Edited by Marc C. E. Van Montagu, University of Ghent, Ghent, Belgium, and approved December 17, 1997 (received for review September 15, 1997) ABSTRACTOver 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and -glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific ␦-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R 0 and R 1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R 0 plants. Bioassays with R 1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.Rice is one of the world's most important food crops, and intense efforts, including use of genetic engineering technologies, must be engaged to increase its yield if the impending global rice shortage is to be avoided (1). Engineering rice for pest resistance is a major challenge, one strategy being the introduction of Bacillus thuringiensis (Bt) crystal insecticidal protein (␦-endotoxin) genes (cry genes). These proteins (Bt toxins) are highly toxic to lepidopteran, dipteran, and coleopteran insects (2), among which are important pests of rice such as striped stem borer (SSB), yellow stem borer (YSB), and leaffolder (Cnaphalocrocis medinalis and Marasmia patnalis) that cause annual losses of an estimated 10 million tonnes (3).Rice plants containing cryIA(b) or cryIA(c) have been obtained by using protoplast (4) or particle bombardment methods (5-7). However, the numbers of plants obtained and levels of the toxin proteins in these studies were unfortunately still very low from a breeder's point of view. In contrast, Ͼ2,600 transgenic plants were produced with the modified cry genes in nine rice strains by using a modified Agrobacteriumbased rice transformation procedure (8). Herein we report that high levels of CryIA(b) and CryIA(c) were detected among these transgenic plants, indicating that many candidate transgenics in this large screen may be the result of optimal position effects. Insect feeding assays with R 1 plant tissues indicated that the transgenic plants were highly toxic to two major rice insects, SSB and YSB, with near 100% mortality within 5 days....
Two transgenic rice (Oryza sativa L.) lines, KMD1 and KMD2 at the R4 generation, transformed with a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, were first evaluated for stem borer resistance in the field during the rice growing season of 1998 in two areas of Zhejiang Province, China. Both KMD1 and KMD2 were highly resistant to the stem borers Chilo suppressalis (Walker) and Scirpophaga incertulas (Walker), and were completely undamaged during the whole rice growing season. In contrast, damage to the plants of the untransformed parental control (Xiushui 11) was in the form of deadhearts or whiteheads. Under natural infestation by the C. suppressalis, the damage to control plants reached a peak of 88.7% of plants and 20.1% of tillers encountered with deadhearts. Under artificial and natural infestation of neonate striped stem borers at the vegetative stage and booting stage, 100% of plants and 25.6% of tillers, 78.9% of plants and 15.6% of productive tillers among artificially infested control plants were observed with the symptom of deadhearts and whiteheads, respectively. Damage to the control plants from artificial infestation by the S. incertulas reached a peak of 97.0% of plants and 22.9% of tillers damaged. The field research indicated that both KMD1 and KMD2 show great potential for protecting rice from attack by these two stem borers.
The inheritance and expression patterns of the cry1Ab gene were studied in the progenies derived from different Bt ( Bacillus thuringiensis) transgenic japonica rice lines under field conditions. Both Mendelian and distorted segregation ratios were observed in some selfed and crossed F(2) populations. Crosses between japonica intra-subspecies had no significant effect on the segregation ratios of the cry1Ab gene, but crossing between japonica and indicainter-subspecies led to distorted segregation of the cry1Ab gene in the F(2)population. Field-release experiments indicated that the cry1Ab gene was stably transmitted in an intact manner via successive sexual generations, and the concentration of the Cry1Ab protein was kept quantitatively stable up to the R(6)generation. The cry1Ab gene, driven by the maize ubiquitinpromoter, displayed certain kinds of spatial and temporal expression patterns under field conditions. The content of the Cry1Ab protein varied in different tissues of the main stems, the primary tillers and the secondary tillers. Higher levels of the Cry1Ab protein were found in the stems, leaves and leaf sheaths than in the roots, while the lowest level was detected in grains at the maturation stage. The content of the Cry1Ab protein in the leaves peaked at the booting stage and was lowest at the heading stage. Furthermore, the Cry1Ab content of cry1Ab expression in different tissues of transgenic rice varied individually with temperature.
The synthesis of two modified genes, Cry IA(b) and CryIA(c), each consisting of 1845 bp, is described in detail. The genes were synthesized using an improved PCR procedure based on recursive principles. The synthetic CryIA(c) gene was put under the control of a maize ubiquitin promoter. This construct was tested in a maize endosperm-derived suspension culture system. The use of maize endosperm culture as a quick and efficient system to test the activity of synthetic genes is described.
Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been reported to express an endogenous insulin-like protein, we also looked for any effect of the human growth factor in transgenic plants, but no developmental or morphological differences with wild type tobacco or rice plants were detected. Since insulin and hIGF-1 share some overlapping roles, hIGF-1 may become a substitute therapeutic agent in subjects with certain defects in their insulin receptor signaling. Hence, if the full beneficial potential of rthIGF-1 is achieved, it is expected that in the future the demand will likely increase significantly.
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