【作者】 徐健；

【导师】 祝树德； 吴进才；

【作者基本信息】 扬州大学 ， 农业昆虫与害虫防治， 2008， 博士

【摘要】 苏云金杆菌(Bacillus thuringiensis,Bt)是研究应用最为广泛的生物杀虫剂,实际使用中存在作用速度慢、防效低等弱点。美洲粘虫颗粒体病毒(Pseudaletia unipuncta granulosis virus , PuGV )是含有增效蛋白( enhancin)的杆状病毒(Baciluvirus),能提高核型多角体病毒(nuclear polyhedrosis virus,NPV)的侵染能力及Bt的毒力。本文以Bt和东方粘虫P. separata转主增殖的美洲粘虫颗粒体病毒(PuGV-Ps)为材料,明确了PuGV-Ps对Bt的增效作用和增效特性;并从PuGV-Ps对杀虫晶体蛋白的酶解活化、昆虫中肠酶活性的变化、昆虫中肠围食膜(peritrophic membrane,PM)结构的破坏等方面探索了增效机理;克隆和测序了PuGV-Ps增效蛋白基因并原核表达了增效蛋白;研制了PuGV-Ps对Bt的增效制剂,并明确了其应用效果。主要结果如下:1明确了PuGV-Ps对Bt的增效作用以小菜蛾Plutella xylostella、甜菜夜蛾Spodoptera exigua、棉铃虫Helicoverpa armigera为试虫,采用生物测定方法测定了PuGV-Ps对Bt制剂的增效作用。结果表明Bt中加入PuGV-Ps对3种鳞翅目害虫都具有增效作用,共毒系数达127-146。高温灭活的PuGV-Ps对Bt同样具有增效作用,对小菜蛾的共毒系数达135.8,说明PuGV-Ps中含有对Bt毒力增强作用的增效因子。PuGV-Ps可以提高Bt对小菜蛾的杀虫速度,250μg/mL浓度的Bt中加入PuGV-Ps较单用Bt致死中时间LT50缩短了37.8%。转Bt基因的抗虫棉叶经PuGV-Ps处理后饲喂棉铃虫死亡率也得到相应提高。PuGV-Ps还能增强Bt对甜菜夜蛾生长发育的抑制作用,表现为幼虫生长量相对减少、蛹重下降、化蛹率降低和化蛹历期延长。2分离纯化了PuGV-Ps增效蛋白并测定其对Bt的增效活性用PuGV感染东方粘虫获得的转寄主粘虫颗粒体病毒PuGV-Ps的包涵体中含有分子量为108 kD的增效蛋白。PuGV-Ps经碱溶、Sephadex G-200凝胶过滤层析分离获得部分纯化的增效蛋白。以小菜蛾、甜菜夜蛾、棉铃虫等3种鳞翅目昆虫为试虫测定部分纯化的增效蛋白对Bt的增效作用,联合作用的共毒系数在116-155之间,表明PuGV-Ps增效蛋白是一种增效因子,可以增强Bt鳞翅目昆虫的毒力。3探明了PuGV-Ps对Bt增效作用的影响因子PuGV-Ps对Bt的增效程度随PuGV-Ps量的变化而不同,试验范围内不同配伍的PuGV-Ps和Bt间的共毒系数在105.3至195.0之间,其中以Bt∶PuGV-Ps为4∶1增效作用最明显,72 h LC50为0.039 mg/mL。不同温度和pH都影响PuGV-Ps对Bt的增效作用,16℃~ 20℃增效程度明显高于24℃~ 32℃,而碱性条件下(pH 8-9)增效作用更显著。PuGV-Ps对Bt的增效作用因小菜蛾龄期不同而变化,2、3龄幼虫试验,小菜蛾死亡率较Bt分别提高了50%和30.31%,而对低龄(1龄)和高龄(4龄)幼虫增效不显著。PuGV-Ps饲喂2 h后接毒Bt,小菜蛾死亡率明显提高,48 h死亡率达66.67%,较Bt+PuGV-Ps处理死亡率提高了53.87%,差异显著。4分析了PuGV-Ps对苏云金杆菌δ-内毒素的酶解活化作用用氨苯磺胺偶氮酪蛋白为底物测定PuGV-Ps中总蛋白酶活力表明,PuGV-Ps在pH 7.38-10.38的碱性条件下均具有一定的蛋白酶活性,且蛋白酶活力随pH的升高而显著提高。4种蛋白酶抑制剂都能抑制PuGV-Ps的蛋白酶活力,以大豆胰蛋白酶抑制剂(STI)的抑制作用最强,表明PuGV-Ps的蛋白酶活性是以胰蛋白酶为主要活力的多种蛋白酶的活性特征。通过SDS-PAGE研究了PuGV-Ps对苏云金杆菌δ-内毒素的降解活化作用,结果表明,碱性条件下Bt晶体蛋白和PuGV-Ps共同孵育,130 kD的δ-内毒素被进一步酶解成分子量为110 kD、87 kD、61 kD、47 kD等多种不同分子量的肽链,其酶解活化程度随缓冲液pH的升高而加深,在pH 10.7的0.1 mol Na2CO3缓冲液中,δ-内毒素被完全酶解,产生具有一定抗蛋白酶继续降解能力的分子量为47 kD、60 kD和61 kD的活性片段。同时PuGV-Ps量的多少和碱解时间都影响δ-内毒素的酶解活化程度,STI能一定程度抑制PuGV-Ps对δ-内毒素的酶解活化。5明确了PuGV-Ps抑制甜菜夜蛾中肠液对δ-内毒素的过度降解PuGV-Ps具有蛋白酶活性,对甜菜夜蛾中肠酶液离体蛋白酶活力及取食PuGV-Ps后总蛋白酶活力影响测定表明,在中肠酶液适宜pH范围(pH 9.38-10.38)内,PuGV-Ps都一定程度抑制了中肠酶液的总蛋白酶活力。SDS-PAGE试验显示,PuGV-Ps影响了甜菜夜蛾中肠酶液对苏云金杆菌δ-内毒素的降解活化作用,表现在对中肠酶液酶解130 kD的δ-内毒素成60 kD-87 kD的较大分子量的活性多肽无明显影响,但对活性多肽的进一步降解具有抑制作用,这种过度降解的抑制作用在25℃-30℃的温度和随酶解时间的延长更为显著。不同缓冲液同样影响甜菜夜蛾中肠酶液对δ-内毒素的降解,Na2CO3盐的存在是影响降解程度的重要因子。6证实了PuGV-Ps并发现Bt对甜菜夜蛾中肠PM结构的破坏作用利用环境扫描电镜和SDS-PAGE电泳技术研究了Bt、PuGV-Ps及其增效蛋白对甜菜夜蛾中肠PM的影响。电镜观察结果表明,正常的甜菜夜蛾PM外壁有韧性,表面较平滑,少皱褶,内壁表面较粗糙,有较厚质感,无孔洞和缝隙;取食PuGV-Ps或增效蛋白后的PM外壁皱缩,内壁平滑、质薄;Bt单独作用于PM同样改变了围食膜结构,但影响程度较小;但不同处理未发现对甜菜夜蛾PM造成穿孔或裂缝。中肠PM蛋白的SDS-PAGE试验表明,取食PuGV-Ps和增效蛋白后,PM上200 kD、150 kD、80 kD的大分子量的蛋白一定程度被降解成78 kD以下的小分子量蛋白带,小分子量27 kD的蛋白也被部分降解,而分子量为28 kD的小分子蛋白同时被完全降解;Bt也影响了PM蛋白的构成,取食Bt的PM蛋白电泳减少了28 kD的小分子蛋白,说明甜菜夜蛾PM上28 kD的蛋白是PuGV-Ps增效蛋白和Bt的共同靶蛋白。离体降解试验进一步证明Bt及增效蛋白对甜菜夜蛾PM上28 kD蛋白具有降解作用。7克隆和测序了PuGV-Ps增效蛋白的全长基因以PuGV-Ps的DNA为模板,参考粉纹夜蛾颗粒体病毒(TnGV)和棉铃虫颗粒体病毒(HaGV)的增效蛋白基因序列设计引物,通过PCR反应扩增出一条2.7 kb的特异性基因片段。纯化的PCR产物克隆到载体质粒pEASY-E2中,构建了重组质粒pEASY-En;用DNA双链测序法测定重组质粒pEASY-En中的外源基因序列,证明PCR扩增的产物是PuGV转宿主病毒PuGV-Ps增效蛋白的全长基因。与原始PuGV基因组增效蛋白的序列比较,两者同源性达99.59%,其中5’端500 nt相似性为98.60%,而3’端500 nt仅1个碱基发生突变。说明PuGV-Ps增效蛋白基因的3’端是基因的保守区域。8原核表达了PuGV-Ps增效蛋白并测定了表达蛋白的增效活性以大肠杆菌BL21(DE3)为感受态细胞,将插入PuGV-Ps增效蛋白基因的重组质粒pEASY-En转化到大肠杆菌中,构建了重组菌,于37℃下通过IPTG诱导表达了108 kD的表达产物。表达的目的蛋白带有6×His标签,能特异性吸附在Ni2+上并得到纯化,证明表达产物是目标增效蛋白。LB培养液中加入0.2%的葡萄糖后有利于增效蛋白基因的表达。生物测定结果表明,粗提的表达产物具有增效活性,可以提高Bt对棉铃虫、甜菜夜蛾的敏感性,600μg/g Bt浓度中加入300μg/g增效蛋白表达产物后甜菜夜蛾死亡率提高了10%,400μg/g Bt浓度中加入400μg/g增效蛋白表达产物后,棉铃虫死亡率由23.67%提高至38.67%,差异显著。随表达产物量的增加,增效作用更为显著。9研制了PuGV-Ps增强Bt制剂,并明确了其应用效果依据PuGV-Ps对Bt的增效作用,采用人工增PuGV-Ps、液体发酵Bt和喷雾干燥加工技术,研制了一种病毒增强Bt可湿性粉剂。该制剂主要成份为PuGV-Ps和Bt,含量为3.0×109OB/g PuGV-Ps·1.0×1010活芽孢/g Bt,共毒系数达162.57。该制剂毒性微毒,大白鼠经口LD50大于5000 mg/kg,无致敏和刺激性,无致病性,对鱼、鸟和蜜蜂均为低毒。田间应用结果表明,对小菜蛾、甜菜夜蛾、水稻纵卷叶螟Canphalocrocis medinalis等多种害虫都有较好防效。2000μg/mL浓度对小菜蛾2 d、7 d的防效达86.74%和79%,较Bt单剂的防效分别提高了23.5%和29.7%,差异显著;对甜菜夜蛾10 d防效达61.22%,与阿维菌素(Abamectin)1000μg/mL防效相当;水稻田使用病毒增强Bt 1500 g/ha对稻纵卷叶螟7 d防效达80.77%,高于Bt单剂71.15%的防效;使用病毒增强Bt对稻田蜘蛛无影响,药后7 d田间蜘蛛减少率为3.92%,而阿维菌素等对蜘蛛杀伤率达34.39%。更多还原

【Abstract】 Bacillus thuringiensis (Bt) has been extensively used for four decades as biopesticide due to its safety to environment and human health. The widespread use of Bt is often challenged by the efficacy of controlling pests. Pseudaletia unopuncta granulovirus (PuGV), which contains a special protein called enhancin, can synergize the infection of nucleopolyhedtovirus and enhance the toxicity of Bt to pests. In this study, with Bt and PuGV-Ps propagated in the larvae of P. separata by PuGV as materials, the synergistic effect and characterization of Bt toxicity to lepidoperous species with PuGV-Ps were investigated. The mechanisms of enhancement were analyzed in respect of proteolytic activity of PuGV-Ps, degradation ofδ-endotoxin, influence of midgut enzyme and damage to peritrophic membrane (PM). The enhancin gene of PuGV-Ps was cloned and expressed. A preparation of Bt enhanced by PuGV-Ps was devised and its application effect was also studied. The main results were as fellows:1 Enhancement of B. thuringiensis Toxicity to Lepidopterous Species with P. unipuncta Granulovirus-Ps: Enhancement of toxicity of B. thuringiensis with PuGV-Ps was demonstrated by bio-assays employing larvae of several lepidopterous species. Combinations of Bt and PuGV-Ps were synergistic and enhanced toxicity against Plutella xylostella, Helicoverpa armigera, and Spodoptera exigua. The co-toxicity coefficients (CTC) of Bt combined with PuGV-Ps to different larvae were diverse from 127 to 146. Denatured PuGV-Ps also enhanced toxicity of Bt to larvae of P. xylostella with CTC value of 136, indicating PuGV-Ps contained some synergistic factors. Besides the increase of mortality to larvae, the rate of larval death of P. xylostella was also accelerated by adding PuGV-Ps in Bt, and the median survival time (LT50) reduced 38 percent compared with the treatment of Bt alone at concentration of 250μg/mL. The toxicity of transgenic Bt cotton to H. armigera were also elevated by adding PuGV-Ps. PuGV-Ps also enhanced the effect of Bt on the larval development of S. exigua, with less weight of larvae and pupae, delay of pupating and lower ratio of pupation.2 Purification of Enhancin from P. unipuncta Granulovirus-Ps and Evaluation of Its Synergistic Effect to Bt: PuGV-Ps was propagated in the larvae of P. separata infected by P. unipuncta granulovirus. SDS-PAGE showed the capsules of PuGV-Ps contained a special protein called enhancin with molecular weight of 108 kD. The capsules was dissolved in alkaline solution of 0.02 mol NaOH, and then filtered through on a column of Sephadex G-200 and enhancin proteins were purified from the concentrated crude protein extract. The synergistic effects of PuGV-Ps enhancin on B. thuringiensis were tested by bio-assays employing larvae of several lepidopteral species, such as P. xylostella, H. armigera, and S. exigua. The CTC of Bt combined with PuGV-Ps enhancin to larvae of different species were range from 116 to 155. The results showed that PuGV-Ps enhancin was a synergistic factor, and could enhance the toxicity of Bt to larvae of Lepidoptera.3 Characterization of B. thuringiensis Enhancement by P. unipuncta Granulouvirus-Ps: The synergistic effects of PuGV-Ps on Bt were tested by bio-assays employing larvae of P. xylostella. The CTC of Bt combined with PuGV-Ps in different ratios were diverse from 105.3 to 195.0, showing a positive synergistic effect of PuGV-Ps on Bt. Among the mixtures, the most significant effect was found in the ratio of Bt and PuGV-Ps being 4:1, in which the LC50 was 0.039mg/mL. When environmental temperature was low (16℃and 20℃), the synergistic effects were statistically significant, while there were no differences at temperature of 28℃and 32℃comparing with the treatment of feeding the insects with Bt alone. The synergistic effect was elevated along with increase of pH value. In higher pH value of 8 and 9, PuGV-Ps elevated mortalities of P. xylostella larvae by Bt up to 16.67% and 23.33%, respectively. The co-effects of Bt and PuGV-Ps were also varied along with the larval age. Mortalities of 2nd and 3rd instar larvae increased 50.00% and 30.31% in the treatment of Bt+PuGV-Ps compared with that of Bt respectively, but there were no significant improvement in that of 1st and 4th instar larvae. Much higher synergistic effect was observed when oral inoculation with PuGV-Ps 2 h prior to Bt treatment. Comparing with the treatment of oral inoculation with Bt and PuGV-Ps simultaneously, The mortality at oral inoculation with PuGV-Ps 2 h prior to Bt increased 66.67% 48 h after treatment .4 Effect of P. unipuncta Granulovirus-Ps on the Degradation ofδ-endotoxin from B. thuringiensis: Total proteolysis of PuGV-Ps was measured by using azocasein under different pH condition from value 7.38 to 10.38, and the protease activity was improved with the rising of pH value. All the four kinds of inhibitors tested inhibited the activity of PuGV-Ps proteolysis, among which soybean trypsin inhibitor (STI) made the greatest affect. That indicated the proteolytic activity of PuGV-Ps was due to several proteases, mainly from trypsin-like enzyme. SDS-PAGE analysis showed that large amounts of activated toxin proteins were yielded fromδ-endotoxin of B. thringiensis incubated with PuGV-Ps under alkaline condition. Theδ-endotoxin of 130 kD degraded into toxic protein fragments with molecular weights from 47 kD to 110 kD. PH value influenced the degradation effect greatly, which elevated along with the increase of pH value. In buffer of 0.1mol Na2CO3 pH value 10.7, theδ-endotoxin completely cleaved into 47 kD, 60 kD and 61 kD activated toxins and resisted further degradation. The amount of PuGV-Ps and the time of incubation influenced the degree of degradation. STI also inhibited the degradation ofδ-endotoxin by PuGV-Ps.5 Elucidation of Inhibition Further Degradation ofδ-endotoxin in Midgut Juice from S. exigua by P. unipuncta Granulovirus-Ps: The proteolytic activity of midgut juice from S. exigua was influenced by PuGV-Ps. Under the suitable pH value, the total proteolysis activity of midgut juice reduced for some degree by PuGV-Ps, but there were some difference between in vitro and in vivo test. SDS-PAGE analysis showed that PuGV-Ps also affected the degradation ofδ-endotoxin of B. thuringiensis in midgut juice from S. exigua. The yield of active toxin proteins molecular weight from 60 kD to 87 kD was not influenced obviously by PuGV-Ps, but the further degradation of activated toxin was inhibited. The inhibiting effect was getting more notable with the degradation time and the rising of incubating temperature. Degradations ofδ-endotoxin by midgut juice in different buffers were disagreed, indicating that the saline of Na2CO3 was an important factor to increase the further degradation ofδ-endotoxin.6 Verification of the Damage of P. unipuncta Granulovirus-Ps and B. thuringiensis to Peritrophic Membrane of S. exigua: Using scanning electron microscope and SDS-PAGE gel, impact of B. thuringiensis, P. unipuncta granulovirus-Ps and enhancin from PuGV-Ps on peritrophic membrane of S. exigua was studied. Scanning electron microscope pictures indicated that exterior PM wall of normal S. exigua was smoothness and few rumple, while inner PM wall texture was thick and with some granulation. There were no hole and crack in the membrane. When insect feed PuGV-Ps or enhancin, the exterior PM wall turned to crimple and inner PM wall to thin and smoothness. When treated with Bt alone, the structure of PM was also changed in some degree. No hole or crack was found in all the treatments. SDS-PAGE gel analysis showed that the proteins in PM, which molecular weights was from 80 kD-200 kD, were partly degraded to small proteins under 78 kD, and a 28 kD protein was degraded completely. This 28 kD protein was also dismissed in the treatment of feeding Bt alone. In vitro tests gave more evidence that several different protiens in PM of S. exigua were degraded by enhancin of PuGV-Ps, and the 28 kD small molecular protein also degraded by PuGV-Ps enhancin andδ-endotoxin from Bt.7 Molecular Cloning and Sequence Analysis of Enhancin Gene from P. unipuncta Granulovirus-Ps: Using the DNA from PuGV-Ps as template and the nucleotide sequences of the enhancin genes from H. armigera granulovirus and Trichoplusia ni granulovirus for reference, we designed a primer for PCR and amplified a 2.7 kb specific fragment by PCR reaction. Purified PCR product was cloned into plasmid pEASY-E2 and a recombinant plasmid pEASY-En was constructed. The PCR product was proved to be the full long fragment of the gene encoding enhancin of PuGV-Ps by DNA sequencing. Sequence analysis revealed that enhancin gene from PuGV-Ps had 99.56% identity to enhancing gene from PuGV, which was reported previously. The different nucleotides were mostly concentrated in 5’terminal half and shared 98.60% identity from 1 to 500 nt, while in 3’terminal half only one nucleotide was changed from 1 to 500 nt, indicating that the 3’terminal half had the greater conservation.8 Expression and bioassay of P. unipuncta granulovirus-Ps Enhancin Gene in Escherichia coli: A 2.7kb enhancin gene of PuGV-Ps cloned into pEASY-E2 vector was recombinanted into Escherichia coli BL21(DE3). When induced by IPTG at 37℃, the target protein with molecular weight of 108kD was expressed successfully. The target protein was linked with 6×His tags and purified by Ni2+ column, indicating the target protein was enhancin, which was expressed by the inserted foreign gene. It was in favor of the expressing of enhancin gene by adding 0.2 percent of glucose. The extracted expressing protein showed synergistic activity to Bt. In the treatment of 600μg/g Bt, the mortality of S. exigua was increased 10.00 percent by adding 300μg/g extracted enhancin. When treating larvae of H. armigera, the mortality was elevated to 38.67 percent from 23.67 percent by adding 400μg/g extracted protein into 400μg/g Bt. The synergistic effect was advanced with the increasing of extracted protein.9 Producing and Application of Preparation of B. thuringiensis Enhanced by P. unipuncta Granulovirus-Ps: According to the toxicity of B. thuringiensis to the larvae of Lepidoptera enhanced by PuGV-Ps, a preparation of Bt+PuGV-Ps was prepared. This preparation was a water powder formulation and produced through the process of propagated PuGV-Ps in cultured P. separata, fermented Bt in liquid substrate and spray drying. It was composed by 3.0×109 OB/g PuGV-Ps and 1.0×1010 spores/g Bt, and the CTC was 162.57. PuGV+Bt almost had no virulence to vertebrate and did no harm to fish, bird and bee. Field trial showed, at the concentration of 2000μg/mL, Bt+PuGV-Ps got control effects of 86.74 percent and 61.22 percent to P. xylostella and S. exigua respectively, exceeding effects by Bt remarkablely. When used to control Canphalocrocis medinalis at dosage of 1500g/ha, the efficacy was up to 80 percent, while efficacy of Bt was 71.15 percent. PuGV-Ps+Bt only reduced 3.92 percent of the amounts of spiders, while Abamactin killed spiders up to 34.39 percent.更多还原