【作者】 杨敏；

【导师】 夏玉先；

【作者基本信息】 重庆大学 ， 微生物学， 2011， 硕士

【摘要】 绿僵菌(Metarhizium acridum)作为重要的昆虫病原真菌之一是控制自然界害虫种群数量的其中一个重要因素。它具有主动穿透昆虫体壁、致病过程复杂不易产生抗性等特点,在日益受到人们重视的生物防控上具有广阔的应用价值。然而它也具有一定的缺点,如架货期短、容易受到环境条件的影响、成本高等。真菌的细胞壁可以根据真菌的整个生活周期和外界环境的变化而不断的改变,并且是真菌和环境接触的媒介,在保护细胞免受外界有害环境的损害以及真菌的整个生活周期中起重要作用。然而,目前对昆虫病原真菌细胞壁的研究还较少。本研究以绿僵菌为实验材料,在已知基因组序列的基础上,克隆了β-1,3-葡聚糖合成酶基因MaFKS和丝状真菌特有的VII类几丁质合成酶基因MaChsVII,并采用基因干扰和基因敲除技术对它们的生物学功能进行了分析。主要研究结果如下:1 MaFKS的克隆与功能分析1.1 MaFKS的克隆及序列分析我们根据已知的基因组DNA序列设计引物克隆得到β-1,3-葡聚糖合成酶基因命名为MaFKS,基因登录号为HQ441252。该序列开放阅读框为5820bp,编码一个1938个氨基酸的蛋白。使用在线工具预测该蛋白分子量为221.7kDa,等电点为7.94。预测MaFKS蛋白含有16次跨膜区域,与其他丝状真菌中葡聚糖合成酶同源性较高。与基因组DNA序列比对发现,MaFKS基因含有两个内含子,分别在N端(177-238 bp)和C端(5539 bp-5600 bp)。1.2利用RNAi技术对MaFKS基因的功能进行分析为研究MaFKS基因的功能我们构建了农杆菌介导的干扰载体(pPK2-pB-MaFKS-RNAi),采用农杆菌介导的丝状真菌的遗传转化方法转化绿僵菌后,获得5个干扰转化菌株。采用定量RT-PCR的方法检测了干扰菌株中MaFKS基因的表达量。结果表明,不同的干扰菌株的干扰效率在40%-64.2%之间。1.3 MaFKS基因与细胞壁的完整性相关我们分析了MaFKS基因干扰菌株在含有不同细胞壁干扰剂的培养基上的菌落生长,发现与野生菌株相比,干扰转化菌株在含有刚果红、荧光增白剂或SDS的PDA平板上的菌落形态明显不同。我们近一步在显微镜下观察了干扰菌株与野生菌株菌丝生长的差别。观察发现,在含有CR、CFW的PDA平板上可观察到野生菌株有大量的分支的气生菌丝。相反,干扰转化菌株仅有少量的没有分支或只有少数分支的气生菌丝。在液体培养基中,转化菌株的大部分菌丝元素与野生型没有差异,但在CR存在的条件下菌丝的尖端或内部隔室出现膨胀。1.4 MaFKS基因与绿僵菌高渗透压的耐受性相关在含有KCl的PDA平板上,干扰转化菌株的基内菌丝比野生型明显减少。并且转化菌株的的菌落表面光滑且呈黄褐色,而野生菌株的菌落表面则褶皱且变绿。干扰转化菌株在含有山梨醇或甘露醇的PDA平板上,与野生型相比转化菌株只有稀疏的气生菌丝。这说明MaFKS影响绿僵菌对高渗透压的耐受性。1.5干扰MaFKS基因后绿僵菌的产孢量降低测定干扰转化菌株和野生菌株在PDA平板和含有CR、CFW、SDS、KCl、山梨醇和甘露醇的PDA平板上接种12天后的产孢量。发现在PDA平板上两菌株的产孢量差别不大,但在含有CR、CFW、SDS、KCl、山梨醇和甘露醇的平板上,干扰菌株的产孢量明显降低。1.6干扰MaFKS基因后菌丝的葡聚糖含量降低测定菌丝壁β-1,3-葡聚糖的含量,发现干扰转化菌株β-1,3-葡聚糖的含量只有野生菌株的51.32%。采用苯胺蓝染色观察菌丝壁β-1,3-葡聚糖的分布发现,转化菌株菌丝间隔带区域荧光信号比野生菌株的要弱。说明减少MaFKS的表达降低了绿僵菌菌丝壁β-1,3-葡聚糖的含量。2 MaChsVII的克隆与功能分析2.1 MaChsVII的克隆及序列分析我们克隆了一个几丁质合成酶基因MaChsVII,序列分析表明该基因含有一个5427bp的ORF,编码1784个氨基酸的蛋白。该蛋白的分子质量为199.2kDa,等电点为5.57,具有6次跨膜区域。预测的MaChsⅦ蛋白序列的N端含有MMD区域,但比V类几丁质合成酶的MMD区域要短,并且不含有第五类几丁质合成酶特有的P-Loop、switchⅠ和switchⅡ。构建进化树分析该基因属于VII类几丁质合成酶基因。2.2利用基因敲除技术研究MaChsVII基因的功能构建了农杆菌介导的基因敲除载体pPK2-pB-MaChsⅦL/R,转化绿僵菌后获得敲除转化菌株,PCR验证转化菌株为同源重组。2.3 MaChsVII基因影响绿僵菌细胞壁的完整性观察了△M aChsⅦ菌株在含有细胞壁干扰剂的培养基上的菌落生长形态。发现在含有CR、CFW、SDS的PDA平板上,△MaChsⅦ菌株菌落大小比野生菌株要稍小,并且转化菌株的气生菌丝的生长明显受到抑制,然而在含有H2O2的PDA平板上,△M aChsⅦ菌株的长势比野生菌株要好。2.4 MaChsVII基因与绿僵菌高渗透压的耐受性相关。观察△M aChsⅦ菌株在不同物质提供高渗透压条件下的菌落表型,发现在含有KCl的PDA平板上MaChsⅦ缺失菌株的菌落较小且呈黄褐色,而野生菌株的菌落表面则褶皱并且明显开始产孢。在含有山梨醇及甘露醇的PDA平板上野生菌株菌落变得褶皱并且呈绿色,但是与野生型相比缺失菌株菌落稍小表面并且菌落呈深绿色。2.5 MaChsⅦ基因与绿僵菌的毒力相关。与野生型相比MaChsⅦ缺失菌株对蝗虫的致死时间有所滞后。野生菌株的半致死时间为5.1天,而MaChsⅦ缺失菌株的半致死时间比野生型延迟了1.2天。说明MaChsⅦ的敲除降低了绿僵菌的毒力,该基因与绿僵菌的致病性有关。更多还原

【Abstract】 Metarhizium, one of the most important genera of entomopathogenic fungi, is an important regulatory factor in pest insect populations in nature and has been considered as a promising alternative or supplement to chemical pesticides. It can invade host directly and low likelihood of the development of insect resistance becaused of the process of Metarhizium acridum infected host is complex, thus in the increasing attention on the importance of biological prevention and control has broad application. However, some disadvantages have retarded widespread application, including short-term storage, sensitivity to environmental conditions and the resources required for mass production of conidia. The dynamic cell wall of fungi is an intermediary between the fungus and the environment and can change, depending on the life cycle stage and environmental conditions. But the study of the entomopathogenic fungal wall is still not clear. In this study, we use the Metarhizium acridum as the material whose whole genomic sequence has known. The gene encodingβ-1,3-glucan synthase and a class VII chitin synthase gene in the entomopathogenic fungus Metarhizium acridum were cloned. To investigate their function, the RNAi and gene knock-out strategies have been used.The main results are as follows:1 cloning and function analysis ofβ-1,3-glucan synthase gene MaFKS1.1 Cloning of MaFKSAccording the genomic sequence, we cloned aβ-1,3-glucan synthase gene named MaFKS (GenBank accession number AY187630). Sequence analysis demonstrated that MaFKS contains a 5820 bp open reading frame (ORF) and two introns located at the N terminus (177–238 bp) and the C terminus (5539–5600 bp). The complete ORF of MaFKS encodes a predicted protein of 1938 amino acids (aa) with an molecular mass of 221.7 kDa and a pI of 7.94. Analysis of the transmembrane domains showed that the deduced MaFKS protein has 16 transmembrane helices. And the putative MaFKS protein is most closely related to the putative FKS protein from two other entomopathogenic fungi, Beauveria bassiana and Cordyceps militaris.1.2 To study the function of MaFKS, an RNAi strategy was applied.To study the function of MaFKS, an RNAi vector was constructed (pPK2-pB-MaFKS-RNAi). Agrobacterium-mediated transformation was carried out, and obtained five RNAi mutants. Transcripts of MaFKS were detected by real-time RT-PCR. In comparison with the WT, MaFKS transcription was inhibited in RNAi transformants, with down-regulation ranging from 40% to 64.2%.1.3 MaFKS is involved in cell wall integrityTo determine the cell wall integrity of FKS-RNAi transformants, the sensitivity of two transformants and the WT to agents that disturb the cell wall or cell membrane was investigated. In comparison with WT, FKS-RNAi transformants had much less aerial mycelium on PDA plates with or without CR, CFW or SDS after 4 days of incubation. To further clarify the effect of agents that disturb the cell wall or cell membrane, we observed mycelial growth in 1/4 SDAY liquid medium supplemented with CR, CFW or SDS. Closer inspection revealed some regions of the mycelium with frequent aberrant conformations, such as blowing out of cell wall elements at hyphal tips and internal compartments in the presence of CR. All the results demonstrate that depleted MaFKS expression affected mycelium growth and increased sensitivity to the agents used, which indicates that MaFKS plays an important role in cell wall integrity in M. acridum.1.4 Depleted MaFKS expression increases the sensitivity to hyperosmotic pressure in M. acridumSubstrate hyphae of the transformants on PDA plates containing KCl were much fewer than for the WT. Moreover, the colony surface for transformants was smooth and tawny, while that for the WT was typically wrinkled and green. On PDA plates containing sorbitol or mannitol, the transformants had sparse aerial hyphae compared with WT.1.5 MaFKS is involved in conidiation in M. acridumConidial yield was measured on PDA and PDA plates containing CR, CFW, SDS, KCl, sorbitol or mannitol after 12 days of incubation. The conidial yield/mm2 was significantly higher for WT than for FKS-RNAi transformants on PDA plates containing CR, CFW, SDS, KCl, sorbitol or mannitol.1.6 FKS-RNAi transformant are defective inβ-1,3-glucan synthesisTheβ-1,3-glucan content of hyphal wall of the transformant was only 51.32% of the WT content.To further investigate changes inβ-1,3-glucan distribution, mycelia were stained with theβ-1,3-glucan-specific fluorochrome aniline blue. Fluorescence microscopy revealed that the septal regions were greatly decreased in transformant compared to WT hyphae.2 cloning and function analysis of class VII chitin synthase gene MaChsVII 2.1 According the genomic sequence, full-length MaChsVII gene was cloned and sequenced. The putative coding sequence which contained a 5427 bp ORF, and encodes a protein of 1784 aa with an molecular mass of 199.2 kDa and a pI of 5.57. The predicted MaChsVII protein has 6 transmembrane helices and a MMD which is short than the class V chitin synthase and without P-loop, switch I and switchⅡ.2.2 To study the function of MaChsVII, a gene knock-out strategy was applied.To study the function of MaChsVII, an gene knock-out vector was constructed (pPK2-pB-MaChsⅦL/R). Agrobacterium-mediated transformation was carried out, and obtained five mutants.2.3 MaChsVII is involved in cell wall integrityWe observed the colonies of the mutants on the PDA with agents that disturb the cell wall or cell membrane. The result demonstrated that the colonies of mutants were smaller than that of WT, and the mutants had much less aerial mycelium.2.4 MaChsVII is involved in tolerance to hyperosmotic pressureColonies of the mutants on the PDA with the KCl and mannitol plates were smaller than that of WT, and the growth of△M aChsⅦmutants on the on the PDA with the KCl plates was hysteretic. The result reveals that MaChsⅦinfluences the hyperosmotic pressure tolerance of M. acridum.2.5 MaChsVII disruption reduced the virulence of the fungiBioassay result showed that the LD50 of MaChsVII-disruption strains to locusts was nearly 1.2 day later than that of the wild-type after inoculation. The result indicated that MaChsVII disruption could impair the infection efficiency of M. acridum in locusts.更多还原