Table 2 - uploaded by Keyu gu
Content may be subject to copyright.
Source publication
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...
Similar publications
The European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), is an economically important insect pest of corn, Zea mays L., in the United States and Canada. The development of genetically modified corn expressing genes derived from Bacillus thuringiensis (Bt) that encodes insecticidal crystalline (Cry) proteins has proven to be ef...
Citations
... The larvae of P. guttata is more voracious than those of C. medialis after the second ecdysis. To explore alternative strategies for control of C. medialis, transgenic rice plants expressing the insecticidal cry genes of Bacillus thuringiensis have been developed (Tu et al., 2000;Ye et al., 2003;Han et al., 2007;Qiu et al., 2010;Xu et al., 2011;Manikandan et al., 2016). A Cry1Ac-Cry2Aa recombinant protein expressed in some transgenic rice lines is reported to likely accelerate the evolution of the leaf roller's resistance because its midgut cadherin features two binding sites specific to Cry1Ac and Cry2Aa (Zhong et al., 2022). ...
... Qiu et al. used a maize-derived PEPC promoter to drive cry1Ab/Ac fusion gene expression to transform the Japonica rice variety Nipponbare. Western Blot analysis of this transgenic rice showed that high expression levels of the Cry1Ab/Ac protein could be detected in the leaves and stems, but the Cry1Ab/Ac protein could not be detected in mature seeds [14]. In the vector constructed by Ye et al., cry1C* gene expression was driven by the rbcS promoter from Nipponbare. ...
As pests are an important factor in reducing crop yields, pest control is an important measure in preventing reductions in crop yields. With the aim of ending the use of chemical pesticides, biological control and genetically modified methods are now considered more reasonable pest control strategies. The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins that have specific toxicity to lepidopteran insects, and so it has been applied as a microbial insecticide in the control of crop pests for several decades. With the development of plant genetic engineering, Bt genes encoding insecticidal crystal protein have been introduced into many crop species for pest control. This article indicates that, after years of experiments and research, Bt transgenic rice is close to becoming a commercial insect-resistant rice, and many studies have shown that transgenic rice has pronounced abilities in the control of pests such as yellow stem borers (Scirpophaga incertulas, YSB), striped stem borers (Chilo suppressalis, SSB), and rice leaf rollers (Cnaphalocrocis medinalis, RLR); moreover, it does not obviously differ from non-transgenic rice in terms of safety. This paper suggests that transgenic Bt rice has application potential and commercial value.
... The cre gene responsible for excision can be under control of constitutive promoters such as the CaMV 35S (Chakraborti et al. 2008;Sengupta et al. 2010), the ubiq-uitin1 promoter (Pradhan et al. 2016) or by the promoters regulated spatio-temporally (Kopertekh et al. 2010;Chen et al. 2017). Several promoters can be activated by heat shock (e.g., Hoenicka et al. 2016;Zheng et al. 2016;Kopertekh et al. 2018), cold (Meszaros et al. 2015;Éva et al. 2018), or chemically with, e.g., β-estradiol Qiu et al. 2010) and salicylic acid (Ma et al. 2009). ...
The Cre/loxP-based self-excision represents a promising strategy for removal of hazardous transgene(s) from genomes of targeted crops prior to their introduction into environment. Here, we applied the Cre/loxP self-excision strategy in which the cre recombinase gene is driven by the embryo-specific CRUC promoter from Arabidopsis thaliana. Besides, the Cre/loxP cassette, the T-DNA also consisted of the β-glucuronidase gene, controlled by the double dCaMV 35S promoter and the selectable neomycin phosphotransferase gene; the latter was aimed to be removed from the transgenic genome. The Cre/loxP self-excision cassette was introduced into genomes of four commercial oilseed rape cultivars Haydn, Heros, Hunter and Topas (Brassica napus L.) via Agrobacterium tumefaciens. Transgenic T0 plants were regenerated from all cultivars. Their detailed molecular analyses revealed premature activation of the CRUC promoter that resulted in partial excision of the nptII gene. Progenies of four self-pollinated T0 lines were further analysed, and the data confirmed complete excision event in 5 out of 105 (4.8%) of T1 transgenic oilseed rape plants. The excision efficiency does not seem to depend on the target cultivar. However, the poor transformation efficiency of rapeseed and the limited specificity of the CRUC promoter are clearly the bottleneck of this approach, and the feasibility of (tissue-specific) self-excision of selectable marker gene from genomes of each commercial rapeseed variety adds to their perspective to cope the increasing negative impacts of climate changes.
... The presence of these selectable marker genes has however, aroused safety concerns amongst the public (Daniell et al., 2001). Therefore, removal of marker genes from transgenic plants would likely hasten the public acceptance of transgenic crops (Qiu et al., 2010). There are several strategies to exclude selectable marker gene in transgenic generations, among them co-transformation has been widely practiced (De Block and Debrouwer, 1991;Depicker et al., 1985;Mcknight et al., 1987). ...
Rice transformation was carried out using Agrobacterium strain C58C1 (pGV2260::pSSJ1A)
containing a binary vector, pC0390-ubi-rtp-cry2AX1 with a view to generate marker-free transgenic
rice plants, resistant to lepidopteran insects. Eight putative transgenic rice plants, positive to
gusA and hpt genes, were generated. Four out of the eight putative transgenic plants were
positive for presence of cry2AX1 gene, indicating co-transformation of the selectable marker
gene, hpt and the gene of interest, cry2AX1 in these plants. Cry2AX1 protein concentration in PCR
positive T0
Plants ranged from 0.010 to 0.022 µg/g of fresh leaf tissue. The T2
generation plants
were subjected to ELISA, and Cry2AX1 protein concentration ranged from 0.016 to 0.057 µg/g of
fresh leaf tissue. Insect bioassay studies on ELISA positive T0 and T2 plants against neonates of
leaffolder resulted in larval mortality ranging from 15 to 30 %
... The presence of these selectable marker genes has however, aroused safety concerns amongst the public (Daniell et al., 2001). Therefore, removal of marker genes from transgenic plants would likely hasten the public acceptance of transgenic crops (Qiu et al., 2010). There are several strategies to exclude selectable marker gene in transgenic generations, among them co-transformation has been widely practiced (De Block and Debrouwer, 1991;Depicker et al., 1985;Mcknight et al., 1987). ...
Rice transformation was carried out using Agrobacterium strain C58C1 (pGV2260::pSSJ1A) containing a binary vector, pC0390-ubi-rtp-cry2AX1 with a view to generate marker-free transgenic rice plants, resistant to lepidopteran insects. Eight putative transgenic rice plants, positive to gusA and hpt genes, were generated. Four out of the eight putative transgenic plants were positive for presence of cry2AX1 gene, indicating co-transformation of the selectable marker gene, hpt and the gene of interest, cry2AX1 in these plants. Cry2AX1 protein concentration in PCR positive T0 Plants ranged from 0.010 to 0.022 μg/g of fresh leaf tissue. The T2 generation plants were subjected to ELISA, and Cry2AX1 protein concentration ranged from 0.016 to 0.057 μg/g of fresh leaf tissue. Insect bioassay studies on ELISA positive T0 and T2 plants against neonates of leaffolder resulted in larval mortality ranging from 15 to 30 %
... Bt rice expressing cry genes have been proven to be effective against various rice pest species, depending on the type of cry genes introduced (Chen et al., 2008;Qiu et al., 2010;Tu et al., 1998;Tu et al., 2000;Wang et al., 2016). Cry protein expression in rice is a very important factor for effective protection against target rice pests. ...
The transgenic rice expressing cry1Ac gene, which is linked to the rice rbcS promoter and its transit peptide sequence (tp), was highly resistant against all instars of Cnaphalocrocis medinalis (Guenetée) (Lepidoptera: Crambidae). In this study, we evaluated the larval mortality, behavior change, and field occurrence of three main rice pests, C. medinalis, Naranga aenescens (Moore) (Lepidoptera: Noctuidae), and Parnara guttata (Bremer & Grey) (Lepidoptera: Hesperiidae) in T4 generations of three Bt rice events (rbcS3:cry1Ac; 608102, 608104 and 608107) and non-Bt rice. All of the three Bt rice events were resistant to C. medinalis which showed significantly higher mortality for all instars compared to non-Bt rice. The resistance of Bt rice events against the larvae decreased gradually as the larvae developed. However, the survived larvae which ingested Bt rice events died eventually without further development. The resistance of three Bt rice events was investigated in the pot test, which was conducted with 3rd instars of C. medinalis, N. aenescens, and P. guttata, showed mortalities of over 70%. In behavioral assay, C. medinalis fed on the Bt rice events showed feeding avoidance and less leaf rolling behavior compared to that of the larvae fed on non-Bt rice. A 2-yr field survey conducted with larvae of C. medinalis and P. guttata also showed that the three Bt rice events significantly had lower damaged on leaves compared to that of non-Bt rice. Overall, the three Bt rice events were highly resistant to the larvae of lepidopteran target rice pests.
... As for the Bt contents in the endosperm, the previously reported tissue-specific promoters could reduce them to very low levels 7,11,14,[39][40][41] but not to zero, due to background expression of the promoters. For instance, the famous Bt rice RJ5, which harbours an rbcS promoter-driven Cry1C construct, can only reduce endosperm Bt protein to 2.6 ng g-1 7 . ...
The presence of genetically modified (GM) protein in the endosperm is important information for the public when considering the biological safety of transgenic rice. To limit the expression of GM proteins to rice green tissues, we developed a modified Cre-lox gene switch using two cassettes named KEY and LOCK. KEY contains a nuclear-localized Cre recombinase driven by the green-tissue-specific promoter rbcS. LOCK contains a Nos terminator (NosT), which is used to block the expression of the gene of interest (GOI), bounded by two loxP sites. When KEY and LOCK are pyramided into hybrid rice, a complete gene switch system is formed. The Cre recombinase from KEY excises loxP-NosT in LOCK and unlocks the GOI in green tissues but keeps it locked in the endosperm. This regulatory effect was demonstrated by eYFP and Bt expression assays. The presence of eYFP and Cre were confirmed in the leaf, sheath, stem, and glume but not in the root, anther or seed of the gene-switch-controlled eYFP hybrids. Meanwhile, gene switch-controlled Bt hybrid rice not only confined the expression of Bt protein to the green tissues but also showed high resistance to striped stem borers and leaffolders.
... (PEPC) gene is also well used. In rice, maize and potato, high levels of Bt were produced exclusively in green tissues under the control of the PEPC promoter (Datta et al., 1998;Hagh et al., 2009;Koziel et al., 1993;Qiu et al., 2010). ...
Using promoters expressed in non-endosperm tissues to activate target genes in specific plant tissues or organs with very limited expression in the endosperm is an attractive approach in crop transgenic engineering. In this paper, five putative non-endosperm tissue expressed promoters were cloned from the rice genome and designated POsNETE1, POsNETE2, POsNETE3, POsNETE4 and POsNETE5. By qualitatively and quantitatively examining GUSplus reporter gene expression in transgenic rice plants, POsNETE1-POsNETE5 were all found to be active in the roots, leaves, stems, sheaths and panicles but not in the endosperm of plants at different developmental stages. In addition, POsNETE2, POsNETE4 and POsNETE5 were also inactive in rice embryos. Among these promoters, POsNETE4 and POsNETE5 exhibited higher activities in all of the tested tissues, and their activities in stems, leaves, roots and sheaths were higher than or comparable to those of the rice Actin1 promoter. We also progressively monitored the activities of POsNETE1-POsNETE5 in two generations of single-copy lines and found that these promoters were stably expressed between generations. Transgenic rice was produced using POsNETE4 and POsNETE5 to drive a modified Bt gene, mCry1Ab. Bt protein expressed in the tested plants ranged from 1769.4 to 4428.8 ng/g fresh leaves, whereas Bt protein was barely detected in the endosperm. Overall, our study identified five novel non-endosperm tissue-expressed promoters that might be suitable for rice genetic engineering and might reduce potential social concern regarding the safety of GMO crops. This article is protected by copyright. All rights reserved.
... SMGs are redundant after transformation, and are not conducive to continuous transformation and safety. Thus, a series of marker-free strategies were developed, such as biolistic method (Tu et al., 2000;Shiva Prakash et al., 2009) and A. tumefaciens-mediated co-transformation, site-specific recombination (Li et al., 2007;Qiu et al., 2010;Sengupta et al., 2010;Yu et al., 2013;Woo et al., 2015b), multi-auto-transformation vector system (Endo et al., 2002;Khan et al., 2006;Scaramelli et al., 2009), transposonmediated method (Gao et al., 2015), intrachromosomal recombination (Zubko et al., 2000), and PCR-based method (de Vetten et al., 2003;Woo et al., 2015a). There are three approaches for the co-transformation: one plasmid harboring two T-DNAs in one Agrobacterium strain (Breitler et al., 2004;Matheka et al., 2013); two T-DNAs located on two plasmids in the same Agrobacterium strain (Parkhi et al., 2005;Sripriya et al., 2008); two T-DNAs in separate Agrobacterium strains (Dutt et al., 2012). ...
Agrobacterium-mediated co-transformation is an efficient strategy to generate marker-free transgenic plants. In this study, the vectors pMF-2A∗ containing a synthetic cry2A∗ gene driven by maize ubiquitin promoter and pCAMBIA1301 harboring hygromycin phosphotransferase gene (hpt) were introduced into Minghui86 (Oryza sativa L. ssp. indica), an elite indica restorer line. Two independent transformants containing both the cry2A∗ gene and hpt gene were regenerated. Several homozygous marker-free transgenic progenies were derived from family 2AH2, and three of them were selected for further insect bioassay in the laboratory and field. Insect-resistance assays revealed that all the three transgenic lines were highly resistant to striped stem borer (Chilo suppressalis), yellow stem borer (Tryporyza incertulas) and rice leaf folder (Cnaphalocrocis medinalis). The measurement of Cry2A protein concentration showed that Cry2A protein was stably expressed in leaves and stems of homozygous transgenic lines and their hybrids. The yields of the marker-free homozygous transgenic lines and their hybrids were not significantly different from those of their corresponding controls. Furthermore, the results of flanking sequence isolation showed that the T-DNA in line 8-30 was integrated into the intergenic region of chromosome 2 (between Os02g43680 and Os02g43690). These results indicate that the marker-free transgenic rice has the potential for commercial production.
... However, constitutive expression of transgenes generally attracted criticisms for their potential to develop target resistance against target insects and more environmental spread of the transgenic protein. Qiu et al. (2010) addressed this issue by expressing hybrid Cry1Ab/Ac Bt protein driven by the maize PEPC promoter. The transgene expressed only in the leaf and stems of rice and provided resistance against RLF. ...
A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34 µg g(-1) leaf fresh weight and with 80-92 % levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7-3.6 ng g(-1)). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.
