Insect Control and Dosage Effects in Transgenic Canola Containing a Synthetic Bacillus thuringiensis cryIAc Gene

Zygotic hypocotyls of canola (Brassica napus L.) cv Oscar, cv Westar, and the breeding line UGA188-20B were transformed with a truncated synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-mediated transformation. Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes. A range of cry expressors was produced from 0 to 0.4% Cry as a percentage of total extractable protein. The Brassica specialists, the diamondback month (Plutella xylostella L.) and the cabbage looper (Trichoplusia ni Hubner), were completely controlled by low-, medium-, and high-expressing lines. Whereas control of the generalist lepidopteran, the corn earworm (Helicoverpa zea Boddie), was nearly complete, the other generalist caterpillar tested, the beet armyworm (Spodoptera exigua Hubner), showed a dose response that had a negative association between defoliation and cry expression. These plants were produced as models for an ecological research assessment of the risk involved in the field release of naturalized transgenic plants harboring a gene (Bt) that confers higher relative fitness under herbivore-feeding pressure.     

转双基因烟草对棉铃虫的杀虫活性评价

以含Ｂｔ杀虫蛋白基因（单基因）烟草和常规烟草为对照,系统测定了含Ｂｔ与豇豆胰蛋白酶抑制剂蛋白基因（双基因）的抗虫烟草对棉铃虫不同龄期幼虫的杀虫活性。结果表明：１￣３龄幼虫取食转双基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食转基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食     

Greenhouse and field evaluations of transgenic canola against diamondback moth, shape Plutella xylostella, and corn earworm, shape Helicoverpa zea

Canola (Brassica napus L.) cultivars Oscar and Westar, engineered with a Bacillus thuringiensis (Bt) cryIA(c) gene, were evaluated for resistance to lepidopterous pests, diamondback moth, Plutella xylostella L. (Plutellidae) and corn earworm, Helicoverpa zea (Boddie) (Noctuidae) in greenhouse and field conditions. In greenhouse preference assays conducted at vegetative and flowering plant stages, transgenic plants recorded very low levels of damage. A 100% diamondback moth mortality and 90% corn earworm mortality were obtained on transgenic plants in greenhouse antibiosis assays. The surviving corn earworm larvae on transgenic plants had reduced head capsule width and body weight. Mortality of diamondback moth and corn earworm were 100% and 95%, respectively, at different growth stages (seedling, vegetative, bolting, and flowering) on the transgenic plants in greenhouse tests. In field tests conducted during 1995–1997, plots were artificially infested with neonates of diamondback moth or corn earworm or left for natural infestation. Transgenic plants in all the treatments were highly resistant to diamondback moth and corn earworm larvae and had very low levels of defoliation. Plots infested with diamondback moth larvae had greater damage in both seasons as compared with corn earworm infested plots and plots under natural infestation. After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non-transgenic plants. Diamondback moth injury caused the most pronounced difference in plant stand and pod and seed number between transgenic and non-transgenic plants. Our results suggest that transgenic canola could be used for effective management of diamondback moth and corn earworm on canola.     

Increased fitness of transgenic insecticidal rapeseed under insect selection pressure

Rapeseed Brassica napus L. transgenic for a Bacillus thuringiensis ( Bt ) transgene was developed and was shown to be insecticidal towards certain caterpillars including the diamondback moth Plutella xylostella L. and the corn earworm Helicoverpa zea Boddie. To simulate an escape of the transgenics from cultivation, a field experiment was performed in which transgenic and nontransgenic rapeseed plants were planted in natural vegetation and cultivated plots and subjected to various selection pressures in the form of herbivory from insects. Only two plants, both transgenic, survived the winter to reproduce in the natural-vegetation plots which were dominated by grasses such as crabgrass. However, in plots that were initially cultivated then allowed to naturalize, medium to high levels of defoliation decreased survivorship of nontransgenic plants relative to Bt -transgenic plants and increased differential reproduction in favour of Bt plants. Thus, where suitable habitat is readily available, there is a likelihood of enhanced ecological risk associated with the release of certain transgene/crop combinations such as insecticidal rapeseed. This is the first report of a field study demonstrating the effect of a fitness-increasing transgene in plants.     

Expression of GFP and Bt transgenes in Brassica napus and hybridization with Brassica rapa

It is possible to monitor the movement of transgenes by tagging them with green fluorescent protein (GFP). In order to develop a model to study transgene flow, canola (Brassica napus cv Westar) was transformed with two GFP constructs, mGFP5er (GFP only) and pSAM 12 [GFP linked to a synthetic Bacillus thuringiensis (Bt) cry1Ac endotoxin gene]. Transformed callus sectors that fluoresced green were preferentially selected in the tissue culture process. Four independent GFP canola events and 12 events of GFP/Bt canola were regenerated through tissue culture. GFP fluorescence was macroscopically detectable throughout the entire life cycle of canola. The GFP/Bt events were insecticidal to neonate corn earworm (Helicoverpa zea) larvae and prevented herbivory damage. Fluorescence intensity at 508 nm varied between the independent transformation events, and ranged from 7.6×10⁵ to 13.8×10⁵ (counts per second) in contrast with the wild-type at 5.3×10⁵ cps. Nine GFP/Bt and three GFP events were hybridized with three wild accessions of B. rapa. The resultant hybrids fluoresced green and were insecticidal to neonate corn earworm larvae to the same degree as the transgenic canola parents. However, fluorescence intensities of the hemizygous F₁ hybrid lines were lower than the respective original homozygous canola parents. Each F₁ hybrid line was backcrossed by hand onto the B. rapa parent, and transgenic backcrosses were produced at rates ranging from 15% to 34%. These data suggest that GFP can be used as a tool to monitor transgene flow from crop species to wild relatives. Received: 11 September 2000 / Accepted: 1 February 2001     

Insect-resistant transgenic brinjal plants

A synthetic cry1Ab gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis (Bt) was transferred to brinjal (eggplant) by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Double-antibody sandwich ELISA analysis revealed Bt toxin protein expression in the transgenic plants. The expression resulted in a significant insecticidal activity of transgenic brinjal fruits against the larvae of fruit borer (Leucinodes orbonalis). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for insect control.     

Resistance to Tecia solanivora (Lepidoptera: Gelechiidae) in three transgenic Andean varieties of potato expressing Bacillus thuringiensis CrylAc protein

Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazúcar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 microg/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7-100% mortality, whereas the mortality levels on nontransgenic lines were 0-2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of CrylAc potatoes in the insect pest management strategies.     

农杆菌介导的苏云金杆菌抗虫基因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达

通过农杆菌介导法用含有抗潮霉素和 Ｇ Ｕ Ｓ 基因的双元载体将杀虫结晶蛋白基因ｃｒｙ Ｉ Ａ（ ｂ） 和ｃｒｙ Ｉ Ａ（ｃ） 导入到籼、粳稻幼穗愈伤组织中,然后经过在含有不同浓度潮霉素的培养基上进行数次筛选,获得一批 Ｂｔ 转基因株。经 Ｐ Ｃ Ｒ、 Ｓｏｕｔｈｅｒｎ 杂交及 Ｗｅｓｔｅｒｎ 印迹分析证实此二基因已整合进水稻中,饲虫试验结果表明,转基因株具有１００ ％ 杀虫率。     

转单、双Bt基因741杨外源基因表达和抗虫性比较

【目的】研究联合使用两种或两种以上的抗虫基因的抗虫效果, 同时鉴定并筛选出转双Bt基因741杨对鳞翅目和鞘翅目害虫有较强抗性的株系。【方法】选取转三基因（Cry3Aa+Cry1Ac+API）741杨8个株系、 转双基因（Cry1Ac+API）741杨1个株系和转单基因（Cry3Aa）741杨3个株系为试材, 从外源基因PCR检测、 毒蛋白表达和抗虫性三方面对转基因株系进行对比分析。【结果】经PCR扩增后各转基因株系出现了预期的电泳条带。ELISA蛋白检测显示转基因株系中都有与所含基因相应的Bt杀虫蛋白表达。用转基因株系新鲜叶片进行柳蓝叶甲Plagiodera versicolora和美国白蛾Hyphantria cunea室内饲虫实验表明： 转入不同抗虫基因的杨树对昆虫的抗性具有选择性, 对非靶标昆虫没有毒杀作用。转双Bt基因741杨具有双抗性, 不同转基因株系表现出高中低的抗性水平： 在对柳蓝叶甲的抗性上, 筛选出的其中5个高抗株系（pCCA1, pCCA2, pCCA5, pCCA6和pCCA9）的抗性水平明显比含Cry3Aa单Bt基因的3个高抗株系（pCC11, pCC53和pCC84）高; 在对美国白蛾的抗性上, 有7个株系（pCCA2~pCCA7和pCCA9）的抗性水平与含Cry1Ac单Bt基因株系（pB29）表现一致, 只有1个株系（pCCA1）对美国白蛾表现出了极低的抗性。【结论】多个抗虫基因在741杨上的联合使用, 不仅扩大了抗虫谱, 其中的高抗株系还具有了更高的抗虫能力, 有效地发挥了基因的叠加效应。     

转Cry1Ac活性杀虫蛋白及慈菇蛋白酶抑制剂B基因的棉花

根据植物基因的结构特征。合成了Cry1Ac活性杀虫蛋白的编码序列并与内质网定位肽编码序列组成嵌合杀虫蛋白基因Bt29K。构建了含Bt29K基因及慈菇蛋白酶抑制剂B（API-B）基因表达框的双抗虫基因植物表达载体。通过根癌土壤杆菌（Agrobacterium trmefaciens(Smith et TOwnsend)ConnLBA4404)介导转化了棉花（Gossypium hirsu-tunL.)的两个生产品种（系）。根据抗棉铃虫（Heliothis armigera)试验及农艺性状的观察调查结果。经6代筛选,获得了抗棉铃虫90.0%_99.7%且农艺性状优良的9个双价抗虫棉纯合品系。分子生物学分析结果表明,两个抗虫基因在棉花基因组中的插入拷贝数为1个或2个,活性Cry1Ac和API-B蛋白在转基因抗虫棉株系中的表达量分别约占总可溶性蛋白的0.17%和0.09%。对双抗纯合系植株及仅转Bt基因的棉花纯合系抗虫性检测结果表明前者的抗虫性明显高于后者,因此推断本研究采用的双抗虫基因表达载体构建策略是合理的。     

转Bt基因抗虫棉与常规棉中几种同工酶的比较——转基因植物安全性评价生理指标初探

经基因改造的转基因抗虫棉在我国已开始进入大规模商业化应用,对此进行安全性评价是一个十分重要的生态学问题。本文比较了抗虫棉和常规棉中过氧化物酶、多酚氧化酶、酯酶等几种重要同工酶的酶活和酶谱的差异,发现在一定生长期中,抗虫棉的可溶性过氧化物酶活性显著高于常规棉,酯酶的酶活和酶谱在两种棉花中也存在着差异。认为这些差异可能影响抗虫棉的次生代谢,进而导致一些综合的生态学效应,提出植物生理上的改变也是转基因植物安全性评价研究的重要内容。     

转Bt基因抗虫水稻的研究进展与生态安全评价

表达杀虫蛋白的转Bt基因植物正在改革着现代农业．综述了国内外转Bt基因水稻及其抗虫性的研究进展及水稻害虫对Bt水稻的抗性风险及抗性管理策略,提出了对转基因Bt水稻进行生态安全风险评价的具体内容．     

转Cry1Ac活性杀虫蛋白及慈菇蛋白酶抑制剂B基因的棉花

根据植物基因的结构特征,合成了CrylAc活性杀虫蛋白的编码序列并与内质网定位肽编码序列组成嵌合杀虫蛋白基因Bt29K.构建了含Bt29K基因及慈菇蛋白酶抑制剂B(API-B)基因表达框的双抗虫基因植物表达载体.通过根癌土壤杆菌(Agrobacteriumtumefaciens(Smith et Townsend)Conn LBA4404)介导转化了棉花(Gossypium hirsu-tun L.)的两个生产品种(系).根据抗棉铃虫(Heliothis armigera)试验及农艺性状的观察调查结果,经6代筛选,获得了抗棉铃虫90.0%～99.7%且农艺性状优良的9个双价抗虫棉纯合品系.分子生物学分析结果表明,两个抗虫基因在棉花基因组中的插入拷贝数为1个或2个.活性Cry1Ac和API-B蛋白在转基因抗虫棉株系中的表达量分别约占总可溶性蛋白的0.17%和0.09%.对双抗纯合系植株及仅转Bt基因的棉花纯合系抗虫性检测结果表明前者的抗虫性明显高于后者,因此推断本研究采用的双抗虫基因表达载体构建策略是合理的.     

Field performance of transgenic elite commercial hybrid rice expressing bacillus thuringiensis delta-endotoxin

Here we describe development of transgenic elite rice lines expressing a Bt fusion gene derived from cryIA(b) and cryIA(c) under the control of rice actinI promoter. The lines used in the study were indica CMS restorer line of Minghui 63 and its derived hybrid rice Shanyou 63. The level of Bt fusion protein CryIA(b)/CryIA(c) detected in Minghui 63 (T51-1) plants was 20 ng/mg soluble protein. The Bt Shanyou 63 was field-tested in natural and repeated heavy manual infestation of two lepidopteran insects, leaffolder and yellow stem borer. The transgenic hybrid plants showed high protection against both insect pests without reduced yield.     

影响苏云金芽孢杆菌基因在转基因植物中表达的因素

苏云金芽孢杆菌（Bacillus thuringiensis,Bt）杀虫晶体蛋白基因是植物抗虫基因工程中应用最广泛的基因资源。影响Bt基因在转基因植物中表达的因素繁多,阐明这些因素的效应对于获得Bt基因在受体植物中的稳定高效表达具有重要意义。现对Bt基因表达的主要影响因子,如Bt基因表达单元、植物发育、外部环境条件、受体植物遗传背景、整合位点及Bt基因沉默现象等进行了综述。     

Overexpression of a novel Cry1Ie gene confers resistance to Cry1Ac-resistant cotton bollworm in transgenic lines of maize

Although transgenic crops expressing either Cry1Ab or Cry1Ac, both derived from Bacillus thuringiensis (Bt), have been used commercially, the evolution of insects resistance to these CRY proteins has become a challenge. Thus, it has been proposed that co-expression of two Bt proteins with different modes of action may delay the development of resistance to Bt. However, few Bt proteins have been identified as having different modes of action from those of Cry1Ab or Cry1Ac. In this study, transgenic lines of maize over-expressing either Cry1Ie or Cry1Ac gene have been developed. Several independent transgenic lines with one copy of the foreign gene were identified by Southern blot analysis. Bioassays in the laboratory showed that the transgenic plants over-expressing Cry1Ie were highly toxic against the wild-type cotton bollworm (Heliothis armigera), producing mortality levels of 50 % after 6 days of exposure. However, the mortality caused by these plants was lower than that caused by the Cry1Ac transgenic plants (80 %) and MON810 plants expressing Cry1Ab (100 %), which both exhibited low toxicity toward the Cry1Ac-resistant cotton bollworm. In contrast, three transgenic maize lines expressing Cry1Ie induced higher mortality against this pest and were also highly toxic to the Asian corn borer (Ostrinia furnacalis) in the field. These results indicate that the Cry1Ie protein has a different mode of action than the Cry1Ab and Cry1Ac proteins. Therefore, the use of transgenic plants expressing Cry1Ie might delay the development of Bt-resistant insects in the field.     

转cry1Ab基因抗虫水稻的田间试验

采用农杆菌介导的遗传转化技术, 将Bt基因cry1Ab导入三系恢复系明恢63获得了一批抗虫性及农艺性状较好的转基因纯合株系。研究中, 进一步检测了这些纯合株系的拷贝数、Bt蛋白含量、抗虫性及主要农艺性状。结果显示: 5个转基因纯合株系均为单拷贝; 纯合株系T_1Ab-10及其杂种的Bt蛋白含量最高; 人工接虫和自然感虫试验中, T_1Ab-10对二化螟、三化螟和稻纵卷叶螟均表现高度抗性; 喷药试验中, 阳性植株与阴性植株、对照植株的主要农艺性状均没有显著差异。这说明转cry1Ab基因纯合株系T_1Ab-10具商品化潜力, 可作基因聚合材料。     

A QTL that enhances and broadens Bt insect resistance in soybean

Effective strategies are needed to manage insect resistance to Bacillus thuringiensis (Bt) proteins expressed in transgenic crops. To evaluate a multiple resistance gene pyramiding strategy, eight soybean (Glycine max) lines possessing factorial combinations of two quantitative trait loci (QTLs) from plant introduction (PI) 229358 and a synthetic Bt cry1Ac gene were developed using marker-assisted selection with simple sequence repeat markers. Field studies were conducted in 2000 and 2001 to evaluate resistance to corn earworm (Helicoverpa zea) and soybean looper (Pseudoplusia includens), and detached leaf bioassays were used to test antibiosis resistance to Bt-resistant and Bt-susceptible strains of tobacco budworm (TBW; Heliothis virescens). Based on defoliation in the field and larval weight gain on detached leaves, lines carrying a combination of cry1Ac and the PI 229358 allele at a QTL on linkage group M were significantly more resistant to the lepidopteran pests, including the Bt-resistant TBW strain, than were the other lines. This is the first report of a complementary additive effect between a Bt transgene and a plant insect resistance QTL with an uncharacterized mode of action that was introgressed using marker-assisted selection.     

Pinellia ternata agglutinin expression in chloroplasts confers broad spectrum resistance against aphid, whitefly, Lepidopteran insects, bacterial and viral pathogens

Broad spectrum protection against different insects and pathogens requires multigene engineering. However, such broad spectrum protection against biotic stress is provided by a single protein in some medicinal plants. Therefore, tobacco chloroplasts were transformed with the agglutinin gene from Pinellia ternata (pta), a widely cultivated Chinese medicinal herb. Pinellia ternata agglutinin (PTA) was expressed up to 9.2% of total soluble protein in mature leaves. Purified PTA showed similar hemagglutination activity as snowdrop lectin. Artificial diet with purified PTA from transplastomic plants showed marked and broad insecticidal activity. In planta bioassays conducted with T0 or T1 generation PTA lines showed that the growth of aphid Myzus persicae (Sulzer) was reduced by 89%-92% when compared with untransformed (UT) plants. Similarly, the larval survival and total population of whitefly (Bemisia tabaci) on transplastomic lines were reduced by 91%-93% when compared with UT plants. This is indeed the first report of lectin controlling whitefly infestation. When transplastomic PTA leaves were fed to corn earworm (Helicoverpa zea), tobacco budworm (Heliothis virescens) or the beet armyworm (spodoptera exigua), 100% mortality was observed against all these three insects. In planta bioassays revealed Erwinia population to be 10,000-fold higher in control than in PTA lines. Similar results were observed with tobacco mosaic virus (TMV) challenge. Therefore, broad spectrum resistance to homopteran (sap-sucking), Lepidopteran insects as well as anti-bacterial or anti-viral activity observed in PTA lines provides a new option to engineer protection against biotic stress by hyper-expression of an unique protein that is naturally present in a medicinal plant.     

A transgenic immunoglobulin mu gene prevents rearrangement of endogenous genes

Transgenic mice containing a microinjected rearranged immunoglobulin (Ig) mu heavy chain gene were examined for the effects on DNA rearrangement of the endogenous Ig genes. Abelson murine leukemia virus (A-MuLV) cell lines were isolated from pre-B cells of transgenic mice and of normal littermates. Microinjected mu gene RNA and a mu heavy chain protein were synthesized in every transgenic A-MuLV cell line. Only 10% of normal mouse A-MuLV transformants synthesized mu protein. A germ-line JH allele was observed in 40% of the transgenic lines, demonstrating that the block to endogenous Ig DNA rearrangement occurred at the first step of heavy chain DNA joining. All alleles were rearranged in normal mouse A-MuLV lines. Germline JH alleles were also detected in 10% of the transgenic hybridomas derived from proliferating B cells. Our results support a model of active prevention of rearrangement by the product of successfully rearranged mu genes.