转基因抗虫作物鉴定质粒标准分子的构建与应用

苏云金芽胞杆菌基因是转基因抗虫作物中通用的外源功能基因,在绝大多数抗虫转基因作物中均有存在,然而Bt基因检测标准样品的缺乏却限制了我国转Bt基因抗虫作物检测工作的发展。为了弥补传统基体标准样品的缺失,首先将Cry1Ab、Cry1Ac、Cry3A 3种常用Bt外源基因克隆到pUC57质粒上,通过测序、酶切和qPCR等技术对质粒的序列和扩增功能进行了验证,然后对扩增效率和实际应用情况加以测试,评价其转基因检测的适用性,构建了质粒标准分子。结果显示,制备的质粒标准分子测序结果与靶标序列完全符合,酶切结果、qPCR扩增结果和扩增效率等均符合预期,在Cry1Ab、Cry1Ac、Cry3A基因特异性检测中的应用符合阳性对照要求,表明制备的阳性质粒标准分子能够作为转Cry1Ab、Cry1Ac、Cry3A基因qPCR基因特异性检测的阳性标准样品。     

家蚕生物钟基因Bmcryl与Bmcry2的克隆及生物信息学分析

隐花色素基因(cryptochrome gene,Cry)是已确认的主要生物钟基因之一,它广泛分布于细菌和真核生物中.昆虫Cry基因分为Cry1,和Cry2两类,果蝇只有Cry1,蜜蜂等膜翅目昆虫只有Cry2.为了研究鳞翅目模式昆虫家蚕Bombyx mori的昼夜生物钟分子调控机制和昆虫CRY蛋白的进化,本研究克隆了家...     

转Cry1Ac+Cry2Ab基因棉花主要生化物质含量变化及其对棉田昆虫的影响

【目的】新型转基因棉花在进入大规模商业化应用前,需对其生态环境安全性进行评价;同时,经基因改造的新型转基因抗虫棉花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,致使棉花生理上发生改变,这也是转基因植物安全性评价研究的重要内容。【方法】比较了不同关键时期新型转Cry1Ac+Cry2Ab基因棉花与转Cry1Ac基因棉花和非转基因棉花叶片的鲜重、干重和干鲜比、主要酶[超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)]活性、营养物质(蛋白质、氨态氮、脯氨酸和可溶性糖)和次生代谢产物(棉酚和单宁)含量的差异及其对棉田不同昆虫营养层昆虫个体总数和物种数的影响。【结果】棉花生长的蕾期、花期和花铃期,转Cry1Ac+Cry2Ab基因棉花、转Cry1Ac基因棉花和非转基因棉花叶片的鲜重、干重和干鲜比呈先升高后降低的趋势;SOD和POD活性在花铃期明显升高,CAT、APX和GR活性无显著变化;蛋白质、氨态氮含量无明显变化,脯氨酸和可溶性糖含量均表现为先升高后下降的趋势;棉酚含量在3个时期无显著变化,而单宁含量呈逐渐升高的趋势。3种棉花叶片中干物质积累、主要酶活性、营养物质和次生代谢产物含量均无显著差异;单株大铃数表现为转Cry1Ac+Cry2Ab基因棉花转Cry1Ac基因棉花非转基因棉花,小铃数则表现为转Cry1Ac基因棉花Cry1Ac+Cry2Ab基因棉花非转基因棉花;昆虫群落和害虫亚群落的昆虫个体总数均表现为转Cry1Ac+Cry2Ab基因棉田转Cry1Ac基因棉田非转基因棉田,天敌亚群落昆虫个体总数无显著变化;3种棉田中昆虫群落、害虫亚群落和天敌亚群落的物种数均未发生显著变化。【结论】转Cry1Ac+Cry2Ab基因棉花叶片干物质积累、产量性状、生化物质含量、酶活性在不同生长期表现不同,但上述参数在3种棉花之间无显著差异;且转Cry1Ac+Cry2Ab基因棉花具有较好的抗虫性,能有效降低棉田害虫数量。     

加强对基因专利的研究

基因专利是生物技术产业知识产权保护的最主要形式,被认为是生物技术产业发展的金钥匙。谁握有基因专利,谁就将占有生物技术产业发展的空间、占有未来的财富。近几年,随着人类基因组、基因功能研究的迅速进展,极大地激发了投资者的想象空间和对获取基因专利的高涨热情。前几年曾出现了以跑马圈地的方式申报基因专利的高潮,我国也曾有过一次申请上百条基因专利的例子。目前,在美国已授权了2万余个基因或基因相关分子的专利.     

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。     

转Cry1Ac+Cry2Ab基因棉对棉蚜生命表参数及种群动态的影响

为研究新型转Cry1Ac+Cry2Ab基因棉对棉蚜Aphis gossypii Glover生命表参数及种群动态的影响。2010—2011年以常规棉中棉所49为对照,对新型转Cry1Ac+Cry2Ab基因棉在室内进行了生物测定和田间进行了系统的调查。结果表明,和常规棉相比,转Cry1Ac+Cry2Ab基因棉花上棉蚜的净增值率降低81.69%,差异达显著水平;内禀增长率和周限增长率分别降低65.00%和13.01%,但差异不显著;平均世代周期和种群加倍时间分别增加5.54%和154.19%,后者差异达显著水平。和常规棉相比,2010年转Cry1Ac+Cry2Ab基因棉花百株苗蚜、伏蚜和秋蚜的数量分别降低10.79%、37.18%和17.49%,差异均未达显著水平;2011年转Cry1Ac+Cry2Ab基因棉花百株苗蚜的数量增加2.03%,伏蚜和秋蚜的数量分别降低37.41%和64.03%,差异均未达显著水平。     

棉铃虫ABC转运蛋白ABCG1的基因克隆、亚细胞定位及其与Cry1Ac毒力的关系

【目的】长期种植转Bacillus thuringiensis(Bt)基因作物使一些靶标害虫产生了Bt抗性,有研究表明小菜蛾Plutella xylostella的white基因表达下调使其产生了Cry1Ac抗性,由于Pxwhite蛋白与ABC转运蛋白ABCG1同属于ABCG亚家族,我们推测棉铃虫Helicoverpa armigera ABCG1基因(HaABCG1)的表达下调可能与棉铃虫对Cry1Ac毒素的抗性有关。【方法】克隆并分析HaABCG1的开放阅读框(open reading frame,ORF),通过构建HaABCG1基因的表达载体检测HaABCG1蛋白在TnH_5细胞中的亚细胞定位;通过细胞毒力实验验证HaABCG1蛋白与Cry1Ac毒素的关系;利用RNAi技术验证HaABCG1表达下调是否会降低棉铃虫对Cry1Ac毒素的敏感性。【结果】棉铃虫ABCG1基因的开放阅读框长1 896 bp,编码蛋白含631个氨基酸残基,分子质量估计为69.63 k D。离体昆虫细胞表达的HaABCG1蛋白主要定位在细胞的核膜和内质网。通过RNA干扰和生物测定实验发现HaABCG1下调表达不能使棉铃虫在Cry1Ac毒素浓度为0.05μg/m L的人工饲料上正常生长,3 d后处理组和对照组棉铃虫幼虫的体重变化无显著差异。经过细胞毒力实验证明HaABCG1蛋白不介导Cry1Ac毒素对TnH_5细胞的毒力,它既不是Cry1Ac毒素的受体,也不是其他3种Bt毒素Cry1Ca,Cry2Aa和Cry1Fa的受体。【结论】HaABCG1基因表达下调与棉铃虫对Cry1Ac的抗性不相关,HaABCG1不是Cry1Ac毒素的受体。这是首次报道ABCG1基因不参与棉铃虫的Cry1Ac抗性。     

Cry1Ie毒素胁迫下亚洲玉米螟的抗性发展及汰选种群对其他Bt毒素的交互抗性

亚洲玉米螟Ostrinia furnacalis (Guenée) 是危害玉米的重要害虫之一, 转Bt基因抗虫玉米为其防治提供了新的途径。然而, 靶标害虫产生抗性将严重阻碍Bt制剂及转Bt基因抗虫玉米的持续应用。明确害虫对转Bt基因玉米表达的毒素蛋白的抗性演化, 对于制定科学有效的抗性治理策略具有重要的理论和实际意义。本实验通过人工饲料汰选法研究了Bt Cry1Ie毒素胁迫下亚洲玉米螟的抗性发展及汰选14代的种群对其他Bt毒素（Cry1Ab, Cry1Ac和Cry1Fa）的交互抗性, 并观察了Cry1Ie蛋白胁迫对亚洲玉米螟生物学的影响。结果表明： 随着汰选压不断提高, 亚洲玉米螟种群对Cry1Ie毒素的敏感性逐渐下降。汰选14代后, 种群对Cry1Ie毒素的抗性水平提高了23倍。然而, Cry1Ab, Cry1Ac和Cry1Fa对所获Cry1Ie汰选种群的毒力与对敏感种群的毒力相比没有显著差异, 说明Cry1Ie汰选没有引起亚洲玉米螟对Cry1Ab, Cry1Ac和Cry1Fa毒素产生交互抗性。同时, 与敏感种群相比, Cry1Ie汰选14代的种群幼虫平均发育历期延长5.7 d, 蛹重减轻13.7%, 单雌产卵量下降40.0%。本研究结果说明, 大面积单一种植转cry1Ie基因抗虫玉米, 可能引起亚洲玉米螟产生抗性; 亚洲玉米螟Cry1Ie抗性种群对Cry1Ab, Cry1Ac和Cry1Fa没有交互抗性, 含有cry1Ie和cry1Ab, cry1Ac或cry1F双/多基因抗虫玉米, 可作为靶标害虫抗性治理的重要策略。     

Effects of transgenic Cry1Ac and Cry2Ab cotton on life table parameters and population dynamics of the cotton aphid

为研究新型转Cry1Ac+Cry2Ab基因棉对棉蚜Aphis gossypii Glover生命表参数及种群动态的影响.2010-2011年以常规棉中棉所49为对照,对新型转Cry1Ac+Cry2Ab基因棉在室内进行了生物测定和田间进行了系统的调查.结果表明,和常规棉相比,转Cry1Ac+Cry2Ab基因棉花上棉蚜的净增值率降低81.69％,差异达显著水平;内禀增长率和周限增长率分别降低65.00％和13.01％,但差异不显著;平均世代周期和种群加倍时间分别增加5.54％和154.19％,后者差异达显著水平.和常规棉相比,2010年转Cry1Ac+Cry2Ab基因棉花百株苗蚜、伏蚜和秋蚜的数量分别降低10.79％、37.18％和17.49％,差异均未达显著水平;2011年转Cry1Ac+Cry2Ab基因棉花百株苗蚜的数量增加2.03％,伏蚜和秋蚜的数量分别降低37.41％和64.03％,差异均未达显著水平.     

抗草甘膦EPSPS基因的专利保护分析

抗草甘膦转基因作物是目前全球播种面积最大的转基因作物,抗草甘膦基因（EPSPS）的克隆、表达及其功能验证等也因此成为现代分子生物育种研究的重点,利用专利等知识产权保护措施将这些功能基因和转化技术转变成自己的独占产权是发达国家和生物技术公司普遍采取的发展策略。通过检索搜集全球范围内EPSPS基因的专利和转化品系信息,分析研究了EPSPS基因在全球的专利保护、核心技术专利分布与产业化运用状况。结果表明,近几年对EPSPS的专利申请量迅速增加,专利申请人主要集中在美国、法国、中国等国家,但是核心技术和产业化应用的绝大部分专利由孟山都、拜耳、先锋、先正达等跨国公司拥有,相关产业的发展主动权也由此被其掌控。     

苏云金芽孢杆菌4.0718菌株的杀虫晶体蛋白基因分析

根据苏云金杆菌(Bacillus thuringiensis)cry1、cry2和cry3型基因的保守区分别设计了3对通用引物Un1(d)/Un1?、Un2(d)/Un2?和Un3(d)/Un3?,以Bt4.0718菌株质粒DNA为模板进行PCR扩增,通过扩增产物片段的分子量大小来确定该菌株所含有的杀虫晶体蛋白基因类型。随后根据上述3类cry基因的高变区设计特异引物再次进行PCR鉴定。结果表明:Bt4.0718菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Cb、cry2Ac和新基因cry4.5等6种基因类型。这一结果为利用该菌株构建高效广谱杀虫工程菌提供了客观依据。     

5种中国苏云金芽孢杆菌的伴孢 晶体蛋白基因分析

利用聚合酶联反应(PCR)和聚丙烯酰胺凝胶电泳(SDS-PAGE)技术分析了5种中国苏云金杆菌制剂菌株的伴孢晶体蛋白及其基因组成。结果发现,5种菌株均含有cry1Aa和/或c和/或d和/或b基因,只有Bt+Virus菌株含有cry1Ab基因,cry1A基因编码的伴孢晶体蛋白分子量约为130 kD;仅有JS-Bt C菌株含有cry1B基因,其编码的伴孢晶体蛋白分子量约为138 kD;除HB Bt C菌株外,其余4个菌株均含有cry2Aa和/或b基因,这类基因编码分子量为70 kD的伴孢晶体蛋白;所有5个菌株都含有cry1I基因,其编码的伴孢晶体蛋白分子量应为81.2 kD,但实验中未曾检测到cry1I基因的表达;所有的菌株都不含有cry1C和cry1D基因。     

RFLP analysis of cry1 and cry2 genes of Bacillus thuringiensis isolates from India

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.     

Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China

Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.     

Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.     

Screening of Bacillus thuringiensis serotypes by polymerase chain reaction (PCR) for insecticidal crystal genes toxic against coffee berry borer

Using PCR,257 isolates of Bacillus thuringiensis(Bt) were screened for cry-type genes. Of 257 isolates/strains, 60 isolates were identified as cry7/8, 10 isolates as cry3 and 36 isolates as cry 1I. One specific strain of B. thuringiensis (sumiyoshiensis; T03B 001) was investigated for the presence of cry7 and cry8 genes. Genes Cry7 and cry8 were first detected in this strain using family primers prior to analysis by exclusion polymerase chain reaction (E-PCR) using specific type primers. E-PCR conducted with the above said primers led to the identification by agarose gel electrophoresis of a remaining 1.5 Kb family band indicating a potentially novel gene. This PCR product, (1.5 Kb), was purified from the gel and cloned in pGEM-T Easy vector. Twenty recombinant colonies bearing 1.5 Kb insert were identified and three randomly selected representatives of the group, clones 7, 8 and 10, were sequenced and compared to all cry7 and cry8 sequences available from Gene Bank. Alignments with available DNA and protein sequences showed that all these clones contained a gene related to cry8Aa1. Analysis using protein sequence alignment showed that the sequence from clone 7 differed from the closest relative, known under the new nomenclature as cry 8Aa1, by 44%. The crystal proteins from B. thuringiensis sumiyoshiensis (T03B 001) was toxic to coffee berry borer larvae.     

cry Genes profiling and the toxicity of isolates of Bacillus thuringiensis from soil samples against American bollworm,Helicoverpa armigera

Aims: The aim of this study was to search for Bacillus thuringiensis (Bt) harbouring cry1A gene which could effectively control cotton pest, American bollworm, Helicoverpa armigera. Methods and Results: cry gene profiling of 50 Bt isolates showed the presence of cry1, cry2, cry3, cry4, cry7, cry8 and cry9 genes. None of the isolates harboured cry1 gene alone. It was always found in combination with cry3. There was no isolate positive for cry10 gene. Considering isolates with single cry genes, the frequency of cry4 was predominant (22%) followed cry2 (6%), cry3 (4%) and cry8 (2%). Isolates having two cry genes in combination had 14% incidence for cry2 + cry4, 12% for cry3 + cry4 and 10% for cry1 + cry3. The most dominant three gene linkage was cry1 + cry3 + cry4. Further profiling of cry1 gene showed that cry1K gene was abundantly present in all combinations such as cry1A, cry1D, cry1F and cry1I. However, cry1C existed independent of other subtypes. Finally, the Bt isolates with cry1A were analyzed for 16S rRNA gene, which showed two distinct groups of isolates on the basis of sequence homology. Bioassays of spore–crystal mixtures of SBS‐Bt4, 8, 17, 21 and 26 harbouring cry1 against neonate larvae of H. armigera showed LC₅₀ 1288, 1202, 467·7, 524·8 and 108·5 μg ml^?1. The SBS‐Bt26 showed fourfold higher toxicity than the cry 1Ac harbouring positive control, HD‐73. Conclusions: None of the isolates harboured single cry 1 gene. They were always in combination of two or three genes. A Bt isolate (Bt26) had fourfold higher toxicity against H. armigera larvae compared with the positive control HD 73 and hence can be commercially exploited to control insect pest. Significance and Impact of the Study: The inter relationship between the cry genes content and the toxicity may allow better understanding of Bt ecology.     

Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain DOR4 Toxic to Castor Semilooper <Emphasis Type="Italic">Achaea janata</Emphasis>: Proteolytic Processing and Binding of Toxins to Receptors

We have isolated a strain of Bacillus thuringiensis (Bt) from Indian soil samples that was shown to be toxic to Achaea janata larvae. The isolate, named B. thuringiensis DOR4, serotypically identified with the standard subspecies kurstaki (H3a3b3c) and produced bipyramidal inclusions along with an amorphous type. Although the plasmid pattern of DOR4 was different from that of the reference strain, a crystal protein profile showed the presence of two major bands (130 and 65 kDa) similar to those of Bt subsp. kurstaki HD-1. To verify the cry gene content of DOR4, triplex PCR analysis was performed; it showed amplification of the cry1C gene in addition to cry1Aa, cry1Ac, cry2A, and cry2B genes, but not the cry1Ab gene. RT-PCR analysis showed the expression of cry1Aa and cry1Ac genes. In vitro proteolysis of DOR4 protoxin with midgut extract generated products of different sizes. Zymogram analysis of DOR4 protoxin as substrate pointed to a number of distinct proteases that were responsible for activation of protoxins. Furthermore, toxin overlay analysis revealed the presence of multiple toxin-binding proteins in midgut epithelium. Based on all these characterizations, we suggest that the Bt DOR4 strain can be exploited for an A. janata control program.     

Association of bioassays and molecular characterization to select new Bacillus thuringiensis isolates effective against Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the main corn pests and Bacillus thuringiensis is important in its control because of its entomopathogenic property. The objective of this study was the molecular characterization of B. thuringiensis isolates for cry1 locus presence and the assessment of the efficiency of these isolates in controlling S. frugiperda caterpillars. Gral-cry1 was used in the PCR analyses to confirm the presence of the cry1 locus in 15 isolates. A 3 x 10(8) spore/ml suspension bathed the diet used to feed 30 caterpillars per isolate, with three replications. The cry1 locus type genes of the different isolates were identified for five gene subclasses; linear regression analyses were carried out to ascertain possible associations between the presence of an individual cry1 locus gene and high levels of toxicity. All the DNAs amplified with Gral-cry1 presented an amplification product with the expected size. Regarding the levels of insecticide efficiency against the cob worm, 41 isolates presented 100% mortality and 16 presented an index between 70% and 90%. The cry1Ab gene was present in 80 isolates, cryb in 69 isolates, cry1Ac in all the isolates and cryv and cry1E in 93 and 27 isolates, respectively. The values regarding the individual effect of each gene on caterpillar mortality were significant at 1% probability for the cry1Ac and cry1E genes.     

Efficient expression of vip184DeltaP gene under the control of promoters plus Shine-Dalgarno (SD) sequences of cry genes from Bacillus thuringiensis

AIMS: To compare vip184DeltaP gene expression time course and Vip184 protein yield under the control of promoters and Shine-Dalgarno (SD) sequences of vip184, cry3A and cry1A gene from Bacillus thuringiensis respectively. METHODS AND RESULTS: Derived from the shuttle vector pHT3101, recombinant plasmids pHPT3, pHTP3A(Delta)P and pHTP1A(Delta)P were constructed with the native vip184 gene and the vip184(Delta)P gene, either under the control of promoters and SD sequences of cry3A or cry1A genes. When the above plasmids were transformed into an acrystalliferous B. thuringiensis strain Cry(-)B, their expression time course were consistent with those of vip184, cry3A and cry1A gene respectively. The maximum yields of Vip184 protein were increased when under the control of promoters plus SD sequences of cry3A and cry1A gene. CONCLUSIONS: The results showed that both cry3A and cry1A promoter/SD sequence combinations were able to enhance synthesis of Vip184 and change its expression time course. SIGNIFICANCE AND IMPACT OF THE STUDY: Both cry3A and cry1A promoter/SD systems offer a method for improving the expression efficacy of the vip184 gene in B. thuringiensis and it is possible to co-express the vip184 gene and cry genes and accumulate Vip184 in the form of inclusion bodies by these systems in order to construct novel useful B. thuringiensis engineered strains.