RESUMO
Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis-resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis-resistant strains of Plutella xylostella and Spodoptera frugiperda. Cry1Bh1 is active against susceptible and B. thuringiensis-resistant colonies of O. nubilalis, P. xylostella, and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda. Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Inseticidas/metabolismo , Lepidópteros/efeitos dos fármacos , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Bioensaio , DNA Bacteriano/química , DNA Bacteriano/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Dados de Sequência Molecular , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Análise de Sequência de DNA , Análise de Sobrevida , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.
Assuntos
Arabidopsis/genética , Sequência Rica em GC , Inativação Gênica , Interferência de RNA , RNA de Plantas/metabolismo , Transgenes , Metilação de DNA , Elementos de DNA Transponíveis , DNA de Plantas/metabolismo , Ácido Graxo Sintase Tipo II/genética , Ácidos Graxos Insaturados/genética , Genes de Plantas , Zea mays/genéticaRESUMO
Matrix Attachment Regions (MARs) are DNA elements that are thought to influence gene expression by anchoring active chromatin domains to the nuclear matrix. When flanking a construct in transgenic plants, MARs could be useful for enhancing transgene expression. Naturally occurring MARs have a number of sequence features and DNA elements in common, and using different subsets of these sequence elements, three independent synthetic MARs were created. Although short, these MARs were able to bind nuclear scaffold preparations with an affinity equal to or greater than naturally occurring plant MARs. One synthetic MAR was extensively tested for its effect on transgene expression, using different MAR orientations, plant promoters, transformation methods and plant species. This MAR was able to increase average transgene expression and produced integration patterns of lower complexity. These data show the potential of making well defined synthetic MARs and using them to improve transgene expression.