes accumulation of trichothecenes within the fungal cells, which inhibits the fungal growth and toxin production, and causes failure to transport trichothecenes from outside into inside of fungal cells, which reduces fungal sensitivity to trichothecenes in surrounding environment.Diffusion of trichothecenes in wheat spike tissues in relation to an efflux pump gene Tril2 in F.graminearum indicate that MT12 was weakly pathogenic on wheat, and only a small amount of DON, 15-acetyldexynivalenol and 3-acetylnivalenol was detected in the MT12-inoculated spikelet. No diffusion of trichothecenes from the inoculated spikelet to other spikelets was observed. In a feeding experiment, a floret in a middle spikelet of a spike was fed with 4 ug of DON daily for 10 days beginning from second day after heading. The day after the last feeding DON was recovered from various parts of the fed spikes. Among the DON recovered, 97% was from the fed spikelet. Among the remaining 3% of recovered DON, 75% was from spikelets and rachis segments above the fed spikelet, and the remainder from spikelets and rachis segments below the fed spiklets and a 1 cm peduncle segment below the spike. In another feeding, experiment, a floret was fed once with 4 ug of DON and was analyzed for DON 10 days later. Among the DON recovered, 97% was from the fed spikelet. Among the remaining 3% of DON, 49% was recovered from one spikelet immediately above and one spikelet immediately below the fed spikelet, 39% from the two top spikelets and 12% from a 1 cm peduncle segment below the spike. These results indicate that Thl2 influences efflux of trichothecenes from trichothecene producing cells of F. graminearum and influx of trichothecenes into wheat spike tissues. Fa更多还原