Bt杀虫基因Cry3A在桑粒肩天牛幼虫肠道优势菌和常驻菌中的转化和表达研究
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摘要
天牛是鞘翅目昆虫中较大的一个类群,在世界各地广有分布,其幼虫大多以木质纤维为食,林木、果树、桑、茶、棉、木建材料、家具等都可以受到天牛的危害,林木的天牛受害率达20-90%,每年造成的经济损失上亿元。由于天牛幼虫营钻蛀性生活,生活隐蔽,活动期长,所以防治工作难度很大。目前的主要防治方法仍是以传统的人工钩杀幼虫、砸卵、堵洞和化学药剂防治等方法为主。其中人工防治费时、费工、成本又高,不适用于规模化大农场生产;利用化学防治药剂防治由于药剂有效期短,不易达到虫体,而且容易导致害虫抗药性增强以及具有污染环境、杀伤天敌的弊端。因此,探索新的防治途径,开发新的防治技术,已成为生产上的迫切要求。新的害虫防治方法和理论完善与发展离不开昆虫生理生化的研究。近年来,研究昆虫肠道正常菌群和肠道微生态以探索新的害虫控制方法正在成为国内外研究的热点之一。
本文用传统培养方法和现代分子生物学方法-16S rDNA分析法对桑粒肩天牛幼虫肠道微生物组成进行了研究,在此基础上,首次探索将杀虫基因转入天牛幼虫肠道常驻的和优势的正常菌群载体菌,以构建在昆虫肠道中高定植率、高生存力、高繁殖并能表达杀虫毒蛋白伴孢晶体的新型杀虫工程菌。研究将可能为害虫的生物防治开辟一条新的途径,为新型转基因生物农药的研发提供理论依据与技术支撑。
主要研究结果如下:
①用传统的培养方法从桑粒肩天牛幼虫肠道中共分离鉴定出18个种的细菌,它们分别是产酸克雷伯氏菌(Klebsilla Oxytoca)、成团杆菌(Enterobacter cloacae)、荧光假单胞菌(Pseudomonas fluorescens)、恶臭假单胞菌(Pseudomonas putida)、弗氏志贺氏菌(Shiqella flexneri)、溶血葡萄球菌(Staphylococcus haemolyticus)、鲍氏志贺氏菌(Shiqella boydii)、人葡萄球菌(Staphylococcus homis)、藤黄微球菌(Micrococcus luteus)、Breneria quercina、无花果沙雷氏菌(Serratia ficaria)、短短芽孢杆菌(Brevibacillus brevis)、苏云金芽孢杆菌(Bacillus thuringiensis)、大肠埃希氏菌(Escherichia coli)、Naxibacter haematophilus、产气长杆菌(Enterobacter aerogens)、克里斯汀微球菌( Micrococcus kristinae)、阿氏肠杆菌(Enterobacter absburiae)。其中溶血葡萄球菌(S. haemolyticus)、人葡萄球菌(S. homis)、短短芽孢杆菌(B. brevis)、苏云金芽孢杆菌(B. thuringiensis)等在幼虫肠道中全年均能分离到,成团杆菌(E. cloacae)、产气肠杆菌(E. aerogens)、阿氏肠杆菌(E. absburiae)可在1～2月份外的全年的大部分时间分离到,其它细菌种类则只能在4～11月份分离到,说明至少溶血葡萄球菌(S. haemolyticus)、人葡萄球菌(S. homis)、短短芽孢杆菌(B. brevis)、苏云金芽孢杆菌(B. thuringiensis)应为天牛肠道常驻菌群,而其余种类微生物则很可能是随进食或与环境接触而进入的过路菌群。根据分离率和肠道菌群培养的数量统计结果表明天牛肠道优势菌群是葡萄球菌属(Staphylococcus)细菌中的溶血葡萄球菌(S. haemolyticus)和人葡萄球菌(S. homis),其菌量分别为7.74±0.61和7.66±0.25,分离率分别为100%和98.09%。
②将按传统方法从桑粒肩天牛幼虫肠道分离、鉴定的18个不同种的细菌作为PCR模板,进行16S rDNA序列的分析,经与数据库中登录序列比对,鉴定结果与分离培养方法所获得的结果一致。所测序列与Genbank中同种细菌的16S rDNA序列相似性分别为98.58%、99.18%、97.86%、99.89%、98.02%、99.19%、99.32%、98.46%、99.30%、98.05%、96.97%、98.33%、99.46%、99.60%、99.03%、99.45%、99.01%、99.52%,均高于98%,说明鉴定结果正确。所得菌株的16S rRNA都已在Genbank中登录注册,系统接受号为EU554427-EU554444。
③将含有红霉素抗性基因和cry3A基因的Escherichia coli -Bacillus thuringiensis穿梭表达质粒pHT305a和pHT7911转入桑粒肩天牛幼虫肠道优势常驻菌溶血葡萄球菌(S. haemolyticus)、人葡萄球菌(S. homis)和常驻内生菌短短芽孢杆菌(B. brevis)、苏云金芽孢杆菌(B. thuringiensis)中,获得的转化子中外源基因能稳定自主复制,而且其中用红霉素抗性平板筛选出的常驻内生工程菌转化子在产伴孢晶体发酵培养基中培养至90%以上芽孢脱落晶体释放时的菌液可提取到经SDS-PAGE分析分子量为65KDa的伴孢晶体蛋白,说明已成功获得了四株转基因杀虫工程菌。对工程菌在天牛幼虫肠道内的定殖能力和生物毒力进一步测定的结果显示此四株工程菌既能在桑粒肩天牛幼虫肠道内定殖,又对天牛幼虫具有一定的杀虫活性。
Longhorn Beetle (Coleopteran: Cerambycidae) is a big group of insects distribuding worldwide and most of the larvae boring and feeding in tree trunks or branches. About 20% to 90% forests are damaged by these pests and millions yuan of money losted every year in China. Forest trees, fruit trees, mulbery trees, tea trees, cotton, wood material, evern furnitures and houses can be damaged. The pest control is very different because of the covert and long time life of the beetle’s larva.Now the control methods for the pests mainly included some traditional strategys such as hooking and kill larvae, smashing eggs, pluging up breath hole and the chemical spray. However, most of traditional means have limitations of low efficancy, high cost and and time taking. And the chemical control often results in grievous environmental pollutions, killing natural enemies or even hard to reach the body of pest. Therefore, new biocontrol strategys and techniques are eagerly demanded in practice. A new pest control theories and approach is always based on the research of insect physiology and biochemique. Recently, a kind of new approach for pest control, based on the research of micro-community and micro-ecosystem of insect intestines, is becoming one of the hot topics all over the world.
In this present, the intestinal microbe flora in Apriona germari (Hope) larvae was analyzed and identified by traditional culture and 16S rDNA sequence analysis. Whith this understanding, a study on the transforming Bt specific insecticidal protein gene cry3A into predominant bacteria and resident bacteria was carried for the first time to construct the recombinant pesticidal engineering bacteria which could settle down and reproduce in intestine of the larva, and express insecticidal crystal protein. The study result would exploit a new approach for covert pest biocontrol and provide a theoretical and technological support for the research of novel genic engineering bio-insecticide development.
The main results were showed as follows:
(1) Eighteen different bacterial strains were isolated and identified from A.germari larvae gut by traditional culture and identification methods. They were Klebsilla Oxytoca, Pseudomonas fluorescen, Pseudomonas putida, Shiqella flexneri, Staphylococcus haemolyticu, Staphylococcus homis, Shiqella boydii, Micrococcus luteus, Micrococcus kristinae, Breneria quercina, Serratia ficaria, Brevibacillus brevis, Bacillus thuringiensis, Escherichia coli, Naxibacter haematophilus, Enterobacter aerogens, Enterobacter cloacae, Enterobacter absburiae. Among them, 4 species bacterial Staphylococcus haemolyticu, Staphylococcus homis, Brevibacillus brevis and Bacillus thuringiensis can be isolated from Apriona germari (Hope) larvae intestine all the year, but other microorganisms just can be isolated from March to November ( the active period). So these 4 species bacteria were presumed resident endogenetic bacteria of Apriona germari larvae intestine, whereas other bacteria may be transient flora which get in intestine byfeeding or contact from enviorment. The data statistic of the bacterial clones showed that S. haemolyticu and S. homis were confirmed the endogenetic predominant flora with count of 7.74±0.61 and 7.66±0.25, and the isolation rate (The number of larvae which had the bacteria /the total number of the larvae had been detected) were 100% and 98.09%. The other isolations were comparatively low. They could be ranked orderly by the measure as Enterobacter aerogens, Escherichia coli, Micrococcus luteus, Brevibacillus brevis, Bacillus thuringiensis,Enterobacter cloacae, Micrococcus kristinae, Shiqella boydii, Shiqella flexneri, Klebsilla Oxytoca, Enterobacter absburiae, Serratia ficaria, Pseudomonas putida, Pseudomonas fluorescens, Breneria quercina, Naxibacter haematophilus according to their isolation rates.
(2) The 18 different bacteria strains, which isolated by traditional cultural means from A. germari larvae’gut, were analyzed by 16S rDNA sequence analysis. The result consists with the results of normal classification by morphology, biochemical methods. The homologous rates between isolations 16S rDNA sequences and the recording 16SrDNA sequences in Genbank were 98.58%, 99.18%, 97.86%, 99.89%, 98.02%, 99.19%, 99.32%, 98.46%, 99.30%, 98.05%, 96.97%, 98.33%, 99.46%, 99.60%, 99.03%, 99.45%, 99.01%, 99.52% respectively. All the 16S rDNA sequeeces abtained were logged in Genbank and the accession number were from EU554427 to EU554444.
(3) The Escherichia coli - Bacillus thuringiensis shuttle plasmid pHT305a and pHT7911, which contained anti- Erythromycin gene (Erythromycinr) and cry3A gene, had been transformed into predominant bacteria strains S. haemolyticus and S. homis and resident bacteria strains B. brevis and B. thuringiensis respectively. Further examinations by the electron microscope observation of the crystal protein and the protein SDS-PAGE analysis approved that the newly transgenic engineering bacterial strains have been constracted successfrlly. Four engineering bacteria were obtained which can colonize and express the target pesticide gene Bt cry3A perfectly in A. germari larvae, which may be developed a newly pesticide to control the pests.
本文用传统培养方法和现代分子生物学方法-16S rDNA分析法对桑粒肩天牛幼虫肠道微生物组成进行了研究,在此基础上,首次探索将杀虫基因转入天牛幼虫肠道常驻的和优势的正常菌群载体菌,以构建在昆虫肠道中高定植率、高生存力、高繁殖并能表达杀虫毒蛋白伴孢晶体的新型杀虫工程菌。研究将可能为害虫的生物防治开辟一条新的途径,为新型转基因生物农药的研发提供理论依据与技术支撑。
主要研究结果如下:
①用传统的培养方法从桑粒肩天牛幼虫肠道中共分离鉴定出18个种的细菌,它们分别是产酸克雷伯氏菌(Klebsilla Oxytoca)、成团杆菌(Enterobacter cloacae)、荧光假单胞菌(Pseudomonas fluorescens)、恶臭假单胞菌(Pseudomonas putida)、弗氏志贺氏菌(Shiqella flexneri)、溶血葡萄球菌(Staphylococcus haemolyticus)、鲍氏志贺氏菌(Shiqella boydii)、人葡萄球菌(Staphylococcus homis)、藤黄微球菌(Micrococcus luteus)、Breneria quercina、无花果沙雷氏菌(Serratia ficaria)、短短芽孢杆菌(Brevibacillus brevis)、苏云金芽孢杆菌(Bacillus thuringiensis)、大肠埃希氏菌(Escherichia coli)、Naxibacter haematophilus、产气长杆菌(Enterobacter aerogens)、克里斯汀微球菌( Micrococcus kristinae)、阿氏肠杆菌(Enterobacter absburiae)。其中溶血葡萄球菌(S. haemolyticus)、人葡萄球菌(S. homis)、短短芽孢杆菌(B. brevis)、苏云金芽孢杆菌(B. thuringiensis)等在幼虫肠道中全年均能分离到,成团杆菌(E. cloacae)、产气肠杆菌(E. aerogens)、阿氏肠杆菌(E. absburiae)可在1～2月份外的全年的大部分时间分离到,其它细菌种类则只能在4～11月份分离到,说明至少溶血葡萄球菌(S. haemolyticus)、人葡萄球菌(S. homis)、短短芽孢杆菌(B. brevis)、苏云金芽孢杆菌(B. thuringiensis)应为天牛肠道常驻菌群,而其余种类微生物则很可能是随进食或与环境接触而进入的过路菌群。根据分离率和肠道菌群培养的数量统计结果表明天牛肠道优势菌群是葡萄球菌属(Staphylococcus)细菌中的溶血葡萄球菌(S. haemolyticus)和人葡萄球菌(S. homis),其菌量分别为7.74±0.61和7.66±0.25,分离率分别为100%和98.09%。
②将按传统方法从桑粒肩天牛幼虫肠道分离、鉴定的18个不同种的细菌作为PCR模板,进行16S rDNA序列的分析,经与数据库中登录序列比对,鉴定结果与分离培养方法所获得的结果一致。所测序列与Genbank中同种细菌的16S rDNA序列相似性分别为98.58%、99.18%、97.86%、99.89%、98.02%、99.19%、99.32%、98.46%、99.30%、98.05%、96.97%、98.33%、99.46%、99.60%、99.03%、99.45%、99.01%、99.52%,均高于98%,说明鉴定结果正确。所得菌株的16S rRNA都已在Genbank中登录注册,系统接受号为EU554427-EU554444。
③将含有红霉素抗性基因和cry3A基因的Escherichia coli -Bacillus thuringiensis穿梭表达质粒pHT305a和pHT7911转入桑粒肩天牛幼虫肠道优势常驻菌溶血葡萄球菌(S. haemolyticus)、人葡萄球菌(S. homis)和常驻内生菌短短芽孢杆菌(B. brevis)、苏云金芽孢杆菌(B. thuringiensis)中,获得的转化子中外源基因能稳定自主复制,而且其中用红霉素抗性平板筛选出的常驻内生工程菌转化子在产伴孢晶体发酵培养基中培养至90%以上芽孢脱落晶体释放时的菌液可提取到经SDS-PAGE分析分子量为65KDa的伴孢晶体蛋白,说明已成功获得了四株转基因杀虫工程菌。对工程菌在天牛幼虫肠道内的定殖能力和生物毒力进一步测定的结果显示此四株工程菌既能在桑粒肩天牛幼虫肠道内定殖,又对天牛幼虫具有一定的杀虫活性。
Longhorn Beetle (Coleopteran: Cerambycidae) is a big group of insects distribuding worldwide and most of the larvae boring and feeding in tree trunks or branches. About 20% to 90% forests are damaged by these pests and millions yuan of money losted every year in China. Forest trees, fruit trees, mulbery trees, tea trees, cotton, wood material, evern furnitures and houses can be damaged. The pest control is very different because of the covert and long time life of the beetle’s larva.Now the control methods for the pests mainly included some traditional strategys such as hooking and kill larvae, smashing eggs, pluging up breath hole and the chemical spray. However, most of traditional means have limitations of low efficancy, high cost and and time taking. And the chemical control often results in grievous environmental pollutions, killing natural enemies or even hard to reach the body of pest. Therefore, new biocontrol strategys and techniques are eagerly demanded in practice. A new pest control theories and approach is always based on the research of insect physiology and biochemique. Recently, a kind of new approach for pest control, based on the research of micro-community and micro-ecosystem of insect intestines, is becoming one of the hot topics all over the world.
In this present, the intestinal microbe flora in Apriona germari (Hope) larvae was analyzed and identified by traditional culture and 16S rDNA sequence analysis. Whith this understanding, a study on the transforming Bt specific insecticidal protein gene cry3A into predominant bacteria and resident bacteria was carried for the first time to construct the recombinant pesticidal engineering bacteria which could settle down and reproduce in intestine of the larva, and express insecticidal crystal protein. The study result would exploit a new approach for covert pest biocontrol and provide a theoretical and technological support for the research of novel genic engineering bio-insecticide development.
The main results were showed as follows:
(1) Eighteen different bacterial strains were isolated and identified from A.germari larvae gut by traditional culture and identification methods. They were Klebsilla Oxytoca, Pseudomonas fluorescen, Pseudomonas putida, Shiqella flexneri, Staphylococcus haemolyticu, Staphylococcus homis, Shiqella boydii, Micrococcus luteus, Micrococcus kristinae, Breneria quercina, Serratia ficaria, Brevibacillus brevis, Bacillus thuringiensis, Escherichia coli, Naxibacter haematophilus, Enterobacter aerogens, Enterobacter cloacae, Enterobacter absburiae. Among them, 4 species bacterial Staphylococcus haemolyticu, Staphylococcus homis, Brevibacillus brevis and Bacillus thuringiensis can be isolated from Apriona germari (Hope) larvae intestine all the year, but other microorganisms just can be isolated from March to November ( the active period). So these 4 species bacteria were presumed resident endogenetic bacteria of Apriona germari larvae intestine, whereas other bacteria may be transient flora which get in intestine byfeeding or contact from enviorment. The data statistic of the bacterial clones showed that S. haemolyticu and S. homis were confirmed the endogenetic predominant flora with count of 7.74±0.61 and 7.66±0.25, and the isolation rate (The number of larvae which had the bacteria /the total number of the larvae had been detected) were 100% and 98.09%. The other isolations were comparatively low. They could be ranked orderly by the measure as Enterobacter aerogens, Escherichia coli, Micrococcus luteus, Brevibacillus brevis, Bacillus thuringiensis,Enterobacter cloacae, Micrococcus kristinae, Shiqella boydii, Shiqella flexneri, Klebsilla Oxytoca, Enterobacter absburiae, Serratia ficaria, Pseudomonas putida, Pseudomonas fluorescens, Breneria quercina, Naxibacter haematophilus according to their isolation rates.
(2) The 18 different bacteria strains, which isolated by traditional cultural means from A. germari larvae’gut, were analyzed by 16S rDNA sequence analysis. The result consists with the results of normal classification by morphology, biochemical methods. The homologous rates between isolations 16S rDNA sequences and the recording 16SrDNA sequences in Genbank were 98.58%, 99.18%, 97.86%, 99.89%, 98.02%, 99.19%, 99.32%, 98.46%, 99.30%, 98.05%, 96.97%, 98.33%, 99.46%, 99.60%, 99.03%, 99.45%, 99.01%, 99.52% respectively. All the 16S rDNA sequeeces abtained were logged in Genbank and the accession number were from EU554427 to EU554444.
(3) The Escherichia coli - Bacillus thuringiensis shuttle plasmid pHT305a and pHT7911, which contained anti- Erythromycin gene (Erythromycinr) and cry3A gene, had been transformed into predominant bacteria strains S. haemolyticus and S. homis and resident bacteria strains B. brevis and B. thuringiensis respectively. Further examinations by the electron microscope observation of the crystal protein and the protein SDS-PAGE analysis approved that the newly transgenic engineering bacterial strains have been constracted successfrlly. Four engineering bacteria were obtained which can colonize and express the target pesticide gene Bt cry3A perfectly in A. germari larvae, which may be developed a newly pesticide to control the pests.
引文
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