Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest,Chilo suppressalis (Lepidoptera: Crambidae)

Two transgenic rice lines (T2A‐1 and T1C‐19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A‐1 > Huahui 1 > T1C‐19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C‐19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C‐19b providing an excellent performance at the maturing stage. By comparison, T1C‐19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A‐1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C‐19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.     

转Cry1Ab和Cry1Ac融合基因型抗虫水稻对田间二化螟和大螟种群发生动态的影响

以Bt水稻华恢1号(Cry1Ac和CryAb融合基因;简称HH1)及其对照亲本明恢63(简称MH63)稻田靶标害虫二化螟Chilosuppressalis和次靶标害虫大螟Sesamia inferens为研究对象,研究了转基因抗虫水稻大田螟虫发生规律及其靶标和次靶标害虫致害力差异。结果表明,Bt水稻及其对照亲本上二化螟或大螟的卵块数量差异不显著,同时,对照亲本上二化螟与大螟的落卵量差异不显著,但Bt水稻上二化螟的落卵量显著大于大螟。与对照亲本相比,Bt水稻上二化螟幼虫发生量显著降低,降幅高达84.9%—100%,但大螟发生量差异不显著;此外,对照亲本上二化螟幼虫发生量显著高于大螟,但Bt水稻上两者差异不显著。同时,Bt水稻上二化螟导致的枯心/白穗率和受害丛率都显著低于其在对照亲本上的致害程度,降幅分别为30.8%—98.3%和11.4%—96.6%,而大螟差异不显著。可见,Bt水稻对靶标害虫二化螟具有较高抗性,而对次靶标害虫大螟的抗性不明显。另一方面,Bt水稻和对照亲本上二化螟导致的枯心/白穗率和受害从率都显著高于大螟。可见,二化螟仍是当前非转基因水稻上的主要害虫,而Bt水稻对二化螟幼虫发生的显著抑制作用以及对大螟幼虫发生的不显著影响,使得其大面积商业化种植下靶标害虫二化螟和次靶标害虫大螟间的竞争替代成为可能。     

Transgenic Rice Plants Expressing a Modified <Emphasis Type="Italic">cry1Ca1</Emphasis> Gene are Resistant to <Emphasis Type="Italic">Spodoptera litura</Emphasis> and <Emphasis Type="Italic">Chilo</Emphasis><Emphasis Type="Italic">suppressalis</Emphasis>

Nucleotide sequence encoding the truncated insecticidal Cry1Ca1 protein from Bacillus thuringiensis was extensively modified based on the codon usage of rice genes. The overall G + C contents of the synthetic cry1Ca1 coding sequence were raised to 65% with an additional bias of enriching for G and C ending codons as preferred by monocots. The synthetic gene was introduced into the Chinese japonica variety, Xiushui 11, by Agrobacterium-mediated transformation. Transgenic rice plants harboring this gene were highly resistant to Chilo suppressalis and Spodoptera litura larvae as revealed by insect bioassays. High levels of Cry1Ca1 protein were obtained in the leaves of transgenic rice, which were effective in achieving 100% mortality of S. litura and C. suppressalis larvae. The levels of Cry1Ca1 expression in the leaves of these transgenic plants were up to 0.34% of the total soluble proteins. The larvae of C. suppressalis and S. litura could consume a maximum of 1.89  and 4.89 mm² of transgenic leaf area whereas the consumption of non-transgenic leaves by these larvae was significantly higher; 58.33 and 61.22 mm², respectively. Analysis of R₁ transgenic plants indicated that the cry1Ca1 was inherited by the progeny plants and provided complete protection against C. suppressalis and S. litura larvae.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Binding Site Concentration Explains the Differential Susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-Producing Rice

Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers.     

Effectiveness in the field of Bt rice lines against target pests under various cultural regimes

China has a long history of rice cultivation, incorporating several cultural practices known to influence damage by insect pests. Transgenic Bt rice expresses lepidopteran‐specific insecticidal proteins that primarily target lepidopteran insect pests. However, the effectiveness of Bt rice against target insect pests under different cultural regimes has not been evaluated. In this study, the effectiveness of Bt rice lines against rice leaffolder, Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), and striped stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae), was evaluated under various transplanting densities, crop establishment methods, and planting times. The results showed that Bt rice lines (T2A‐1 and T1C‐19, containing Cry2A and Cry1C, respectively) could prevent damage by these target pests under a range of cultural practices. Injury by C. medinalis or C. suppressalis on rice did not differ with the rice lines under various transplanting densities. Direct‐seeded non‐Bt rice MH63 suffered heavier injury by C. medinalis and C. suppressalis than it did with transplanting, whereas injury to the two Bt rice lines did not differ with planting methods. Planting time significantly affected injury by C. medinalis or C. suppressalis on non‐Bt rice, whereas injury to Bt rice lines did not differ with planting time. These results suggest that transplanting density, planting method, and planting time did not significantly affect the resistance of two Bt rice lines, due to their high insecticidal activity against target insects.     

Effects of Bacillus thuringiensis δ-endotoxins Cry1Ab and Cry1Ac on the coccinellid beetle,Cheilomenes sexmaculatus (Coleoptera,Coccinellidae) under direct and indirect exposure conditions

Genes encoding cry1Ab and cry1Ac δ-endotoxins from the bacterium, Bacillus thuringiensis (Bt) that have been incorporated in several crops to enhance their resistance to insect pests may possibly influence the activity and abundance of natural enemies of insect pests. The ladybird beetle, Cheilomenes sexmaculatus (L.) might ingest Bt toxins expressed by genetically modified plants by feeding on aphids, early instar larvae of lepidopterans, and other soft bodied insects feeding on transgenic plants. Therefore, we studied the effects of Cry1Ab and Cry1Ac Bt toxins on C. sexmaculatus under direct and indirect exposure conditions. For direct exposure, the neonate C. sexmaculatus larvae were fed either pure 2M sucrose (control) or sucrose solution containing Cry1Ab or Cry1Ac (0.1%), and on alternate days with aphids till pupation. Direct exposure of C. sexmaculatus larvae to Bt toxins resulted in reduced larval survival and adult emergence as compared to the controls, which might be due to long-term direct exposure. However, there were no adverse effects of the Bt toxins on C. sexmaculatus when the larvae were reared on Aphis craccivora Koch fed on different concentrations of Cry1Ab or Cry1Ac in the artificial diet. A significant and positive correlation was observed between the presence of Bt toxins in aphids, and coccinellid larvae and adults (r=0.53** to 0.86**). The results suggested that a direct exposure to Bt toxins expressed in transgenic plants or predation on H. armigera on Bt-transgenic plants will have little effect on the activity and abundance of the ladybird, C. sexmaculatus.     

Production of marker-free transgenic rice expressing tissue-specific Bt gene

The hybrid Bacillus thuringiensis (Bt) δ-endotoxin gene Cry1Ab/Ac was used to develop a transgenic Bt rice (Oryza sativa L.) targeting lepidopteran insects of rice. Here, we show the production of a marker-free and tissue-specific expressing transgenic Bt rice line L24 using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L24 carries a single copy of marker-free T-DNA that contains the Cry1Ab/Ac gene driven by a maize phosphoenolpyruvate carboxylase (PEPC) gene promoter. The marker-free T-DNA was integrated into the 3′ untranslated region of rice gene Os01g0154500 on the short arm of chromosome 1. Compared to the constitutive and non-specific expression of the P _Actin1 :Cry1Ab/Ac:T _Nos gene in the control Bt rice line T51-1, the P _Pepc :Cry1Ab/Ac:T _Nos gene was detected only in the leaf and stem tissues of L24. More importantly, compared to high levels of CRY1Ab/Ac proteins accumulated in T51-1 seeds, the CRY1Ab/Ac proteins were not detectable in L24 seeds by Western blot analysis. As demonstrated by insect bioassay, L24 provided similar level of resistance to rice leaffolder (Cnaphalocrocis medinalis) as T51-1. The marker-free transgenic line L24 can be used directly in rice breeding for insect resistance to lepidopteran insects where absence of Bt toxin protein in the seed is highly desirable.     

Development of insect-resistant transgenic indica rice with a synthetic cry1C* gene

Stemborers and leaffolders are two groups of lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, a cry1C* gene encoding Bacillus thuringiensis (Bt) δ-endotoxin was synthesized by codon optimization as the first step towards gene stacking in our resistance management strategy of transgenic rice. Agrobacterium-mediated transformation of this gene into Minghui 63 (Oryza sativa L.), an elite indica CMS restorer line, produced 120 independently transformed plants, 19 of which had a single-copy transgene. Preliminary screening of T₁ families of these 19 transformants in the field identified five lines showing a high level of resistance to leaffolders (Cnaphalocrocis medinalis) and stemborers. Hybrids were produced by crossing these five lines with Zhenshan 97A, the male-sterile line for Shanyou 63, the most widely cultivated hybrid in China. These five lines and their hybrids were highly resistant to yellow stemborer (Tryporyza incertulas) as revealed by an insect bioassay. The content of Cry1C* protein varied considerably among the five lines as well as among the corresponding hybrids. T1c-19, a line showing the highest content of Cry1C* protein, and its hybrid were tested in the field for insect resistance and agronomic performance and found to be highly resistant to stemborers and leaffolders throughout the growth period, resulting in a significantly increased grain yield compared with the respective controls. These results indicate that T1c-19 can be used for production of insect-resistant hybrid rice and as a germplasm for gene stacking to produce rice plants with two toxins.     

Effects of seed mixture sowing with transgenic Bt rice and its parental line on the population dynamics of target stemborers and leafrollers,and non‐target planthoppers

The widespread planting of insect‐resistant crops has caused a dramatic shift in agricultural landscapes, thus raising concerns about the potential impacts on both target and non‐target pests. In this study, we examined the potential effects of intra‐specific seed mixture sowing with transgenic Bt rice (Bt) and its parental non‐transgenic line (Nt) (100% Bt rice [Bt₁₀₀], 5% Nt+95% Bt [Nt₀₅Bt₉₅], 10% Nt+90% Bt [Nt₁₀Bt₉₀], 20% Nt+80% Bt [Nt₂₀Bt₈₀], 40% Nt+60% Bt [Nt₄₀Bt₆₀] and 100% Nt rice [Nt₁₀₀]) on target and non‐target pests in a 2‐year field trial in southern China. The occurrence of target pests, Sesamia inferens, Chilo suppressalis and Cnaphalocrocis medinalis, decreased with the increased ratio of Bt rice, and the mixture ratios with more than 90% Bt rice (Bt₁₀₀ and Nt₀₅Bt₉₅) significantly increased the pest suppression efficiency, with the lowest occurrences of non‐target planthoppers, Nilaparvata lugens and Sogatella furcifera in Nt₁₀₀ and Nt₀₅Bt₉₅. Furthermore, there were no significant differences in 1000‐grain dry weight and grain dry weight per 100 plants between Bt₁₀₀ and Nt₀₅Bt₉₅. Seed mixture sowing of Bt rice with ≤10% (especially 5%) of its parent line was sufficient to overcome potential compliance issues that exist with the use of block or structured refuge to provide most effective control of both target and non‐target pests without compromising the grain yield. It is also expected that the strategy of seed mixture sowing with transgenic Bt rice and the non‐transgenic parental line would provide rice yield stability while decreasing the insecticide use frequency in rice production.     

Molecular characterization of lepidopteran pest-resistant transgenic rice events expressing synthetic Cry1Ac

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is one of the most commonly used in the development of genetically modified (GM) crops. In this research, we analyzed Bt rice showing lepidopteran pest-resistance. The Bt gene is a synthetic Cry1Ac composed of optimal codons for plants, and the Bt protein is targeted to the chloroplast by a transit peptide. Three Cry1Ac rice events (C103-3, C127-1, and C7-1) were analyzed for molecular characterization. C103-3 contains two copies of T-DNA where the left border (LB) region is truncated. Both C7-1 and C127-1 have a single copy of T-DNA, but a part of the vector backbone DNA is inserted into the genome of C127-1; thus, only C7-1 had intact T-DNA. Progenies of C7-1 crossed with the original cultivar, Nakdong, and double-haploid lines from anther culture of lines crossed with the elite cultivar, Dongjin, were analyzed for T-DNA flanking genomic DNA and genotyping. Results showed that an intact T-DNA region without the vector backbone was inserted into the genome and was stably inherited through generations. The C7-1 homozygous event could be used as breeding material to develop GM rice with pest resistance.     

A UV Tolerant Mutant of Bacillus thuringiensis subsp. kurstaki Producing Melanin

A UV-resistant mutant (Bt-m) of Bacillus thuringiensis subsp. kurstaki, producing a dark brown pigment, identified as melanin, was studied. Bt-m had higher larvicidity against Heliothis armigera than its parent. Survival of Bt-m spores and their insecticidal activity to irradiation at 254 nm and 366 nm were higher than those of the parent. The only toxic polypeptide produced by Bt-m was Cry1Ac (130 kDa); it lost cry1Aa, cry2Aa, and cry2Ab. Received: 2 April 2001 / Accepted: 14 May 2001     

The effect of Bt crops on soil invertebrates: a systematic review and quantitative meta-analysis

The ongoing debate about the ecological effects of Bt-crops calls for thorough reviews about the impact on soil biodiversity and their ecosystem services. Transgenic Bt-crops have been genetically modified by inserting a Bacillus thuriengensis gene so the plant expresses a Cry toxin aimed for insect crop pests. Non-target soil invertebrates are particularly recognized for their contribution to plant nutrient availability and turnover of organic matter and it is therefore relevant to protect these invertebrate taxa. A number of studies have compared the population abundance and biomass of soil invertebrates in agricultural fields planted with genetically modified Bt crops and their conventional counterparts. Here, were review and analyze a selection of studies on Protista, nematodes, Collembola, mites, enchytraeids, and earthworms systematically to empower the evidence for asking the question whether population abundances and biomasses of soil invertebrates are changed by Bt crops compared to conventional crops. 6110 titles were captured, of which 38 studies passed our inclusion criteria, and a final number of 22 publications were subject to data extraction. A database with 2046 records was compiled covering 36 locations and the Bt types Cry1Ab, Cry1Ac, Cry3Bb1 and Cry3Aa. Comparative effect sizes in terms of Hedges’ g were calculated irrespectively of statistical significance of effects of the source studies. Cry effects on populations were compared across the studies in a meta-analysis employing a hierarchical Bayesian approach of weighted data according to the level of replication. The temporal development of effect sizes was modelled, thereby taking into account the variable duration of the field experiments. There was considerable variation among soil invertebrate orders, but the sample size was insufficient and the sample heterogeneity too large to draw any credible conclusions on the effect of Cry at the order level. However, across orders there was no significant effect of Cry on soil invertebrates.

     

Bt rice expressing Cry1Ab does not stimulate an outbreak of its non-target herbivore, Nilaparvata lugens

In this study, the non-target effects of Bt rice “KMD2” expressing a Cry1Ab protein on the performance of the brown planthopper (BPH), Nilaparvata lugens, over multiple generations were evaluated under laboratory and field conditions. In the laboratory, BPH was reared to observe the impact of the Bt rice as compared to its parental non-Bt cultivar Xiushui 11, while the population dynamics and oviposition performance of BPH were investigated in the field. The survival of BPH nymphs fed Bt and non-Bt rice did not differ significantly. The nymph developmental duration of BPH was significantly delayed by the Bt rice by comparison with the non-Bt rice for the 1st and 2nd but not the 4th generation. Most importantly, the fecundity of BPH on the Bt rice was significantly decreased in every generation when compared with the non-Bt rice. In the field investigations, the population density of BPH nymphs was significantly lower in the Bt rice field. However, the temporal pattern of population dynamics of BPH adults was similar between the Bt and non-Bt rice, presumably due to migratory interference of the adults. In the Bt rice field, the percentage of tillers with eggs and the number of eggs per tiller were also significantly lower from tillering to mature stage. Additionally, Cry1Ab protein could not be detected in guts from single BPH adults. In general, our results suggest that the Bt rice “KMD2” could not stimulate an outbreak of BPH.     

Effects of Transgenic cry1Ca Rice on the Development of Xenopus laevis

In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 μg L^–1 or 10 μg L^–1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development.     

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking Cry1Ab expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the cry1Ab gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of cry1Ab gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%–30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low concentration of 5-azacytidine (45 mg/L for 1 d and 2 d) could lead to the highest reactivation ratio and the highest expression level of the cry1Ab gene.     

Normal expression of insect-resistant transgene in progeny of common wild rice crossed with genetically modified rice: its implication in ecological biosafety assessment

Transgene outflow from genetically modified (GM) rice to its wild relatives may cause undesirable ecological consequences. Understanding the level of transgene expression in wild rice following gene flow is important for assessing such consequences, providing that transgene escape from GM rice cannot be prevented. To determine the expression of a transgene in common wild rice (Oryza rufipogon), we analyzed the content of Cry1Ac protein in three GM rice lines containing a Bt transgene, their F₁ hybrids with common wild rice and F₂ progeny at different growth stages, using the sandwich enzyme-linked immunosorbent assay. The average content of Cry1Ac protein in leaf samples of the wild rice lines ranged between 0.016 and 0.069% during the entire growth period, whereas that in stems varied between 0.12 and 0.39%. A great variation in Cry1Ac protein content was detected among individuals of F₁ hybrids and F₂ progeny, with some wild individuals showing higher level of Bt toxin than the cultivated GM rice. The results suggest that the Bt transgene can express normally in the interspecific hybrids between insect-resistant GM rice and common wild rice, and may have similar effects on the target insects as in GM rice.     

Effect of plant age, larval age, and fertilizer treatment on resistance of a cry1Ab-transformed aromatic rice to lepidopterous stem borers and foliage feeders

The resistance of vegetative, booting, and flowering stage plants of a variety of an aromatic rice, Oryza sativa L., transformed with a Bacillus thuringiensis Berliner cry1Ab gene under control of the maize phosphoenolpyruvate carboxylase (PEPC) promoter was evaluated against four lepidopterous rice pests--the stem borers Chilo suppressalis (Walker) (Lepidoptera: Crambidae) and Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae), and the foliage feeders Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae) and Naranga aenescens Moore (Lepidoptera: Noctuidae). Plants of the cry1Ab-transformed line (no. 827) were more resistant to young larvae of S. incertulas, C. suppressalis, and C. medinalis than control plants at the vegetative stage but not at the flowering stage. Survival of 10-d-old stem borer larvae did not differ on cry1Ab plants and control plants at either the vegetative or flowering stage, but the development of 10-d-old C. suppressalis larvae was retarded on the vegetative stage cry1Ab plants. Immunological analysis also showed an apparent decline in Cry1Ab titer in leaf blades and leaf sheaths at the reproductive stage. In experiments comparing three fertilizer treatments (NPK, PK, and none), there was a significant interaction between fertilizer treatment and variety on larval survival only in whole-plant assays at booting stage with C. suppressalis. On cry1Ab plants, larval survival did not differ significantly among the three fertilizer levels, whereas on control plants survival was highest with the NPK treatment. cry1Ab plants tested at the sixth and seventh generations after transformation were more resistant than control plants to N. aenescens and C. suppressalis, respectively, suggesting that gene silencing will not occur in line 827. The results of the experiments are discussed in terms of resistance management for B. thuringiensis toxins in rice.     

二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合

【目的】二化螟是水稻的重要害虫之一,钙黏蛋白(cadherin,CAD)是一类重要的Bt杀虫蛋白受体,在获得二化螟钙黏蛋白基因(Cs CAD1)的基础上,明确Cs CAD1蛋白与Cry1Ac和Cry2Aa蛋白的结合能力。【方法】利用PCR技术克隆Cs CAD1基因片段,将构建的p ET-28a-(+)-Cs CAD1重组质粒转入原核表达菌株BL21(DE3)中,IPTG诱导表达。目的蛋白经Ni柱亲和纯化后SDS-PAGE电泳检测,利用western blot和ligand blot技术分析其与Cry1Ac和Cry2Aa蛋白的结合能力。【结果】重组载体可在表达菌株BL21中表达一个约44 ku的蛋白,原核表达载体构建成功。SDS-PAGE显示该蛋白条带单一,且纯度较好。Ni柱亲和层析纯化该目的蛋白后进行Ligand blot分析,结果显示Cs CAD1重组蛋白可以与Cry1Ac和Cry2Aa蛋白结合。【结论】Cs CAD1蛋白可以与Cry1Ac和Cry2Aa蛋白结合,是潜在的Cry蛋白受体,所得结果有助于阐明Cry1Ac和Cry2Aa蛋白对二化螟的作用机制。