转Bt玉米叶面微生物区系及Bt蛋白表达规律的研究
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摘要
随着转基因作物全球化的应用和推广，转Bt基因作物释放后对生态环境产生的潜在影响受到广泛重视。转Bt基因玉米的释放对土壤微生态的影响、与野生近缘种的基因流、对非靶标昆虫的影响和靶标害虫抗性问题是近年研究的热点，然而转Bt基因玉米对叶面微生物的影响鲜有报道，因此研究田间自然条件下转Bt基因玉米对叶面微生物群落及多样性的影响对Bt玉米的生态风险评价具有重要意义。
2010年和2011年，在农业部转基因植物环境安全监督检验测试中心（济南）专用试验基地内种植转Bt基因玉米和亲本玉米，于苗期、拔节期、喇叭口期、抽雄期、抽丝期、乳熟期和完熟期采样，研究大田自然状态下转Bt基因玉米对叶面微生物群落结构和功能的影响。主要研究方法为Biolog微平板法、传统微生物分离培养法和酶联免疫法。
利用传统培养法，于2010和2011连续两年测定转Bt基因玉米MON810及其亲本玉米DK647叶面细菌、真菌和放线菌的数量变化，并对细菌生理群的数量和多样性进行分析，结果表明：虽然不同年份和生育期两个玉米品种叶面微生物数量存在差异，但是，年度间和相同的发育时期叶面微生物的数量变化趋势一致，一般由苗期开始增多，到生育后期达到数量高峰。转Bt基因玉米对叶面细菌和真菌影响较大，大部分生育期内Bt玉米叶面真菌和细菌数量与亲本玉米有显著差异，而放线菌数量与亲本相比差异不显著。2010年的抽雄期Bt玉米叶面细菌生理群Shannon-Wiener群落多样性指数、Simpson优势集中性指数、和均匀度指数显著低于亲本，2011年苗期和拔节期Bt玉米细菌生理群的三种群落特征参数显著大于对照。
利用Biolog微平板法对转Bt基因玉米及其亲本玉米叶面微生物的单一碳源利用水平进行分析，探讨转基因玉米对其叶面微生物群落功能多样性的影响。结果表明：玉米叶面微生物总体活性苗期最低，抽丝期最强，完熟期有所减弱；与非转基因亲本相比，苗期、喇叭口期和完熟期转Bt基因玉米叶面微生物活性大于非转基因玉米；叶面微生物对碳源的利用主要集中在糖类及其衍生物类上，代谢中间产物和次生代谢物类碳源利用程度最低；整个生育期内Bt玉米叶面微生物群落Shannon多样性指数和丰富度指数均高于常规玉米，其中喇叭口期差异显著（P＜0.05），均匀度指数和优势度指数在整个生育期无显著差异（P＞0.05）。主成分分析表明苗期、喇叭口期和抽丝期，转Bt基因玉米与非转基因玉米叶面微生物群落的碳源利用模式相似，不同生育期分异明显；完熟期转Bt基因与非转基因玉米叶面微生物碳源利用模式在PC2轴上存在显著差异。PC1轴和PC2轴分别对不同生育期和不同基因型玉米叶面微生物碳源利用模式造成主要分异。
应用Biolog方法研究了转Bt基因玉米粉碎叶对土壤微生物群落结构功能多样性的影响。取腐解10 d、25 d、40 d、55 d的土样分析土壤微生物群落多样性指数及对不同类碳源的利用情况。结果表明：在腐解的第25 d、40 d、55 d，转Bt基因玉米粉碎叶处理土壤微生物群落丰富度指数（H）、优势集中性指数（D）和均匀度指数（E）与常规玉米处理均无明显差异，腐解10d抽雄期转Bt基因玉米粉碎叶处理的土壤微生物群落多样性和均匀度显著高于常规玉米（P＜0.05），而乳熟期的优势集中性显著低于常规玉米（P＜0.05）。主成分分析表明，转Bt基因玉米粉碎叶对土壤微生物碳源利用模式的影响随腐解时间延长而减小，腐解第10 d影响最大，第55 d已基本无影响。
利用酶联免疫吸附测定法(ELISA)，对转Bt基因玉米不同发育时期和不同组织器官Bt晶体蛋白表达量进行了测定。结果表明，叶、茎、花丝、雌穗轴，雄穗小花和雄穗秆中均可检测到Bt杀虫蛋白，外源Bt蛋白占可溶性蛋白的比例随着作物的生长呈上升的趋势。相关分析表明，Bt蛋白表达量与玉米生长时间呈显著正相关，Bt玉米对叶面微生物群落结构没有显著影响，作物生长时间比玉米基因型对叶面微生物的影响大。
With the large scale release of transgenic crops, their possible ecological risks have beenpaid an increasing attention. Transgenic maize containing gene to produce the insecticidal toxinhas been widely planted. Most research on Bt maize has focus on invasiveness, gene flow toindigenous organisms, development of resistance in target pests, and direct or indirect effects onnon-target organisms and ecosystem. A few studies have been published to evaluate theecological effects of Bt maize on epiphytes. The aerial habitat colonized by these microbes istermed the phyllosphere, and the inhabitants are called epiphytes. While there has been someinvestigation of the colonists of buds and flowers, most work on phyllosphere microbiology hasfocused on leaves, a more dominant aerial plant structure. Bacteria are by far the most numerouscolonists of leaves, often being found in numbers averaging 106to 107cells/cm2(up to 108cells/g) of leaf. Because of their numerical dominance on leaves, and because more informationis available on the process of bacterial colonization of leaves, we focus on this group of microbesin this review. Therefore, in this study impact of Bt maize on the diversity of epiphytescommunities under field conditions is evaluated for the ecological risk of Bt maize.
The transgenic Bt maize MON810 and non-transgenic maize were grown in field condition(The Ministry of Agriculture, genetically modified plants Environmental Safety Supervision,Inspection and Testing Center special test base) for two years from 2010 to 2011.The leaves ofmaize seedling stage, jointing stage, trumpet stage, tasseling stage, silking stage, milking stageand full-ripe stage were examined. Epiphytes community diversity and functional diversity undernatural conditions were analyzed to make out whether the maize genotypes affect thephyllosphere microbes. The main research methods were the Biolog micro-plate method and thetraditional isolation culture methods. Enzyme-linked immunosorbent assay (ELISA) was alsoused to detect the Bt protein.
1 The traditional separation of culture results showed that although there were differencesin the colony-forming unites of culturable bacteria, actinomycetes and fungi of phyllospherebetween Bt maize and non-Bt maize in different growing periods and different years, the trend ofannual differences was basically consistent at the same growth period: the quantity of microbesin seeding stage is fewest and reaches the peak in the late of growing period. Compared with the controls, transgenic Bt maize seemed to stimulate the reproduction of phyllosphere fungi.However, no significant effect on the populations of actinomycete was observed. Significantdifferences were detected for bacteria at jointing stage, trumpet stage, silking stage and milkingstage. In 2010，the Shannon-Wiener index, Simpson index and evenness index of bacteriaphysiological groups were higher at seeding, jointing and full-ripe stage, but lower at trumpetstage, tasseling stage, silking stage and milk stage. In 2011, the population characteristicparameters of the microorganisms of Bt-corn are more than the ones of non-Bt corn in the wholegrowing period except trumpet stage. It may be concluded there was some difference in terms ofimpacts between transgenic maize and non-transgenic maize，while the main impacts onmicrobial community composition were likely due to different years and different growthperiods．
2 Biolog method was used to find the difference of carbon used by non-Bt maize and Btmaize, and to study the microbial differences of the metabolic functions of microbial activity inresponse to whether the influence by the Bt maize or not. The results showed that the activity ofPhyllosphere microorganism was weakest at seeding stage, highest at silking stage, and declinedat full-ripe stage. The carbon sources utilization ability of microorganisms on the Bt maizeleaves higher than that on the non-Bt maize leaves. The main carbon sources of epiphytes werecarbohydrate and its derivatives, while intermediate and secondary metabolites were used least.In the whole growth period, the Shannon functional diversity index and richness index of Btmaize were higher than non-Bt maize: compared with non-transgenic maize, transgenic maizehad less effects on Shannon functional diversity index, and richness index of Phyllospheremicrobes at seedling, silking and full-ripe stages, but had significant influence (P<0.05)ondiversity and richness index at trumpet stage. There were no significant difference been found inShannon evenness index and Simpson index during the whole growth stages. Principalcomponent analysis (PCA) indicated that there existed greater differentiation in the carbonsource utilization by the microbes on leaf surface of non-transgenic and transgenic maize atfull-ripe stage, suggesting the significant difference in the carbon source utilization pattern of themicrobes at this stage.
3 The diversity of soil microbial community and utilization pattern of different carbonsources were assessed via Biolog method on the 10th, 25th, 40th and 55th day of transgenic Bt maize leaves decomposition. The results showed that there were no differences in soil microbialcommunity with ground leaves evennness, diversity, and concentration index between transgenicBt maize and no transgenic Bt maize on the 25th, 40th and 55th. Compared with non-transgenicmaize, transgenic maize had a higher influence (P<0.05) on soil microbial community ondiversity and evenness index at tasseling stage, but a lower influence (P<0.05) on simpsonconcentration index at milking stage. Principal component analysis indicated that transgenic Btmaize ground leaves had a obviously effects on indigenous soil microbial community structureand function on the 10th. It, however, had no effects on soil microbial community on the 55th.
4 Temporal and spatial dynamic of Bt gene expression in Bt transgenic maize was studiedby testing the Bt crystal toxin protein in different growing stages and tissues of plant. The resultsshowed that with the growth of Bt maize, the proportion of Bt protein to total soluble proteins inleaves, stalk, filament, tassel stalk, tassel florets and female corncobs continuously increased.Ingeneral, the contents and proportion were lower in the young tissues or organs, but higher in themature. The correlation analysis showed that, the content of Bt protein was positively related toBt maize growth periods. The insecticidal crystal proteins has no significant infuence onepiphytes, and it has a lower infuence on epiphytes compared with growing stages.
2010年和2011年，在农业部转基因植物环境安全监督检验测试中心（济南）专用试验基地内种植转Bt基因玉米和亲本玉米，于苗期、拔节期、喇叭口期、抽雄期、抽丝期、乳熟期和完熟期采样，研究大田自然状态下转Bt基因玉米对叶面微生物群落结构和功能的影响。主要研究方法为Biolog微平板法、传统微生物分离培养法和酶联免疫法。
利用传统培养法，于2010和2011连续两年测定转Bt基因玉米MON810及其亲本玉米DK647叶面细菌、真菌和放线菌的数量变化，并对细菌生理群的数量和多样性进行分析，结果表明：虽然不同年份和生育期两个玉米品种叶面微生物数量存在差异，但是，年度间和相同的发育时期叶面微生物的数量变化趋势一致，一般由苗期开始增多，到生育后期达到数量高峰。转Bt基因玉米对叶面细菌和真菌影响较大，大部分生育期内Bt玉米叶面真菌和细菌数量与亲本玉米有显著差异，而放线菌数量与亲本相比差异不显著。2010年的抽雄期Bt玉米叶面细菌生理群Shannon-Wiener群落多样性指数、Simpson优势集中性指数、和均匀度指数显著低于亲本，2011年苗期和拔节期Bt玉米细菌生理群的三种群落特征参数显著大于对照。
利用Biolog微平板法对转Bt基因玉米及其亲本玉米叶面微生物的单一碳源利用水平进行分析，探讨转基因玉米对其叶面微生物群落功能多样性的影响。结果表明：玉米叶面微生物总体活性苗期最低，抽丝期最强，完熟期有所减弱；与非转基因亲本相比，苗期、喇叭口期和完熟期转Bt基因玉米叶面微生物活性大于非转基因玉米；叶面微生物对碳源的利用主要集中在糖类及其衍生物类上，代谢中间产物和次生代谢物类碳源利用程度最低；整个生育期内Bt玉米叶面微生物群落Shannon多样性指数和丰富度指数均高于常规玉米，其中喇叭口期差异显著（P＜0.05），均匀度指数和优势度指数在整个生育期无显著差异（P＞0.05）。主成分分析表明苗期、喇叭口期和抽丝期，转Bt基因玉米与非转基因玉米叶面微生物群落的碳源利用模式相似，不同生育期分异明显；完熟期转Bt基因与非转基因玉米叶面微生物碳源利用模式在PC2轴上存在显著差异。PC1轴和PC2轴分别对不同生育期和不同基因型玉米叶面微生物碳源利用模式造成主要分异。
应用Biolog方法研究了转Bt基因玉米粉碎叶对土壤微生物群落结构功能多样性的影响。取腐解10 d、25 d、40 d、55 d的土样分析土壤微生物群落多样性指数及对不同类碳源的利用情况。结果表明：在腐解的第25 d、40 d、55 d，转Bt基因玉米粉碎叶处理土壤微生物群落丰富度指数（H）、优势集中性指数（D）和均匀度指数（E）与常规玉米处理均无明显差异，腐解10d抽雄期转Bt基因玉米粉碎叶处理的土壤微生物群落多样性和均匀度显著高于常规玉米（P＜0.05），而乳熟期的优势集中性显著低于常规玉米（P＜0.05）。主成分分析表明，转Bt基因玉米粉碎叶对土壤微生物碳源利用模式的影响随腐解时间延长而减小，腐解第10 d影响最大，第55 d已基本无影响。
利用酶联免疫吸附测定法(ELISA)，对转Bt基因玉米不同发育时期和不同组织器官Bt晶体蛋白表达量进行了测定。结果表明，叶、茎、花丝、雌穗轴，雄穗小花和雄穗秆中均可检测到Bt杀虫蛋白，外源Bt蛋白占可溶性蛋白的比例随着作物的生长呈上升的趋势。相关分析表明，Bt蛋白表达量与玉米生长时间呈显著正相关，Bt玉米对叶面微生物群落结构没有显著影响，作物生长时间比玉米基因型对叶面微生物的影响大。
With the large scale release of transgenic crops, their possible ecological risks have beenpaid an increasing attention. Transgenic maize containing gene to produce the insecticidal toxinhas been widely planted. Most research on Bt maize has focus on invasiveness, gene flow toindigenous organisms, development of resistance in target pests, and direct or indirect effects onnon-target organisms and ecosystem. A few studies have been published to evaluate theecological effects of Bt maize on epiphytes. The aerial habitat colonized by these microbes istermed the phyllosphere, and the inhabitants are called epiphytes. While there has been someinvestigation of the colonists of buds and flowers, most work on phyllosphere microbiology hasfocused on leaves, a more dominant aerial plant structure. Bacteria are by far the most numerouscolonists of leaves, often being found in numbers averaging 106to 107cells/cm2(up to 108cells/g) of leaf. Because of their numerical dominance on leaves, and because more informationis available on the process of bacterial colonization of leaves, we focus on this group of microbesin this review. Therefore, in this study impact of Bt maize on the diversity of epiphytescommunities under field conditions is evaluated for the ecological risk of Bt maize.
The transgenic Bt maize MON810 and non-transgenic maize were grown in field condition(The Ministry of Agriculture, genetically modified plants Environmental Safety Supervision,Inspection and Testing Center special test base) for two years from 2010 to 2011.The leaves ofmaize seedling stage, jointing stage, trumpet stage, tasseling stage, silking stage, milking stageand full-ripe stage were examined. Epiphytes community diversity and functional diversity undernatural conditions were analyzed to make out whether the maize genotypes affect thephyllosphere microbes. The main research methods were the Biolog micro-plate method and thetraditional isolation culture methods. Enzyme-linked immunosorbent assay (ELISA) was alsoused to detect the Bt protein.
1 The traditional separation of culture results showed that although there were differencesin the colony-forming unites of culturable bacteria, actinomycetes and fungi of phyllospherebetween Bt maize and non-Bt maize in different growing periods and different years, the trend ofannual differences was basically consistent at the same growth period: the quantity of microbesin seeding stage is fewest and reaches the peak in the late of growing period. Compared with the controls, transgenic Bt maize seemed to stimulate the reproduction of phyllosphere fungi.However, no significant effect on the populations of actinomycete was observed. Significantdifferences were detected for bacteria at jointing stage, trumpet stage, silking stage and milkingstage. In 2010，the Shannon-Wiener index, Simpson index and evenness index of bacteriaphysiological groups were higher at seeding, jointing and full-ripe stage, but lower at trumpetstage, tasseling stage, silking stage and milk stage. In 2011, the population characteristicparameters of the microorganisms of Bt-corn are more than the ones of non-Bt corn in the wholegrowing period except trumpet stage. It may be concluded there was some difference in terms ofimpacts between transgenic maize and non-transgenic maize，while the main impacts onmicrobial community composition were likely due to different years and different growthperiods．
2 Biolog method was used to find the difference of carbon used by non-Bt maize and Btmaize, and to study the microbial differences of the metabolic functions of microbial activity inresponse to whether the influence by the Bt maize or not. The results showed that the activity ofPhyllosphere microorganism was weakest at seeding stage, highest at silking stage, and declinedat full-ripe stage. The carbon sources utilization ability of microorganisms on the Bt maizeleaves higher than that on the non-Bt maize leaves. The main carbon sources of epiphytes werecarbohydrate and its derivatives, while intermediate and secondary metabolites were used least.In the whole growth period, the Shannon functional diversity index and richness index of Btmaize were higher than non-Bt maize: compared with non-transgenic maize, transgenic maizehad less effects on Shannon functional diversity index, and richness index of Phyllospheremicrobes at seedling, silking and full-ripe stages, but had significant influence (P<0.05)ondiversity and richness index at trumpet stage. There were no significant difference been found inShannon evenness index and Simpson index during the whole growth stages. Principalcomponent analysis (PCA) indicated that there existed greater differentiation in the carbonsource utilization by the microbes on leaf surface of non-transgenic and transgenic maize atfull-ripe stage, suggesting the significant difference in the carbon source utilization pattern of themicrobes at this stage.
3 The diversity of soil microbial community and utilization pattern of different carbonsources were assessed via Biolog method on the 10th, 25th, 40th and 55th day of transgenic Bt maize leaves decomposition. The results showed that there were no differences in soil microbialcommunity with ground leaves evennness, diversity, and concentration index between transgenicBt maize and no transgenic Bt maize on the 25th, 40th and 55th. Compared with non-transgenicmaize, transgenic maize had a higher influence (P<0.05) on soil microbial community ondiversity and evenness index at tasseling stage, but a lower influence (P<0.05) on simpsonconcentration index at milking stage. Principal component analysis indicated that transgenic Btmaize ground leaves had a obviously effects on indigenous soil microbial community structureand function on the 10th. It, however, had no effects on soil microbial community on the 55th.
4 Temporal and spatial dynamic of Bt gene expression in Bt transgenic maize was studiedby testing the Bt crystal toxin protein in different growing stages and tissues of plant. The resultsshowed that with the growth of Bt maize, the proportion of Bt protein to total soluble proteins inleaves, stalk, filament, tassel stalk, tassel florets and female corncobs continuously increased.Ingeneral, the contents and proportion were lower in the young tissues or organs, but higher in themature. The correlation analysis showed that, the content of Bt protein was positively related toBt maize growth periods. The insecticidal crystal proteins has no significant infuence onepiphytes, and it has a lower infuence on epiphytes compared with growing stages.
引文
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