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Immature embryos of rice varieties "Xiushuill" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant calli were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56% approximately 72% measured as numbers of Geneticin (G418)-resistant calli produced and 36% approximately 60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38% approximately 61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16% approximately 75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1 and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues, respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159wΔ mutant that was deficient in 3-ketoacyl-CoA reductase activity. Wild-type growth rate was restored in ybr159wΔ cells that expressed either GhKCR1 or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p.

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.