Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product

Conditions for hyperexpression, in Escherichia coli, of the Bacillus thuringiensis var, kurstaki gene, cryIA9(c)73, encoding an insecticidal crystal protein, CryIA(c)73, were investigated by varying the promoter type, host cell, plasmid copy number, the second codon and number of terminators. The cryIA(c)73 gene was cloned into three E. coli expression vectors, pKK223-3 (Ptac promoter), pET-3a (P phi 10 promoter), and pUC19 (Ptac promoter). The level of cryIA(c)73 expression was measured by ELISA and compared to total cellular protein over growth periods of 24 and 48 h. Maximum expression levels of 284 microgram CryIA(C)73/ml (48% of cellular protein) were obtained in shake flasks with the Ptac promoter in E. coli JM103. Optimal conditions were found to be low-copy-number plasmid (pBR322 ori), 48 h of growth, in lon+ cells. A change of the gene's second codon to AAA can improve expression by two to three fold but is undetectable in the presence of a strong E. coli promoter. The cryIA(c)73 gene product, in E. coli, formed crystals with the same lattice structure as the native crystals formed in B. thuringiensis (as visualized by electron microscopy). Bioassay results (insect toxicity and specificity) of the crystal produced in E. coli were similar to that produced in B. thuringiensis.     

Expression of cryIA(c) gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod-borer (Heliothis armigera) larvae

Two strains of chickpea (Cicer arietinum L.) ICCV-1 and ICCV-6, were used for transgenic plant generation. Embryo axis of mature seed devoid of the root meristem and the shoot apex was used as experimental material. The explants were cultured in medium containing MS macro salts, 4 × MS micro salts, B5 vitamins, 3.0 mg l–1 BAP, 0.004 mg l–1 NAA, 30 mg l–1 sucrose and cultured at 26 °C in dark, 24 h prior to bombardment. Gene delivery to the explants was carried out using a Bio-Rad Biolistic 1000/He particle gun. A chimaeric, truncated bacterial cryIA(c) gene construct was developed for plant expression with the CaMV35S promoter, nos terminator, an initiatory kozak sequence and a translational enhancer (STAR-P) sequence of tobacco mosaic virus. This cryIA(c) gene was cotransferred with a plasmid containing nptII gene as the selection marker. Transgenic kanamycin resistant chickpea plants were obtained through multiple shoot formation and repeated selection of the bombarded explants. Molecular analyses of the transformants revealed the presence of the transferred functional cryIA(c) gene in plant. Insect feeding assay indicated that the expression level of the cryIA(c) gene was inhibitory to the development of the feeding larvae of Heliothis armigera Hubner, the chickpea pod-borer     

Arabidopsis thaliana small subunit leader and transit peptide enhance the expression ofBacillus thuringiensis proteins in transgenic plants

The expression of the modified gene for a truncated form of thecryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein fromBacillus thuringiensis var.kurstaki (B.t.k.) HD73, under control of theArabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunitats1A promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that theats1A promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10-fold to 20-fold increase incryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-En35S) was used to express the samecryIA(c) gene. Transient expression assays in tobacco protoplasts and the whole plant results support the conclusion that the transit peptide plus untranslated sequences upstream of that region are both required for the increase in expression of the CryIA(c) protein. Furthermore, the CaMV-En35S promoter can be used with theArabidopsis ats1A untranslated leader and transit peptide to increase expression of this protein. While subcellular fractionation revealed that the truncated CryIA(c) protein fused to theats1A transit peptide is located in the chloroplast, the increase in gene expression is independent of targeting of the CryIA(c) protein to the chloroplast. The results reported here provide new insight into the role of 5 untranslated leader sequences and translational fusions to increase heterologous gene expression, and they demonstrate the utility of this approach in the development of insect-resistant crops.     

Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer

The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality. The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein. We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment. The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3) was directly cloned into the BglII site of plant transformation vectors. The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin. Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment. DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses. ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein. Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight. A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect. Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer     

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。     

A Brassica S locus gene promoter directs sporophytic expression in the anther tapetum of transgenic Arabidopsis

The S locus glycoprotein (SLG) gene of Brassica encodes stigmatic glycoproteins that are implicated in the pollen-stigma interaction of self-incompatibility. We have transformed the related plant Arabidopsis thaliana with a chimaeric gene consisting of the promoter region of an SLG gene fused to the reporter gene beta-glucuronidase (GUS). In transgenic plants the gene was expressed in two cell types of the flower. In stigmas, the timing and distribution of GUS activity was similar to that previously described for SLG expression in Brassica. In anthers, expression was detected at an earlier stage of flower development with GUS activity restricted to the tapetal cell layer. The novel finding of SLG-promoter activity in the anther supports the hypothesis that sporophytic control of self-incompatibility is a result of SLG-gene expression in the tapetum.     

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.     

A promoter from the loblolly pine PtNIP1;1 gene directs expression in an early-embryogenesis and suspensor-specific fashion

The PtNIP1;1 gene encodes an aquaglyceroporin that is expressed early in embryogenesis and appears to be expressed preferentially in the suspensor [V.T. Ciavatta et al. (2001) Plant Physiol 127:211-224]. An 899-bp fragment 5' to the PtNIP1;1 open reading frame (NIP(-899)) was cloned from loblolly pine (Pinus taeda L.) genomic DNA and fused to the beta-glucuronidase (GUS) reporter gene. The resulting plasmid, pNIP-GUS, was transformed into Norway spruce (Picea abies L.) embryogenic cultures by co-bombarding with a plasmid containing a bar gene construct as a selectable marker. The identity of lines selected on medium containing the herbicide Basta and showing beta-glucuronidase activity was confirmed by polymerase chain reaction as harboring GUS. Histochemical GUS assays of these lines revealed GUS activity in all cells of proembryogenic masses. During early embryogeny, GUS staining was intense in the suspensor region but not detectable in embryonal masses. GUS staining was absent by mid-embryogeny. By contrast, a control transgenic line, transformed with EuCAD-GUS, expressed GUS throughout embryo development. These results suggest that NIP(-899) contains elements that drive early embryogenesis-specific expression and suspensor-specific expression. This is the first example of a suspensor-specific promoter in conifers.     

Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest

 The truncated chimeric Bt gene, cryIA(b) of Bacillus thuringiensis, driven by two constitutive promoters, 35S from CaMV and Actin-1 from rice, and two tissue-specific promoters, pith tissue and pepcarboxylase (PEPC) for green tissue from maize, was introduced into several varieties of rice (indica and japonica) by microprojectile bombardment and protoplast systems. A total of 1800 putative transgenic Bt rice plants could be produced. Southern analysis revealed that more than 100 independently transformed plants could be confirmed for integration of the cryIA(b) gene. High levels of CryIA(b) proteins were obtained in the green tissue (leaves and stem) of many plants using the PEPC promoter. There was little difference in Bt protein level in leaves and stems from transgenic plants with the 35 S or Actin-1 promoter. Out of 800 Southern-positive plants that were bioassayed, 81 transgenic plants showed 100% mortality of insect larvae of the yellow stem borer (Scirpophaga incertulas). The transgene, cryIA(b), driven by different promoters showed a wide range of expression (low to high) of Bt proteins stably inherited in a number of rice varieties with enhanced yellow stem borer resistance. This first report of transgenic indica Bt rice plants with the PEPC or pith promoter either alone or in combination should provide a better strategy for providing rice plants with protection against insect pest resistance, minimizing the expression of the CryIA(b) protein in seeds and other tissues. Received: 12 November 1997 / Accepted: 25 November 1997     

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

AtSTP3启动子控制的外源基因在转基因水稻中的特异表达研究

利用PCR技术从哥伦比亚型拟南芥基因组DNA中分离了AtSTP3绿色组织特异表达的启动子,序列分析表明,扩增片段(1774bp)与已报道序列的相应区域同源性达99.9%。将其与GUS报告基因融合在一起,构建了植物表达载体,并由农杆菌介导法导入水稻品种‘中花11’中。对转基因水稻植株中的GUS活性进行定性与定量测定结果表明,AtSTP3启动子可驱动GUS报告基因在转基因水稻植株叶片中特异性表达,而在根和种子等器官中不表达或表达活性极弱,AtSTP3启动子表现出明显的组织特异性。     

Expression of the crystal protein gene under the control of the alpha-amylase promoter in Bacillus thuringiensis strains.

The expression of an insecticidal crystal protein gene of Bacillus thuringiensis under the control of the alpha-amylase gene promoter was investigated. The cryIC gene, which encodes a protein known to have a unique activity against Spodoptera (armyworm) species, was used in this investigation. The cryIC gene was placed, along with the alpha-amylase promoter from B. subtilis, in a B. thuringiensis-derived cloning vector, generating a pair of recombinant plasmids, pSB744 and pSB745. The cloning vector that contains the minimal replicon of B. thuringiensis subsp. kurstaki HD73 is stably maintained in a variety of B. thuringiensis strains, as previously reported by Gamel and Piot (Gene 120:17-26, 1992). The present study confirmed that the recombinant plasmids are also stably maintained in B. thuringiensis subsp. kurstaki Cry-B and HD73 growing in media without selection pressure for at least 48 h. The cryIC gene on the recombinant plasmids were notably expressed at high levels in both recombinant strains. Expression of the introduced cryIC gene on the recombinant plasmid in B. thuringiensis subsp. kurstaki HD73 did not impair expression of the resident cryIA(c) gene. The CryIA(c) protein is known to have a high level of activity against loopers such as Trichoplusia ni (the cabbage looper). As a result of coexpression of the introduced cryIC gene and the resident cryIA(c) gene, recombinant strain HD73 acquired an additional insecticidal activity against Spodoptera exigua (the beet armyworm) whereas the original activity level against T. ni was maintained.     

Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.