Decomposition of Bacillus thuringiensis (Bt) transgenic rice residues (straw and roots) in paddy fields

Background, aim, and scope  

Genetic modification of commercial crops may affect their decomposition and nutrient cycling processes in agricultural ecosystems. Intensive rice cultivation under partially submerged conditions (paddy rice) is an important and widespread cropping system, particularly in the tropics, yet there is little data on the decomposition of Bt rice residue under field conditions. We investigated straw and root decomposition of rice modified to express the Cry1Ab protein of Bacillus thuringiensis (Bt) to kill lepidopteran pests, compared with a parental non-Bt isoline. The objective of this study was to assess the possible impacts of cry gene transformation of rice on residue decomposition under intensive rice cultivation with long period of submergence.     

转Bt基因作物对丛枝菌根真菌的影响研究进展

在过去的十年里，世界范围内转基因作物尤其是抗虫性转Bt基因作物的品种和种植面积迅速增加。同时，转Bt基因作物的环境安全性评价问题成为人们关注和研究的热点。丛枝菌根真菌（AMF）是生态系统中普遍存在的土壤微生物，能与绝大多数植物种类形成共生关系，在农业生态系统中起重要作用。转Bt基因作物环境释放后，其与AMF问的共生关系是否受所转入Bt基因的影响，以及影响机制需要及时研究。为此，综述了转Bt基因作物与AMF共生特征方面的研究进展，并根据Bt毒素发生的空间和时间规律提出了危害机制以及转Bt基因植物的规模化种植将降低农田系统中的AMF的生物多样性的观点。     

Soil microbial properties in Bt (Bacillus thuringiensis) corn cropping systems

Growing Bt crops reduces the use of insecticides applied to them, but these crops could affect soil microorganisms and their activities. We evaluated the effects of Bt (Cry1Ab) corn (Zea mays L.) and deltamethrin ([S]-α-cyno-3-phenoxybenzyl [1R, 3R]-3-[2,2-dibromovinyl]-2,2-dimethylcyclopropane-1-carboxylate) insecticide application on soil microbial biomass C (MBC), β-glucosidase enzyme activity (final season only), bacterial functional diversity, and bacterial community-level physiological profiles (CLPPs) in corn monoculture in five seasons. We also determined if growing Bt corn in crop rotation would alter these effects. Statistical analysis of pooled data across seasons did not show any effects of Bt technology, insecticide application or crop rotation on soil microbial biomass or diversity even though differences between seasons and between the rhizosphere and bulk soil were observed. Annual analyses of results also showed that neither the Bt technology nor insecticide application affected soil MBC, enzyme activity, or functional diversity of bacteria in corn rhizosphere, but shifts in bacterial CLPPs due to Bt trait were observed in one year. Crop rotation effects on soil microbial properties were not observed in most cases. Where effects were observed, Bt corn grown in rotation resulted in greater MBC, enzyme activity and functional diversity than Bt corn grown in monoculture or conventional corn grown in rotation, and these effects were observed only in bulk soil. Therefore, the Bt technology is safe with respect to the non-target effects measured in this study. However, the effects of repeated use of Bt crops over many years on the soil environment should continue to be monitored.     

苏云金芽孢杆菌分子伴侣基因p19的克隆和含有该基因的Bacillus thuringiensis表达载体的构建

苏云金芽孢杆菌（Bacillus thuringiensis,Bt)中的分子伴侣p19蛋白能否提高杀虫晶体蛋白（insecticidal crystal proteins,ICPs)的表达量及促进晶体形成，目前尚不确定。根据已知序列，了一对引物（p19-5N,p19-3N)，利用这对引物从苏云金芽孢杆菌以色列亚种（B.thuringiensis subsp.israelensis)HD-567中扩增出一个1.0kb的DNA片段。序列测定及分析结果表明，这个DNA片段包含cry11Aa操纵子启动子序列，全长p19基因及其偶联的下游基因cry11Aa的ATG起始密码子。将这个片段克隆到Bt-E.coli穿梭载体pHT3101上，构建成一个含有p19基因的Bt表达载体pHP19。其特点是在p19基因下游的ATG处顺序插入了5个限制性酶切位点NdeI,SacI,KpnI,SacI和Eco R I，为下一步研究p19基因的功能奠定了基础。     

Litterbag decomposition of residues from Bacillus thuringiensis (Bt) rice hybrids and the parental lines under multiple field conditions

Purpose

The cultivation of genetically modified (GM) crops has raised environmental concerns, since large amounts of plant materials remain in the field after harvesting. Specific proteins of GM crops might negatively impact soil ecosystem by changing residue decomposition dynamics. Particularly, the residue decomposition of crop-wild hybrids, which were formed through transgene escape to wild population, remains unexplored.

Materials and methods

We used litter bags to assess residue (leaves, stems and roots) decomposition dynamics of two stacked genes from Bacillus thuringiensis (Bt) Cry1Ac and the sck (a modified CpTI gene encoding a cowpea trypsin-inhibitor) (Bt/CpTI) rice lines (Kefeng-6 and Kefeng-8), a non-transgenic rice near isoline (Minghui86), wild rice (Oryza rufipogon) and Bt wild rice at three sites. The enzyme-linked immunosorbent assay (ELISA) was used to monitor the changes of the Cry1Ac protein in Bt rice residues.

Results and discussion

Mass remaining, total N and total C concentrations of rice residues declined over time and varied among plant tissues, with significant differences among cultivar, crop-wild hybrids and wild rice, but no differences between Bt and non-Bt rice cultivars. The initial concentration of Cry1Ac was higher in leaves and stems than in roots and was different between rice types. The degradation dynamics of Cry1Ac fitted best to a first-order kinetics model and correlated with the level of total nitrogen in residues but did not correlate with the mass decomposition rate. The predicted DT50 (50 % degradation time) of the protein ranged from 10.7 to 63.6 days, depending on plant types, parts and burial sites. By the end of the study (~170 days), the protein was present in low concentration in the remaining residues.

Conclusions

Our results suggest that the impacts of the stacked Bt/CpTI gene inserts on the decomposition dynamics of rice residues are insignificant.     

煤矿中苏云金芽胞杆菌(Bt)菌株的分离及其鉴定

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)是一种已被广泛用于农业害虫生物防治的昆虫病原细菌。本研究从永春天湖山矿区采集的159份样品中分离出1株Bt,光学显微镜检测发现菱形伴孢晶体。以cry1/7/9、cry2、cry3、cry4/10、cry5、cry6、cry8、cry11和cry1I的通用引物对该Bt分离菌进行Cry毒素基因分析,利用SDS-PAGE和双向电泳-质谱法(2-dimensional electrophoresis/mass spectrometry,2-DE/MS)对Bt分离菌进行蛋白鉴定。结果表明,分离菌含有cry1基因型,经克隆、测序后可知,菌株中的cry1亚基因型与cry1Ac有极高的同源性。分离株表达的晶体蛋白约为130 k D,通过蛋白质谱分析表明,杀虫蛋白为Cry1Ac,与PCR-RFLP所得出的结果一致;通过质谱分析还鉴定出分子伴侣Gro EL、F0F1型ATP-β亚基合成酶、延伸因子Tu和丙酮酸脱氢酶。生物信息学分析结果表明,这些蛋白主要参与帮助新生肽的正确折叠、能源生产和转换,以及提高细胞的应激能力以抵御外界不良刺激。本研究从煤矿中分离鉴定出Bt菌,为发现新资源、新型杀虫蛋白基因以及利用微生物治理重金属污染提供基础资料。     

Evaluation of Bt (Bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry

The health safety of Bt (Bacillus thuringiensis) corn (Zea mays L.) was studied using mouse testes as a sensitive biomonitor of potential toxic effects. Pregnant mice were fed a Bt corn or a nontransgenic (conventional) diet during gestation and lactation. After they were weaned, young male mice were maintained on the respective diets. At 8, 16, 26, 32, 63, and 87 days after birth, three male mice and an adult reference mouse were killed, the testes were surgically removed, and the percentage of germ cell populations was measured by flow cytometry. Multigenerational studies were conducted in the same manner. There were no apparent differences in percentages of testicular cell populations (haploid, diploid, and tetraploid) between the mice fed the Bt corn diet and those fed the conventional diet. Because of the high rate of cell proliferation and extensive differentiation that makes testicular germ cells highly susceptible to some toxic agents, it was concluded that the Bt corn diet had no measurable or observable effect on fetal, postnatal, pubertal, or adult testicular development. If data from this study were extrapolated to humans, Bt corn is not harmful to human reproductive development.     

Fate and effects of insect-resistant Bt crops in soil ecosystems

Recent applications of biotechnology, especially genetic engineering, have revolutionized crop improvement and increased the availability of valuable new traits. A current example is the use of the insecticidal Cry proteins from the bacterium, Bacillus thuringiensis (Bt), to improve crops, known as Bt crops, by reducing injury from various crop pests. The adoption of genetically modified (GM) crops has increased dramatically in the last 11 years. However, the introduction of GM plants into agricultural ecosystems has raised a number of questions, including the ecological impact of these plants on soil ecosystems. Crop residues are the primary source of carbon in soil, and root exudates govern which organisms reside in the rhizosphere. Therefore, any change to the quality of crop residues and rhizosphere inputs could modify the dynamics of the composition and activity of organisms in soil. Insect-resistant Bt crops have the potential to change the microbial dynamics, biodiversity, and essential ecosystem functions in soil, because they usually produce insecticidal Cry proteins through all parts of the plant. It is crucial that risk assessment studies on the commercial use of Bt crops consider the impacts on organisms in soil. In general, few or no toxic effects of Cry proteins on woodlice, collembolans, mites, earthworms, nematodes, protozoa, and the activity of various enzymes in soil have been reported. Although some effects, ranging from no effect to minor and significant effects, of Bt plants on microbial communities in soil have been reported, using both culturing and molecular techniques, they were mostly the result of differences in geography, temperature, plant variety, and soil type and, in general, were transient and not related to the presence of the Cry proteins. The respiration (i.e., CO₂ evolution) of soils cultivated with Bt maize or amended with biomass of Bt maize and other Bt crops was generally lower than from soils cultivated with or amended with biomass of the respective non-Bt isolines, which may have been a result of differences in chemical composition (e.g., the content of starch, soluble N, proteins, carbohydrates, lignin) between Bt plants and their near-isogenic counterparts. Laboratory and field studies have shown differences in the persistence of the Cry proteins in soil, which appear to be the result primarily of differences in microbial activity, which, in turn, is dependent on soil type (e.g., pH, clay mineral composition, other physicochemical characteristics), season (e.g., temperature, water tension), crop species (e.g., chemical composition, C:N ratio, plant part), crop management practices (e.g., till vs. no-till), and other environmental factors that vary with location and climate zones. This review discusses the available data on the effects of Cry proteins on below-ground organisms, the fate of these proteins in soil, the techniques and indicators that are available to study these aspects, and future directions.     

Microbial response of soils with organic and conventional management history to the cultivation of Bacillus thuringiensis (Bt)-maize under climate chamber conditions

An experiment was carried out in a climate chamber to analyse if Bt-maize may cause particular changes in soils with different levels of microbial biomass and activity due to long-term management history. Among the soils selected, the ones managed organically for 30 years exhibited twice the microbial biomass and 2.6 times the dehydrogenase activity (DHA) of the soil from a field with long-term conventional maize monoculture. Soils were cultivated twice in a row with Bt-maize, its near-isogenic line and a conventional breeding line. We tested the hypotheses that (a) soil microbial biomass and activity are affected by the cultivation of Bt-maize and that (b) the influence of Bt-maize depends on the level of soil microbial biomass and activity. Shoot and root yield and shoot C-content of Bt-maize were higher than the ones of the near-isogenic line. DHA under Bt-maize was 6 % higher, and the metabolic quotient for CO₂ (qCO₂) was 9 % lower than under its near-isogenic line, giving some support to hypothesis (a). No significant interactions of the soils and the varieties used were found in this study, thus hypothesis (b) was not confirmed, and soils with different microbial biomass and activity appear to react in a similar way to the cultivation of Bt-maize.     

苏云金芽孢杆菌vip2A(c)基因在昆虫细胞中的表达

利用苜蓿丫纹夜蛾核多角体病毒(Autographacalifornicamulticapsidnucleopolyhedrovirus,AcMNPV)Bac-to-Bac表达系统在粉蚊夜蛾(Trichoplusiani)TnHi5细胞中表达了GFP与Vip2A(c)酶活成分区域的融合蛋白(GV2融合蛋白),研究了该蛋白对昆虫细胞和病毒产生的影响。Bac-gfpDNA转染48h后部分TnHi5细胞已经检测到荧光,48￣120h出现荧光的细胞数目增加,最后70%左右的细胞都有较强的荧光。而Bac-gv2DNA转染后2￣5d仅有极少TnHi5细胞出现荧光且多呈零散分布,推测这可能是由于杆状病毒极晚期表达的GV2融合蛋白中的Vip2A对细胞骨架成分肌动蛋白进行ADP-核糖基化,从而影响了芽生型病毒粒子(BV)的正常产生或限制了BV在其它细胞间的传播。     

苏云金芽胞杆菌N-酰基高丝氨酸内酯酶基因(aiiA)在毕赤酵母中的优化表达

苏云金芽胞杆菌(Bacillus thuringiensis)N-酰基高丝氨酸内酯酶基因(auto inducer inactivation A,aiiA)编码的N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AiiA)能够水解植物病原菌群体效应的信号分子N-酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL),进而使革兰氏阴性细菌群体感应受到抑制,减弱病原菌的致病性。本研究将aiiA基因连接至分泌型穿梭表达载体pPICZαB,获得重组表达质粒p PICZαB-aiiA,线性化后电击转化毕赤酵母(Pichia pastoris)GS115,获得重组工程菌GS115-p PICZαBaiiA。利用定点突变技术,对aiiA基因进行密码子优化,获得重组工程菌GS115-p PICZαB-MaiiA。以终浓度为1%的甲醇、28℃条件下诱导表达,重组工程菌GS115-p PICZαB-aiiA和GS115-p PICZαB-MaiiA均成功表达并分泌出AiiA蛋白。抗病性分析表明,分泌表达的AiiA蛋白能有效抑制胡萝卜软腐欧文氏杆菌(Erwinia carotovora)的致病性。AiiA蛋白在毕赤酵母中的分泌表达,拓宽了AiiA蛋白的获取途径,为AiiA蛋白的产业化提供理论依据。密码子优化为今后改造AiiA蛋白,提高AiiA蛋白的表达效率提供了新的思路。     

Soil persistence of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Bt) toxin from transgenic Bt cotton tissues and its effect on soil enzyme activities

A silty loam soil was incubated with the leaves and stems of two transgenic Bacillus thuringiensis (Bt) cotton varieties and nontransgenic Bt cotton to study the soil persistence of the Bt toxin from the decomposing transgenic Bt cotton tissues and its effect on soil enzyme activities. The results showed that after Bt cotton tissue amendment, Bt toxin was introduced into soil upon decomposition; about 50% of the introduced Bt toxin persisted in soil for at least 56 days. No Bt toxin was detected in the nontransgenic Bt cotton-amended soil; the amount of Bt toxin was the highest in the soil treated with the residue with the higher Bt toxin content. Activities of soil urease, acid phosphomonoesterase, invertase, and cellulase were stimulated by the addition of Bt cotton tissues, whereas activity of soil arylsulfatase was inhibited. Probably cotton tissue stimulated microbial activity in soil, and as a consequence, enzyme activities of soil were generally increased. This effect can mask any negative effect of the Bt toxin on microbial activity and thus on enzyme activities.     

Biomimetic extraction of Bacillus thuringiensis insecticidal crystal proteins from soil based on invertebrate gut fluid chemistry

Quantitative monitoring of Bacillus thuringiensis (Bt) insecticidal crystal proteins in soil has been hampered by the lack of efficient extraction/detection methods. A novel approach for simple and effective Bt protein extraction was explored by evaluating extraction solutions from invertebrate gut fluids. Marine worm gut fluids were identified as promising for extracting Bt protein from soil. An artificial gut fluid based on these marine worm gut fluids was developed using commercially available chemicals and was evaluated for its ability to extract Bt proteins from soil. On the basis of experiments with Cry1 proteins, the artificial gut fluid in combination with ELISA was highly effective for protein extraction and analysis in a variety of soil types and was well-correlated with bioassay results. Coupling of immunoassay with this extraction method provides, for the first time, an efficient, accurate, and quantitative assay for routine measurement of Bt protein residues in soil.     

酞酸酯在土壤中的环境行为与健康风险研究进展

酞酸酯(PAEs)又称邻苯二甲酸酯,是环境激素类有机化合物,作为增塑剂在塑料、树脂和橡胶制品中的添加量一般为20%~60%。土壤中PAEs的主要来源有农用化学品、污水灌溉和大气沉降。PAEs在土壤中有较强的富集作用,并能通过一系列的环境地球化学过程进入不同的环境介质,引起环境污染和人类健康风险。本文结合国内外土壤PAEs的相关研究成果,综述了我国土壤PAEs的污染现状,分析了PAEs在土壤-大气界面(挥发、沉降)、土壤-植物系统(植物吸收、植物修复)、土壤-水界面下的环境行为(吸附-解吸)及土壤PAEs污染的环境健康风险,并指出国内土壤PAEs研究中存在的不足。研究结果显示,我国土壤环境总体上已遭受不同程度的PAEs污染;同时,土壤PAEs通过不同界面之间的迁移转化过程,也面临较高的生态环境健康风险。提出今后土壤PAEs研究应以区域土壤污染与环境行为为重点,深入研究土壤PAEs的时空传输与演变规律、多介质迁移转化机制和风险削减与修复措施,为保障土壤生态环境与健康提供理论依据。     

苏云金芽孢杆菌cry1Aa14基因的分离、克隆及其表达

Bt25是中国自行分离的对小菜蛾(Plutella xylotella)具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis),经PCR-RFLP鉴定含有cry1Aa基因。以全长基因PCR产物的粘端定向克隆的方法,设计1对特异引物,以Bt25质粒DNA为模板扩增cry1Aa全长基因。序列测定结果表明,该基因编码区为3552bp,编码1183个氨基酸,分子量为133．7kD,pI4．755。该基因序列已在GenBank注册,登记号为AY197341,并获得正式命名cry1Aa14。在氨基酸序列918～1180间,和已知的11种cry1Aa存在22-23个氨基酸的差异(其中1094～1097的4个氨基酸无对应序列),而这段区域和Cry1Ab氨基酸序列的对应区域无差异。cry1Aa14全长基因插入Bt表达载体,获得了重组表达质粒pBYB1,转化Bt无晶体突变株HD73cry-,经过抗性筛选、DNA酶切分析和PCR检测,证实转化成功。SDS-PAGE分析表明,该基因在上述受体中能正常表达133kD蛋白。杀虫生物测定结果表明,cry1Aa14表达产物对小菜蛾幼虫具有显著的毒杀作用,与cry1Aa12进行比较,毒力无明显差异。这种单基因菌株的发现及其基因的获得,为害虫抗性研究和高效工程菌的构建提供了重要实验材料。     

盆栽条件下溶磷菌对花生生长的影响

利用本实验室在红壤茶园土中筛选出的高效溶磷菌B1,以红壤为基质进行花生盆栽试验,研究溶磷菌B1对花生生长的影响。试验设置接种溶磷菌(B1,苏云金芽孢杆菌)、直接添加草酸(OA)、磷酸二氢钾(KP)以及空白对照(CK)处理。结果表明溶磷菌在红壤中定殖能力强、存活性较好;与对照相比,接种溶磷菌能显著提高土壤有效磷含量,促进花生根瘤的形成以及地下部生长发育,增加根面积、根体积和根尖数,花生果实的双荚果比例提高5%,百仁重高出3.8 g,果实中粗蛋白的含量增加2.5%。各项指标都显示,接种溶磷菌的效果要显著好于直接添加草酸的效果,而与直接添加可溶性磷源的效果相当。因此开发有效的溶磷菌剂应用于大田生产是可行的。     

转三价抗虫基因棉花遗传特性研究

研究采用农杆菌介导法将外源三价抗虫基因(Bt CpTI GNA)导入常规棉花品种中获得转基因再生株,分子检测表明外源基因已在棉花体内表达并遗传给后代材料。其转化再生株后代材料经抗性检测,进行大田选育已至F8代。PCR分子检测与转化的标记基因和外源目的基因抗性三者极有规律性,转化后代材料分离有的符合孟德尔规律,有的则较偏离,其所携带的基因在转基因棉花低代材料中分离规律较为含糊,高代材料则较稳定。     

苏云金芽胞杆菌WB5中营养期杀虫蛋白3Aa基因(vip3Aa)的克隆、表达与纯化

营养期杀虫蛋白(vegetative insectcidal protein,Vip)是由苏云金芽胞杆菌(Bacillus thuringiensis,Bt)在营养生长指数中期至稳定期期间分泌产生的一类新型杀虫因子,分为Vip1、Vip2和Vip3三种,以Vip3的研究最为深入。Vip3A对鳞翅目(Lepidoptera)害虫具有广谱杀虫活性,具有重要研究意义。本研究以Bt WB5菌株总DNA为模板,采用PCR方法扩增vip3Aa(Gen Bank登录号:AAM22456)全长,将该片段纯化回收后与p MD18-T连接并转入大肠杆菌(Escherichia coli)DH5α,进行酶切验证和序列测定。将vip3Aa基因片段与经同样酶切的表达载体p Czn1连接,构建重组表达载体p Czn1-vip3Aa,转入大肠杆菌Arctic ExpressTM(DE3),异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)进行诱导表达。SDS-PAGE结果表明,在沉淀和上清中均有约88 k D VIP3Aa蛋白质表达产物;采用Ni亲和树脂对上清中的VIP3Aa融合表达蛋白进行了纯化,获得了纯化蛋白。本研究成功亚克隆了vip3Aa基因,并表达纯化了VIP3Aa蛋白,为后续研究VIP3Aa蛋白在昆虫中肠的作用受体提供了基础资料。