【作者】 徐飞；

【导师】 夏玉先；

【作者基本信息】 重庆大学 ， 生物学， 2011， 硕士

【摘要】 绿僵菌（Metarhizium anisopliae）是一种重要的昆虫病原真菌,它能侵染50多个科中的200多种昆虫:如蝗虫、白蚁、蚂蚱、牧草虫等,是目前广泛应用的微生物农药来源之一,关于其侵染与致病机制目前已经得到广泛的研究和了解。孢子是其生物防治中的毒力单位,在疾病循环的繁殖和致病性上起着重要的作用。在模式生物粗糙脉胞菌、构巢曲霉等丝状真菌中,两种主要的产孢模式已经被阐述:正常产孢和微循环产孢。绿僵菌作为一种丝状病原真菌,也具有这两种典型的产孢模式。与正常产孢相比,绿僵菌的微循环产孢具有繁殖快速、抗逆性强等诸多优势。因此,较好的理解微循环产孢的分子机制,将会极大的提高绿僵菌的商业化生产,扩大昆虫防治中的病原真菌应用范围。但是,以前的微循环产孢研究主要集中于诱导条件的探索,关于其详细分子机制研究较少。基于本实验室先前构建了绿僵菌微循环产孢时期的差减文库,获得了221条特异的EST（表达序列标签）基因序列,本研究选取了其中的3个高表达基因:MaAGA,Pyk,PP5,克隆了这3个重要基因并进一步进行了相关的分子水平研究,其主要研究内容为: （一）MaAGA调控绿僵菌的微循环产孢过程及耐热性1.克隆了MaAGA基因的cDNA（Genebank登录号:HM068369）,并进行了生物信息分析。MaAGA基因cDNA片段含有2个内含子,开放阅读框为978bp,编码325个氨基酸,预测编码蛋白大小为35.05KDa,等电点为5.1。与粗糙脉胞菌中的精氨酸酶基因具有72%的同源性,E-Value值为6e^-12。并且,在Genebank数据库进行Blast比对,显示绿僵菌中该基因具有精氨酸酶蛋白典型的二价锰离子结合位点、精氨酸结合及催化位点等。同时,进一步的进化树分析显示,该基因具有非常强的保守性,与植物具有21%-23%的相似性,与细菌具有30%-38%的相似性,与真菌和动物分别有40%以上的相似性2.通过RNAi手段干扰该基因的表达。设计该基因的最佳干扰区域,连接至高效的RNA干扰载体中,基因枪转化野生型绿僵菌,通过筛选培养基及PCR双重验证,筛选获得5个稳定的转化菌株。进一步,通过定量PCR和酶学水平检测该基因在转录水平和翻译水平的受干扰程度,其干扰效率均在22%-62%之间。3.MaAGA基因的对产孢模式的影响。对干扰效率最高的转化菌株进行后续功能研究,在微循环产孢培养基上发现该基因被干扰后微循环产孢过程不能正常发生,产生的子代孢子无法从亲代孢子上脱落,而形成一种树状形态。接种5天后的菌落形态也具有显著性的差异,主要体现为干扰菌株形成的菌落更小,周边白色菌丝更多。精氨酸酶的干扰导致了这些异常表现,进一步的回复实验显示,当微循环产孢培养基中添加精氨酸酶的产物鸟氨酸时,干扰菌株的异常表型能得到恢复,直接证明了MaAGA在这种表型中的作用。为了进一步检测干扰菌株中的孢子分离机制,半定量PCR检测了丝状真菌中9个产孢分离相关基因的表达特征,发现一关键基因Mob2表达量显著性的降低>2倍。但是,在正常产孢培养基上该基因干扰后野生型和突变型并无显著性差异。4.MaAGA基因干扰对产孢量的影响。分别分析了干扰菌株和野生菌株在正常产孢培养基及微循环产孢培养基上的产孢量区别,发现在微循环产孢培养基上,干扰菌株的10d内的产孢总量及产孢速度均显著性降低。与野生型相比,干扰菌株的10d内的产孢总量和产孢速度分别为野生型的0.27、0.32倍。但是,在正常产孢培养基上,干扰菌株的产孢量及产孢速度并未发生显著性差异。5.MaAGA基因干扰对菌株抗逆性的影响。测试了干扰菌株在紫外、干热、湿热等逆境下的孢子萌发率,与野生菌株相比,发现干扰菌株对干热、湿热等比较敏感,孢子萌发率显著性降低。而在紫外环境下并无显著性差异产生。海藻糖在真菌孢子的耐热机制中起着非常重要的作用,为此我们检测了干扰菌株和野生菌株中海藻糖的含量,结果显示干扰菌株的海藻糖含量为3.31pg/孢子,显著低于野生菌株的3.86pg/孢子。进一步在海藻糖的合成机制上,定量PCR证明其分解酶基因ntl受到了抑制,显著的降低了41.6%。6.MaAGA基因干扰对菌株毒力的影响。我们检测了MaAGA干扰后菌株在毒力上的一些变化,通过附着孢观察、蝗虫体内注射、蝗虫体表侵染等方法,发现干扰菌株的毒力并无显著性变化,推测该基因并不参与绿僵菌的毒力机制。（二）Pyk调控绿僵菌的微循环孢子形态1.克隆了Pyk基因的cDNA（Genebank登录号:HQ153828）,并进行了生物信息分析。该基因全长cDNA全长1934 bp,开放阅读框长1752 bp,无内含子。编码583个氨基酸,预测相对分子量为47.8 KDa,等电点为9.73,为碱性蛋白。预测并无信号肽序列。进化树分析显示Pyk基因与子囊菌门中的17个物种具有较强的相似性63%-77%。2.通过RNAi手段干扰该基因的表达。设计该基因的最佳干扰区域,连接至高效的RNA干扰载体,基因枪转化野生型绿僵菌,通过筛选培养基及PCR双重验证,筛选获得3个稳定的转化菌株。进一步,通过定量PCR检测该基因的干扰程度,其干扰效率均在33%-56%之间。3.Pyk基因的对微循环产孢模式的影响。观察了该基因被干扰后菌株0-32h中的微循环产孢过程,发现干扰菌株的微循环产孢模式未受影响。但是干扰菌株产生的孢子形态发生了变化,子孢子大小分布更不规则,具有椭圆状及长棒状两种形态,而野生菌株只有椭圆状孢子产生。在菌落大小上,干扰菌株与野生菌株并无显著性差异产生,但是干扰菌株的周边具有更多的白色菌丝。（三）PP5基因的克隆及其在两种产孢模式中的表达特征分析1.克隆了PP5基因的DNA及cDNA（Genebank登录号:HQ317137）,并进行了生物信息分析。PP5基因DNA长2100 bp,含有7个外显子和6个内含子,开放阅读框长1428 bp。该基因编码蛋白大小为38.1 KDa,等电点为9.66,为碱性蛋白,无信号肽序列。2.PP5编码产物蛋白结构分析。PP5编码产物在一级结构上显示了第五类Ser/Thr保守的结构区域,如TRP序列及催化结构域,与所选5个亲近物种也具有较高的相似性（73%-82%）。在二级结构上约含46.32%的α螺旋, 7.16%的β转角, 14.74%的延伸串,31.79%的不规则卷曲。Swiss-Model进行三级结构同源建模显示该酶的核心结构类型为α/β型,Procheck对建模结果可信度进行检测,算得出Ramachandran图,证实了该建模结果具有较强的可信度。3.定量PCR检测该基因在两种产孢模式中的表达特征。结果显示,PP5基因在绿僵菌微循环产孢的不同阶段表达水平具有显著性差异,特别是在孢子接种16、24、32小时高表达,而在正常产孢模式下表达量非常少。更多还原

【Abstract】 Metarhizium anisopliae is a significant filamentous pathogenic fungus, because it infects ca. 200 insect species from more than 50 taxonomic families, including termites, grasshoppers, thrips and mosquitoes. The mechanisms by which the insect pathogenic fungus M. anisopliae infects the host insect are relatively well understood. Conidiation, as the efficient propagule used in biocontrol programs, is a key step for survival and transmission in the disease cycle. Just like two representative conidiation elucidated in detail for two model organisms: Neuropora crassa and Aspergillus nidulans, our previous work found that M. anisopliae also exhibited two different conidiation programs: normal conidiation on non-inducing medium and microcycle conidiation on inducing medium. Compared with normal conidiation, microcycle conidiation showed remarkable advantages in the speed of conidia production and resistance to stress factors. Therefore, better understanding of the molecular mechanism of microcycle conidiation would improve conidial commercial production of M. anisopliae, which would greatly facilitate expansion of the scope of insect fungal pathogen applications in pest control.Previously, genes preferentially expressed during microcycle conidiation vs. in normal conidiation hyphae were isolated by suppression subtractive hybridization in M. anisopliae. Among these candidates, we identified and described three key genes (AGA, Pyk, PP5), which are highly expressed during microcycle conidiation. The results provide the ideal candidates for further studies of microcycle conidiation and its molecular regulation. The main achievements are as follows:(一)MaAGA regulates microcycle conidiation and thermotolerance in M. anisopliae1.A full-length arginase gene MaAGA was cloned and sequenced, and deposited in Genbank under accession number (HM068369). The ORF (open reading frame), which was interrupted by two introns, was 978 bp long. Analysis of the predicted MaAGA protein indicated that it was composed of 325 amino acid residues (35.05 kDa) with a predicted pI of 5.10. The predicted protein encoded by MaAGA contained two manganese ion binging domain, arginine-specific binding domain and other arginase-conserved amino acid residues. Identities with the MaAGA sequence were 21–23% (plant) and 30–38% (bacterium) compared to 40–53% and 40–49% for fungal and animal arginases, respectively.2.A gene interference strategy was used and the RNAi vector containing a 367 bp fragment of MaAGA was transformed into Metarhizium wild type. Five putative RNAi-MaAGA mutants were identified. To investigate that RNA interference efficiency of MaAGA in mutants, we verified mRNA expression and arginase activity of MaAGA in mutants. These results demonstrated a successful suppression of MaAGA in mutants (22%-62%)3.On SYA medium, the conidiation processes of RNAi mutant obviously differed from the wild type and exhibited an unexpected phenotype, in which daughter cells had partly failed to separate from their mother cells. After further rounds of budding, the mutant formed tree-like structure. The growth of the RNAi mutant-3 (2.01±0.10 cm) was significantly slower than that of the wild type (2.56±0.9 cm) (P < 0.05). Besides, more similar white hyphae could be observed after 5 day postinoculation on SYA medium. In the phenotype restore assay, when the medium was amended with ornithine, the fungal phenotype could be restored, which supports a direct role for arginase in microconidial formation. Further, we investigated nine daughter-special genes expression in RNAi mutant. Mob2 gene, a pivotal protein involved cell separation system, was down-regulated >2-fold in the RNAi mutant. However, normal conidiation seemed to be unaffected in RNAi mutant compared with the wild type.4.We investigate the difference of conidial production. Both the total conidial yield (at day 11, P= 0.008) and the speed of conidiation (at day 2, P= 0.005) /cm~2 of the RNAi mutant-3 were significantly lower. The mean total conidial yield and speed of the RNAi mutant-3 were 0.27-fold and 0.32-fold higher than those of wild-type, respectively. On normal conidiation medium, the total conidial yield (at day 12, P = 0.593) and the speed of conidiation (at day 2, P= 0.674) in RNAi mutant-3 and wild-type were not discernibly different.5.The conidial thermotolerances were analyzed with dry-heat, wet-heat and UV-B treatment. The germination of RNAi mutant was significantly lower than that of wild type under dry-heat, wet-heat treatment, except UV-B. Trehalose was an important component in fungal conidia and had been widely reported to protect the cells against several stresses. We investigated conidial accumulation of trehalose and found trehalose content was about 3.31 pg/conidia in RNAi mutant and 3.86 pg/conidia in wild type, showing about 14.3% reduction compared with the wild type Furthermore, RT-PCR showed the decreasing expression of ntl (Neutral trehalase) resulted in lower trehalose content in RNAi mutants.6 . No significant difference was found in appressorium formation and pathogenicity between the wild-type and RNAi mutant-3, indicating that MaAGA was dispensable to conidial tolerance to UV-B, appressorium formation and pathogenicity.(二)Pyk regulates conidial shape of microcycle conidiation in M. anisopliae1.A full-length arginase gene Pyk and its upstream and downstream regulatory sequences was cloned and sequenced, and deposited in Genbank under accession number (HM068369). The ORF with no interruption was 978 bp long. Analysis of the predicted Pyk protein indicated that it was composed of 583 amino acid residues (47.8 kDa) with a predicted pI of 9.73. No signal peptide was found in the prediceted protein. The phylogenetic tree showed 63%-77% identies between Pyk and the selected 17 species.2.The optimal RNA interference area of Pyk was designed and inserted into the RNAi vector, and then the expression vector was transformed into Metarhizium wild type. Three putative RNAi-Pyk mutants were identified. To investigate that RNA interference efficiency of Pyk in mutants, we verified mRNA expression of Pyk in mutants. These results demonstrated a successful suppression of Pyk in mutants (33%-56%)3.Furthermore, we analyzed suppression effect to Pyk in mutations. On SYA, The wild type has only Ellipsoid conidia. But the mutants showed more varied shapes of conidia: Ellipsoid conidia and Rod-like conidia. Moreover less white hypha in colonial morphology could be observed compared to wild type.(三)Cloning and expression analysis of Ser/Thr protein phosphatase type 5 during microcycle conidiation in M. ansopliae1.A full-length cDNA and DNA of PP5 was cloned and sequenced, and deposited in Genbank under accession number (HM068369). The genomic DNA was 2100bp. The ORF of cDNA with six interruptions was 1428 bp long.2.The predicted PP5 protein was composed of 475 amino acid residues (38.1 kDa) with a predicted pI of 9.66. No signal peptide was found in the prediceted protein. In protein structure analysis, Ser/Thr protein phosphatase type 5 in M. anisopliae showed a conserved structure features.3.Quantitative real time PCR analysis showed that PP5 expression varied obviously in different stages of microcycle conidiation. Expression was sharply up-regulated after 16 h, with the highest transcript levels at 24 h in microcycle conidiation, but lowly expressed in normal conidiation.更多还原