【作者】 张蕾；

【导师】 冯明光；

【作者基本信息】 浙江大学 ， 微生物学， 2013， 博士

【摘要】 金龟子绿僵菌(Metarhizium anisopliae)和球孢白僵菌(Beauveria bassiana)等昆虫病原真菌被广泛用于农林害虫防治,因而称为害虫虫生真菌。它们的杀虫机理无一例外地都是通过昆虫体壁侵入血腔而诱发昆虫病害和死亡,而不能通过口器摄入孢子的途径从消化道侵入寄主。虫生真菌的生防潜能既取决于侵染体对靶标害虫的侵染力或毒力,也取决于侵染体对夏季高温、阳光紫外辐射等胁迫因素的耐受力。本研究的目标,一是解决表达异源昆虫肠毒蛋白转基因工程菌的绝对口服毒力评价的技术难题,在本实验室前期工作基础之上进一步夯实虫生真菌通过遗传改良获得胃毒杀虫活性而研制新一代双途径侵染的安全高效广谱真菌杀虫剂的科学基础；二是解析球孢白僵菌自噬相关基因ATG5(BbATG5)的功能,首次探索细胞自噬可能对虫生真菌生长发育及生防潜能的影响,为虫生真菌细胞自噬的深入研究积累经验。主要结果总结如下。表达昆虫肠毒蛋白的金龟子绿僵菌工程菌构建及其口服毒力测定以高效侵染蚜虫、飞虱、叶螨等刺吸式口器害虫但不能经体壁有效侵染斜纹夜蛾(Spodoptera litura)低龄幼虫的金龟子绿僵菌野生菌株Ma456为受体菌,运用根癌农杆菌(Agrobacterium tumefaciens)介导的遗传转化技术,将苏云金芽孢杆菌(Bacillus thuringiensis)营养生长期微量分泌的非晶体昆虫肠毒蛋白Vip3Aa1基因导入受体菌株中组成性表达,筛选获得一株生长产孢性状良好且稳定表达目标基因的工程菌株MaVipB01.将野生菌株与工程菌株的分生孢子配制成1×106、1×107和1×108个孢子/mL的悬液,在自动喷塔中将各菌株低、中、高浓度的孢子悬液依次等量(1mL)喷雾到直径约为13cm的甘蓝叶圆片上,空气干燥数分钟后供斜纹夜蛾一至四龄幼虫取食,每天按需更换相同喷菌处理的新鲜叶片。如此供食8天,每天观察记录试虫死亡率、存活幼虫的平均叶片取食量(cm2/头)及体重(mg/头),并根据不同喷菌处理叶片上的孢子沉降量(孢子数/mm2)估计各处理存活幼虫的孢子累计摄入量(孢子数／头),每浓度处理重复三次,每重复试虫28～40头,以综合评价表达Vip3Aa1毒蛋白的工程菌株对试虫的胃毒杀虫活性和口服毒力。结果显示,一至四龄幼虫通过取食野生株高浓度喷菌叶片的摄入量分别达到2.28×105、1.88×106、5.22x106和8.11×106个孢子／头,而无论虫龄高低其死亡率始终与对照无异(5%左右)。所获少量虫尸经保温培养,也未见真菌病害致死的典型症状,而且同一虫龄的各处理间在幼虫的叶片取食量和体重方面均无显著差异。显然,野生株无法通过大量摄入孢子诱发试虫的实质性感染,与喷菌叶片接触也未能诱发孢子经体壁附着的实质性感染。与此形成鲜明对比的是,取食工程菌株喷菌叶片的各龄幼虫,其累计死亡率、叶片摄入量、体重及摄孢子入量,均随供食叶片的喷菌浓度而变化。其中,一龄幼虫取食高浓度喷菌叶片第2-4天的死亡率分别为44%、87%和97%,叶片取食量分别比对照降低69%、76%和83%,期间存活幼虫体重分别下降47%、57%和80%,相对应的孢子摄入量仅为2.8、4.2和5.2x103个／头。随着个体和虫龄的增大,带菌叶片取食量或孢子摄入量更大才能诱发与较小个体或较低虫龄相同的死亡率。取食高浓度喷菌叶片的四龄幼虫第6-8天摄入7.9、9.9和12.1×105个孢子后才分别死亡45%、62%和69%,期间存活幼虫的叶片取食量和体重分别下降～85%和84～91%。取食工程菌分生孢子而死亡的幼虫,无论虫龄高低,均表现典型的中毒症状,如个体显著变小、食量减少及行动迟缓。工程菌株对各龄幼虫的生测数据经时间—30量一死亡率模型的拟合分析,获得随取食带菌叶片时间而变化的半致死浓度LC5o和随供食叶片喷菌浓度而变化的半致死时间LT50,即为口服毒力的定量指标。据此,叶片上工程菌孢子达到1293个/mm2,初孵一龄幼虫取食第2天即可死亡50%,取食第3-5天死亡一半所需的理论喷菌浓度分别降为103、28和8个孢子/mm2,事实上该龄幼虫取食高浓度处理叶片的第5天即全部死亡。二龄幼虫需要取食1500个/mm2的带菌叶片4天才死亡一半,第5-8天死亡一半的叶片带菌量分别降为537、152、83和45个孢子/mm2。杀三龄幼虫50%所需的喷菌浓度与二龄幼虫的接近,或许因为其增加的体重刚好抵销摄食量提高的影响。进入暴食期的四龄幼虫个体更大,取食带菌叶片第6-8天死亡一半所需的喷菌浓度分别为1760、730和410个孢子/cm2。自噬因子BbATG5对球孢白僵菌生长发育及毒力的影响从球孢白僵菌野生株Bb2860中克隆获得自噬相关基因BbATG5的全长ORF(905bp)及其2382、1129bp的上下游侧翼序列。其ORF带一个95bp的内含子,编码由269个氨基酸组成的蛋白质,以单拷贝的形式存在于基因组中。将BbATG5的部分片段(49bp)用带bar标记基因的表达盒(942bp)进行替代而同源重组,构建靶基因敲除突变株ABbATG5。随后在敲除株中转入由BbATG5全长编码序列及其启动子(上游2.6kb)和磺酰脲抗性基因sur组成的表达盒进行靶标基因的拯救而构建回补株ΔBbATG5::BbATG5。将野生株、敲除株和回补株的液培菌丝在含50mM LiCl的除氮察氏培养液Cz-N(氮饥饿胁迫)中诱导培养3h后,收集菌丝并用单丹磺酰戊二胺MDC染色。荧光显微镜观察显示,野生株和回补株的菌丝细胞中均有大量自噬体和溶酶体出现,而敲除株细胞中虽可见较明显的溶酶体,但自噬体稀有。透射电镜观察进一步证实诱导后的野生株和回补株菌丝细胞的液泡中存在大量自噬体,而同样诱导的敲除株细胞中少有自噬体。这些结果证实球孢白僵菌在氮饥饿条件下会发生细胞自噬现象,而BbATG5的缺失可阻碍自噬发生。用流式细胞仪进行各菌株分生孢子的前向散射(FSc)和侧向散射(SSc)检测,发现敲除株分生孢子增大约20%,且伴随明显的形态畸变。在察氏培养基(CzA)上,野生株和回补株的孢子萌发率基本保持100%,而敲除株的却降低～20%。在无氮源的CzA-N平板和无营养的吐温-80水琼脂平板上,野生株和回补株的孢子萌发率分别降低60%和43%左右,而敲除株的分别仅有～20%和不足5%。将各菌株在萨氏培养基SDA、燕麦培养基OMA、CzA和CzA-N等不同营养的平板上培养7天,发现敲除株在CzA上的营养生长显著慢于野生株和回补株。而其在CzA-N上的营养生长则减慢50%左右,且敲除株菌落中的菌丝变得稀疏。在富营养的SDA和OMA上,敲除株的营养生长虽与野生株和回补株无明显差异,但其产孢量却大幅度下降80%左右。在富营养的培养液SDB中接种106个分生孢子/mL进行36h摇床培养,敲除株仅产0.52×107个芽生孢子/mL,比野生株和回补株少产芽生孢子94～97%。用斜纹夜蛾二龄幼虫对各菌株进行同一浓度(107个孢子/mL)等量孢子悬液的标准化喷菌生物测定,野生株、敲除株和回补株对试虫的LT50分别为5天、7天和5天,即敲除株的毒力降低40%。结语以上结果表明,组成性表达异源肠毒蛋白的金龟子绿僵菌工程菌株,通过获得胃毒杀虫活性,不仅拓宽了真菌的寄主范围,而且使原本对斜纹夜蛾无侵染力的菌株的口服毒力达到可应用的水平。这充分说明利用现代生物技术将虫生真菌与苏云金芽孢杆菌丰富多样的Vip3A家族蛋白结合起来的科学意义和应用前景。另一方面,通过解析自噬相关基因BbATG5的生物学功能,证明BbATG5不仅是球孢白僵菌细胞正常自噬所必须的,而且参与调节营养吸收、孢子萌发、营养生长、产孢及毒力等一系列重要表型而影响真菌的生防潜能。这为深化虫生真菌细胞自噬现象及其意义的科学认识奠定了基础。更多还原

【Abstract】 Entomopathogenic fungi, such as Beauveria bassianaandMetarhizium anisopliae, have been widely used as biocontrol agents against agricultural and forestry insect pests. Their killing action unexceptionally stems from the capability of their infecting target pests by integument penetration into host hemocoel for proliferation but rarely from the ingestion of infective cells by chewing insects. The fungal biocontrol potential depends not only on cell infectivity to target pests but also on cell tolerance to outdoor stresses, such as high temperature and solar UV irradiation during the summer of pest infestation. This study aimed at:(1) solving technical problems with the assessment of absolute oral virulence in transgenic fungiwith per os infectivity being acquired byexpressing a heterogeneous insect midgut-specific toxin; and (2) characterizing the function of the autophage-relating geneBbATG5in B. bassiana and probing possible effect of cell autophage on the fungal growth, development, and biocontrol potential. The results are summarized below.Construction of a transgenic M. anisopliae strain expressing a midgut toxin and assessment of its oral virulence.As an insect midgut-specific toxin gene from Bacillus thuringiensis, the vegetative insecticidal proteigeneVip3Aa1was integrated into Ma456, a wild-type M. anisopliae strain that can kill sucking pests, such as aphids, planthoppers and spider mites, but is unable to infect Spodoptera litura neonatesby cuticlar infection, via Agrobacterium-mediated transformation. A transformant (MaVipB01) selected from several putative mutant colonies was confirmed expressing constitutively the target protein. To assess per osvirulenceacquired by the transformant, conidia preparations of parental Ma456and transgenic MaVipB01were suspended in0.02%Tween80respectively and stardardized to the low, median and high concentraions of1×106,1×107and1×108conidia per milliliter. For each strain, equal-volume (1ml) aliquots of the conidial suspensions (treatments) and aqueous0.02%Tween80(control) were separately sprayed onto cabbage leaf discs (～13cm diameter) in an automatic spray tower at the uniform working pressure of0.7kg/cm2. Air-dried for minutes, sprayed leaf discs were supplied with the larvaeof S. lituraInstars IV in large Petri dishes (15cm diameter) for their feeding. During8-day rearing, leaf disc residues were daily changed with fresh leaf discs sprayed as above in advance. Daily observations and meaurements in each dish included larval mortality, mean leaf consumption (cm2) and mean body weight (mg) per survivor, and the number of conidia ingested per larva in terms of consumed leaf area and conidial deposit (no. conidia per mm2) on the leaf disc sprayed. The bioassay of each strain against each instar was repeated three times and each replicate (dish) of the treatments including2840larvae.Consequently, all the larvae showed consistently null responses to the wild-type strain althoughInstars ⅡⅤ ingested up to2.28×105,1.88×106,5.22x106and8.11×106conidia per capita from theleaf discs with high conidial deposits respectively. Larval mortalities fell in a range of-5%observed in the controls irrespective of different larval instars or fungal sprays. Notypical mycosis syndrome was observed from a very few cadavers in each treatment after incubation for several days under saturated humidity. Nor were significant differences in both leaf consumption and body weight of a given instar found between the fungal sprays. Thus, the wild-type strain failed to cause substantial infection by either conidial ingestion or cuticle attachment during the ingestion. In contrast, the larvae of each instar feeding on theleaf discs under differentsprays of transgenic conidia showed significant differences in mortality, leaf consumption and body weight. The first-instar larvae ingesting2.8,4.2and5.2x103conidia per capita suffered from the mortalities of44%,87%and97%after feeding the high-spray leaf discs for42days respectively, accompanied with the reductions of69%,76%and83%in leaf comsuption and of47%,57%and80%in survivor body weight. Similar mortalities of larger and older larvae required longer leaf feeding or more conidial ingestion. For Instar IV, for instance, cumulative ingestions of7.9,9.9and12.1×105conidia per capita on days68resulted in the mortalities of45%,62%and69%respectively and the reductions of-85%in leaf consumption and8491%in survivor body weight during the period. Interestingly, all the larvae died of the ingestion of transgenic conidia exhibited typical poisoning syndrome, such asbody shrunkage, much less leaf consumption and drastic body weight decrease.Time-concentration-mortality modeling analysis of the transgenic strain against Instars IIVgenerated the trends of median lethal concentration (LC50) over thefeeding time of sprayed leaf discs and of median lethal time (LT50) over the conidial deposits on the leaf discs. Based on the trends for the oral virulence of the transgenic strain, ingestion of leaves with1293conidia/mm2could cause50%neonate mortality on day2and the sprays decreased drastically to103,28and8conidia/mm2for the same mortality if the feeding time was allowed for3to5days. For the second-instar larvae, LC50required the leaf spray of1500conidia/mm2for4-day feeding and decreased to537,152,83and45conidia/mm2on days58respectively. The oral virulence trend for Instar Ⅲ was close to that for Instar Ⅱ perhaps due to the deducted effects of theirlarger bodiesand more leaf consumption. The50%mortality of the fourth-instar larvae on days68required the leaf sprays of1760,730and410conidia/cm2for their feeding respectively.Function of autophage-relating gene BbATG5and its contributions to the growth, asexual development and virulence of B. bassiana.Autophagy is a highly conserved process, representing the major eukaryotic degradative pathwayof cellular components. Autophagy-mediated recycling of cellular materials contributes to celldifferentiation, tissue remodelling and proper development. In fungi, autophagy is required fornormal growth and cell differentiation. The filamentous entomopathogen Beauveria bassiana and itsinvertebrate targets represent a unique model system with which to examine host-pathogeninteractions. The ATG5gene is one of17involved in autophagosome formation, and the B.bassiana homologue (BbATG5) with the coding sequence of905bp and the upstream and downstream flanking regions of2382and1129bp was identified. The role of autophagy in B. bassiana growth andvirulence was investigated via construction of a targeted gene knockout of BbATG5. The mutant ΔBbATG5displayed increased sensitivity to nutrient limitation, with decreased germination and growthas compared with the wild-type parent. Conidiation became severely defective and conidiaderived from the ΔBbATG5mutant were altered in morphology. Cell differentiation intoblastospores was also reduced by a great deal. Apart from the significant defects in growth and development,insect bioassays using the oriental leafworm moth, Spodoptera litura, indicated～40%decrease in virulence in the ΔBbATG5strain. All the phenotypic defects of the ΔBbATG5straincould be restored by integration of an intact copy of BbATG5into the delta mutant. These data suggest that unlikeseveral plant and animal pathogenic fungi, where ATG5is required for infection, in B. bassiana itis dispensable for pathogenesis despite its significant influence on the fungal virulence.Conclusive remarks.The aquirement of per os infectivity by the transgenic strain MaVipB01expressing constitutively the insect midgut-specific toxin Vip3Aal broadens a spectrum of M. anisopliae against insect pests.The high oral virulence of the transformant to S. litura larvae which cannot be killed by the parental wild-type stain through normal cuticle infection indicates a potential for its commercial development and application. This highlights prospects for developing a newgeneration of green mycoinsecticidesby the engineering of various entomopathogenic fungi with diverse midgut-specific toxins in the Vip3A family fromB. thuringiensis. On the other hand, the functional characterization of BbATG5demostrates for the first time that BbATG5is not only essential for the normal autophage of B. bassiana but also involved in regulating the fungal nutrition uptake, conidial germination, vegetative growth, conidiation and virulence, thereby affecting the biocontrol potential of B. bassiana against insect pests.更多还原