【作者】 汤强；

【导师】 黄勃； 宋礼华； 樊美珍；

【作者基本信息】 安徽农业大学 ， 微生物学， 2009， 博士

【摘要】 玫烟色棒束孢Isaria fumosorosea是一种子囊菌类虫生真菌,它广布全世界,寄主范围广泛,已记载有25个不同科的昆虫,包括小菜蛾Plutella xylostella、麦双尾蚜Diuraphis noxia和烟粉虱Bemisia tabaci等一些重要农业害虫,主要通过分泌多种胞外水解酶降解昆虫体壁从而侵入寄主,其分泌的水解酶主要包括蛋白酶、几丁质酶、脂酶等。在生防菌中,球孢白僵菌Beauveria bassiana在害虫的生物防治中发挥着重要作用,是目前研究运用最多的虫生真菌之一,我国应用球孢白僵菌防治马尾松毛虫达到每年200万公顷的规模,已成为世界上应用规模最大和最成功的害虫生防项目之一。由于真菌杀虫剂存在杀虫效果慢、受环境影响较大、效果不稳定和杀虫谱过窄等缺点,直接导致其广泛应用受到限制。因此利用基因工程和细胞工程等技术提高菌株毒力,遗传改造获得适合市场需要的真菌杀虫剂具有重要的应用价值。本研究以上述两种昆虫病原真菌作为出发菌株进行了如下几项研究:1高产蛋白酶菌株的筛选及菌株几丁质酶产酶条件的优化本文通过对本实验室已保存的几十株玫烟色棒束孢进行总结分析,经过不同的寄主与不同的采集地初筛出16株玫烟色棒束孢,再通过测定蛋白酶酶活的方法复筛选出蛋白酶活性较高的一株菌株RCEF3304,并对该菌株产几丁质酶条件与酶学性质做了进一步的研究,结果表明该菌株产几丁质酶的适宜条件为:胶体几丁质为2%,NaNO₃为3%,Mg²⁺为0.05%,培养基初始pH值6.0,接种量12%,100 mL装液量为10 mL,温度25℃,培养时间36 h。2玫烟色棒束孢蛋白酶基因、几丁质酶基因及其上游调控序列的克隆与分析以玫烟色棒束孢RCEF3304作为出发菌株,首次通过SMART RACE法和DNAwalking技术从玫烟色棒束孢中成功克隆得到与其毒力相关的两种重要基因:蛋白酶基因与几丁质酶基因,序列递交国际权威基因数据库GenBank登录,序列号分别为FJ423001和FJ377733。添补了国际权威基因数据库GenBank在玫烟色棒束孢蛋白酶基因与几丁质酶基因这一内容上的空白。通过对序列进一步的分析表明:1)玫烟色棒束孢蛋白酶cDNA基因序列全长为1704bp,编码阅读框由1287个核苷酸组成,编码了428个氨基酸。5′非翻译区（5′-UTR）与3’非翻译区（3’-UTR）分别为233个核苷酸和198个核苷酸,其信号肽长度为16个氨基酸。成熟的蛋白理论分子量为46.3kDa,理论等电点为7.37。428个氨基酸中含强碱氨基酸50个（K,R）,强酸氨基酸52个（D,E）,疏水氨基酸146个（A,I,L,F,W,V）,极性氨基酸105个（N,C,Q,S,T,Y）,分别占总氨基酸的11.8%,12.2%,34.3%和24.7%。利用生物分析软件DNAman将7种不同来源的真菌进行氨基酸序列同源性分析表明,该蛋白酶属于类枯草杆菌蛋白酶,并且和粉棒束孢Isaria farinosa（AAY87460）,里氏木霉Hypocrea lixii（ABI84117）,深绿木霉Trichoderma atroviride（ABG57252）,金龟子绿僵菌小孢变种Metarhizium anisopliae var.anisopliae（CAD13274）同源性分别为88%、71%、71%和70%。2)玫烟色棒束孢几丁质酶cDNA基因序列全长为1542bp,编码阅读框由1275个核苷酸组成,编码了425个氨基酸。5′非翻译区（5′-UTR）与3′非翻译区（3′-UTR）分别为33个核苷酸和234个核苷酸,其信号肽长度为24个氨基酸。成熟的蛋白理论分子量为47.6kDa,理论等电点为4.89。425个氨基酸中含强碱氨基酸39个（K,R）,强酸氨基酸54个（D,E）,疏水氨基酸139个（A,I,L,F,W,V）,极性氨基酸118个（N,C,Q,S,T,Y）,分别占总氨基酸的9.2%,12.7%,32.6%和27.8%,与GenBank中已登录的一些真菌序列进行比较发现,和蜡蚧轮枝菌Lecanicilliumlecanii（AAX56960）,里氏木霉Hypocrea lixii（DAA05855）,绿木霉Hypocrea virens（AAL78814）,爪哇束孢Isaria javanicus（AAZ83728）,同源性分别为90%、71%、71%和70%。通过Blast对保守区进行预测,这些几丁质酶都属于糖基水解酶18家族,并包含许多保守序列,其中2个高度保守序列SIGG和DGIDVDWE是糖基水解酶18家族水解活性中心位点。3)对玫烟色棒束孢蛋白酶Ifuprl结构基因及上游启动子序列分析表明,其结构基因中未有内含子的出现,该序列不仅包含启动子的核心结构序列CAAT框,亦包含一些特异的反应元件和转录因子结合位点（包括GATA-1、GATA-2、CdxA等）。CAAT框是调控转录速率的元件,而其他一些转录因子结合位点（包括GATA-1、GATA-2、CdxA等转录因子结合位点）则可能具有特异性调节基因转录的功能。多个GATA转录因子结合位点共同调控会显著增加报告基因的转录活性,但也有研究表明GATA会抑制基因的表达。CdxA元件一般位于基因的5′-端,作为启动子的一部分起作用,在表达CdxA蛋白的报告基因表达系统中,载体中含有的CdxA元件能够增强基因的转录活性。4)获得了玫烟色棒束孢几丁质酶Ifuchil结构基因和上游启动子序列,其结构基因中含有2个内含子,上游启动子序列没有明显的TATA框和CAAT框。但包含了多个转录因子结合位点,如GATA-1等。该上游序列还含多个富集嘧啶和富集嘌呤相连的序列,可能是葡萄糖抑制元件Mig1和Mig2的结合位点。Mig1和Mig2受到葡萄糖的直接或间接激活,从而抑制Ifuchila的表达。启动子序列Ifuprlb和Ifuchilb均含有可能的压力反应元件STREs（CCCCT）。这可能是因为玫烟色棒束孢寄生生活的基因转录或表达受到昆虫营养的诱导获得毒力基因。3玫烟色棒束孢蛋白酶与几丁质酶转基因球孢白僵菌菌株的获得及其对马尾松毛虫毒力增效作用本实验选用了制备芽生孢子遗传转化方法对球孢白僵菌生产菌株Bb13进行遗传改造,获得了单价工程菌株Bb13-Ifuprl和Bb13-Ifuchil,通过对其酶活的测定表明,转基因中的酶活相对于野生型菌株得到显著的提高,分别为野生型菌株酶活的2.65倍和1.73倍。对马尾松毛虫的生物测定中,在孢子浓度为1×10⁷个/ml时,与野生型菌株Bb13相比,重组菌株Bb13-Ifuprl的LT₅₀平均缩短了平均缩短了33.7%,而重组菌株Bb13-Ifuchil的LT₅₀平均缩短了平均缩短了29%;同时相对于野生型菌株Bb13,重组菌株Bb13-Ifuprl的LC₅₀降低了4.2倍的孢子用量,而重组菌株Bb13-Ifuchil的LC₅₀降低了1.7倍的孢子用量,大大降低了生物防治的经济成本。4玫烟色棒束孢蛋白酶基因Ifuprl和几丁质酶基因Ifuchil在毕赤酵母中表达用逆转录聚合酶链式反应（RT-PCR）方法扩增出带有EcoRI和NotI双酶切位点的Ifuprl和Ifuchil结构基因,经序列证实后,将该结构基因引入载体pPIC9K中,将重组质粒pPIC9K转化大肠杆菌DH5α细胞,质粒提取后分别采用菌落PCR扩增、双酶切和克隆测序的方法对重组质粒进行了验证,将正确构建的重组质粒转化毕赤酵母GS115细胞,得到酵母工程菌株。在酵母α-Factor及AOX1基因启动子和终止信号的调控下,Ifuprl和Ifuchil在甲醇酵母中大量表达并分泌到胞外,该两种酶的表达受甲醇的严格调控和诱导,随着诱导培养时间的增加,在培养基上清液中的单位体积酶活力相应上升,GS115-Ifuprl在诱导培养48h后酶活力达到最大值40.5U/mL;GS115-Ifuchil在诱导培养72h后酶活力达到最大值25.5U/mL.SDS-PAGE分析显示:1两表达产物的分子量大小约为47kD。该研究为构建可大量生产蛋白降解酶和几丁质降解酶的酵母工程菌提供了重要理论依据。更多还原

【Abstract】 Isaria fumosorosea is a kind of entomogenous fungi belongs to Ascomycota and have a worldwide distribution.It was recorded to be able to infect many species insects belonging to 25 families including some important agricultural insects,plutella xylostella, diuraphis noxia and bemisia tabaci,etc.In order to invade the hosts,the fungus mostly excretes several varieties of ectoenzyme hydrolases for degradating the body wall of the insect.Those hydrolases mainly contain protease,chitinase,lipase,and many others.The biocontrol fungus Beauveria bassiana plays an important role in biological control against insect pests,and it also has been studied and applied widely recently.In China,it has been mass-produced in metric tons and applied in large scale against Masson’s pine caterpillar, Dendrolimus punctatus（Walker）,making this one of the largest,successful microbial pest control programs in the world.However,the fungal insecticide has such shortcomings for example,slow kill,susceptible to environment,unstable kill effect,and relative narrow insecticidal range,resulting in the limit for being used widely.Thus in ordor to gain the commercial fungal insecticide,it is necessary to apply genetic and cell engineering technologies to improve strain virulence.This thesis conducts the following several researches on Isaria fumosorosea and Beauvewria bassiana:1.The screening of high yielding protease strain and the optimization of conditions for chitinase producing by the corresponding strain.By analyzing a couple of tens of Isaria fumosorosea preserved in this lab,we firstly selected 16 strains of Isaria fumosorosea with different hosts and different habitats,and then screened out the strain RCEF3304 which has a relatively high protease activity by protease activity assay.The further study on the optimization enzymatic kinetics study of yielding chitinase from this strain demonstrates that the appropriate condition are:2%（m/v） of colloid chitin,3%（m/v） of NaNO₃,0.05%（m/v） of Mg²⁺,pH6.0 as the initial value of the culture medium is,inoculum concentration is 12%,temperature is 25℃,time is 36 h.2.The clone and analysis of Isaria fumosorosea protease gene,chitinase gene and upstream regulatory elementsWith SMART RACE and DNA walking technology,we successfully cloned two important new genes from Isaria fumosorosea RCEF3304,protease gene and chitinase gene,which are associated with its virulence.And we deposited the two genes to the GenBank database coded as FJ423001 and FJ377733 respectively.The subsequent studies was conducted as follows:1) The full-length cDNA of lfuprl gene is 1704bp,with 1287bp,233bp and198 bp of open reading frame（ORF）,5’-untranslated region（5’-UTR） and 3’-untranslated region （3’-UTR）,respectively.The open reading frame（ORF） encodes 428 amino acids.The theoretical molecule quantity of mature protein is 46.3kDa with a calculated pI of 7.37.In the 428 amino acids,there are 50 with strong alkali（K,R）,52 with strong acid（D,E）,146 hydrophobic amino acids（A,I,L,F,W,V）,and 105 polar amino acids（N,C,Q,S,T,Y）, taking up 11.8%,12.2%,34.3%and 24.7%respectively.Using of the biological sofeware DNAman,Alignments with the deduced amino acid of mature proteins in 4 species of fungi showed 88%,71%,71%and 70%,respectively,identical with those of Isaria farinosa（AAY87460）,Hypocrea lixii（ABI84117）,Trichoderma atroviride（ABG57252）, and Metarhizium anisopliae var.anisopliae（CAD13274）,respectively.Meanwhile,the active site residues for serine proteases could be readily identified in the mature protease.2) The full-length cDNA of IfuchilI gene is 1704bp,with 1542bp,33 bp and 234bp of open reading frame（ORF）,5’-untranslated region（5’-UTR） and 3’-untranslated region （3’-UTR）,respectively.The open reading frame（ORF） encodes 425 amino acids.The theoretical molecule quantity of mature protein is 47.6 kDa with a calculated pI of 4.89.In the 425 amino acids,there are 39 with strong alkali（K,R）,54 with strong acid（D,E）,139 hydrophobic amino acids（A,I,L,F,W,V）,and 118 polar amino acids（N,C,Q,S,T,Y）, taking up 9.2%,12.7%,32.6%and 27.8%respectively.Alignments with the deduced amino acid of mature proteins in 4 species of fungi showed 90%,71%,71%and 70%, respectively,identical with those of Lecanicillium lecanii（AAX56960）,Hypocrea lixii （KAA05855）,Hypocrea virens（AAL78814）,and Isaria javanicus（AAZ83728）, respectively.It is prognosticated that,on the guarding area by Blast,these chitinases all belong to the family of Glycosyl hydrolase 18,and contain many conserved regions, including 2 highly- conserved regions SIGG and DGIDVDWE,which are the centred point of active hydrolysis of 18 family.3) It is shown that,by analysis to the structural gene of Ifuprl and the upstream promoter region,there is no intron existing in the structural gene,and this sequence not only contains the central stucture order CAAT frame of the promoter,but also covers some differential response components and the binding sites of transcription factors（including GATA-1,GATA-2,CdxA,etc.）.The CAAT frame is a component of controlling transcription speed,but other binding sites of transcription factors（including GATA-1, GATA-2,CdxA,etc.） may take on distinct controlling function.The team work of several GATA binding sites of transcription factors can notably enhance the report of genetic transcription activity,however,there is also research illustrating that GATA is able to restrain gene.As a part of the promoter,the CdxA component usually lies at the end of 5’, and it can increase the genetic transcription activity within the carrier in the system of delivering CdxA protein report.4) It is shown that,by gaining the structural gene of Ifuchil and the upstream promoter regions,there is 2 introns existing in the structural gene,and the uptream promoter has no obvious TATA frame and CAAT frame,but includes many binding sites of transcription factors,eg.GATA-1,etc..The uptream region also has the conjoint sequence between pyrimidine-rich and purine-rich,and it may be the binding site of the Glucose restraining component Mig1 and Mig2,which are activated by Glucose directly or indirectly so as to restrain the expression of Ifuchila.The promoter region lfuprlb and Ifuchilb both have the potential pressure-response componet STREs（CCCCT）.The reason might be the genetic transcription or expression of Isaria fumosorosea’s parasitic life is induced by insect nutrient to gain virulence gene.3.Generation of transgenic Beauveria bassiana strains with Protease gene and chitinase gene from Isaria fumosorosea and its increased virulence against Dendrolimus punctatus.In order to obtain Bb13-Ifupr1 and Bb13-Ifuchi1,the blastospore preparation methods of genetic transformation was resorted to transformate strain Bb13 produced by Beauveria bassiana—The enzymatic activity measurement assay,result shows that activity in transgenic compared with wild-type strains has been improved significantly.Wild-type strains were 2.65 times and 1.73 times of activity.In the biometrics study on Dendrolimus,the average LT₅₀ of recombinant strain Bb13-Ifuprl was reduced by 33.7%and the average LT₅₀ of recombinant strain Bb13-Ifupr1 was reduced by 29%by comparing with wide-type strain while the spore concentration is Bb13 is 1×10⁷ /ml,In addition,the average LT₅₀ of recombinant strain Bb13-Ifupr1 was reduced 4.2 times of spore usage,and Bb13-Ifuchi1 was reduced 1.7 times of spore usage comparing with wide-type.The cost of biological control,therefore,has been decreased dramatically.更多还原