The development history and fundamental experience of transgenic crops (Genetically modified crops) breeding in China for near 30 years were reviewed. It was illustrated that a scientific research, development and industrialization system of transgenic crops including gene discovery, transformation, variety breeding, commercialization, application and biosafety assessment has been initially established which was few in number in the world. The research innovative capacity of transgenic cotton, rice and corn has been lifted. The research features as well as relative advantages have been initially formed. The problems and challenges of transgenic crop development were discussed. In addition, three suggestions of promoting commercialization, speeding up implementation of the Major National Project of GM Crops, and enhancing science communication were made.
The interaction between ethylene and osmotic stress pathways modulates the expression of the genes relating to stress adaptation; however, the mechanism is not well understood. In this paper, we report a novel ethylene responsive factor, tomato ethylene responsive factor 1 (TERF1), that integrates ethylene and osmotic stress pathways. Biochemical analysis indicated that TERF1 binds to the GCC box (an element responsive to ethylene) and to the dehydration responsive element, which is responsive to the osmoticum. Expression of TERF1 was induced by ethylene and NaCl treatment. Under normal growth conditions, overexpression of TERF1 in tobacco activated the expression of GCC box‐containing pathogen related genes and also caused the typical ethylene triple response. Further investigation indicated that transgenic TERF1 tobacco exhibited salt tolerance, suggesting that TERF1 might function as a linker between the ethylene and osmotic stress pathways.
Agrotis ypsilon is a destructive pest in agricultural production.The way to improve crop resistance against Agrotis ypsilon by genetic manipulation is available.Previously,the cry2Ab4 gene and vip3Aa11 gene were cloned from Bacillus thuringiensis.Two plant expressive vectors pCS2Ab and pCSvip3A harboring cry2Ab4 gene and vip3Aa11 gene were constructed and transferred into tobacco(Nicotiana tabacum L.) NC89 by Agrobacterium mediated method.32 and 35 positive regenerated plants were obtained and confirmed by PCR,RT-PCR and Western blot,respectively.The insect-resistant capability in the transgenic tobacco plants with pCS2Ab and pCSvip3A vectors had also been detected by bioassay.The mortality of Agrotis ypsilon larvae fed with leaves from transgenic plants was above 82%,which was significantly more than the contrast.The insecticidal activity of transgenic tobacco harboring vip3Aa11 gene is higher than that of transgenic tobacco with cry2Ab4 gene.
Biotechnology in conjunction with the traditional technology and promoting industrialization of GMO will play an important role in developing modern agriculture in China. The progress and achievements of transgenic crop breeding home and abroad were reviewed. The active policy for industrialization of GMO and correct understanding of biosafety were suggested. The basic and innovation research should be further enhanced.
We have developed a parallel, rapid, high-throughput oligonucleotide microarray-based assay for the reliable detection and genotyping of three cry genes (cry1, cry2 and cry9) in Bacillus thuringiensis (Bt).After the nonpolymerase chain reaction (PCR), amplified Bt genomic DNA were fluorescent-labeled using a random primer.The corresponding oligonucleotide probes were designed for the different cry genes that can hybridize Bt genomic DNA after cluster analysis and were printed on glass slides.This microarray has unambiguously detected and identified the cry genes in 10 isolates and reference Bt.Our data demonstrates that the microarray assay is simple and rapid for the detection and genotyping of genes.This type of assay is also a potentially valuable tool for identification and characterization of bacterial functional genes in general.
Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100 x g 2 min and the protoplasts yield was 7x106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, 100 x g 2 min and the protoplasts yield was 6 x 10(6) cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000 x g 2 min and the protoplasts yield was 6x10(6) cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%-80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.
Over the past two decades, Zea mays (maize) has been established as a model system for the study of indirect plant defense against herbivores. When attacked by lepidopteran larvae, maize leaves emit a complex blend of volatiles, mainly composed of sesquiterpenes, to attract the natural enemies of the herbivores. This is associated with a swift transcriptional induction of terpene synthases such as TPS10; however, the molecular components controlling the complex transcriptional reprogramming in this process are still obscure. Here, by exploiting the finding that the maize TPS10 promoter retained its full responsiveness to herbivory in Arabidopsis, we identified the region from -300 to -200 of the TPS10 promoter as both necessary and sufficient for its herbivore inducibility through 5' deletion mapping. A high-throughput screening of an Arabidopsis transcription factor library using this promoter region as the bait identified seven AP2/ERF family transcription factors. Among their close homologs in maize, EREB58 was the only gene responsive to herbivory, with a spatiotemporal expression pattern highly similar to that of TPS10. Meanwhile, EREB58 was also responsive to Jasmonate. In vivo and in vitro assays indicated that EREB58 promotes TPS10 expression by directly binding to the GCC-box within the region from -300 to -200 of the TPS10 promoter. Transgenic maize plants overexpressing EREB58 constitutively over-accumulate TPS10 transcript, and also (E)-β-farnesene and (E)-α-bergamotene, two major sesquiterpenes produced by TPS10. In contrast, jasmonate induction of TPS10 and its volatiles was abolished in EREB58-RNAi transgenic lines. In sum, these results demonstrate that EREB58 is a positive regulator of sesquiterpene production by directly promoting TPS10 expression.
The double T-DNA border expression vector pCS1Ⅰa-LR harboring Bacillus thuringiensis cry1 Ⅰ a gene was introduced into the cotyledon petioles and hypocotyls of cauliflower(Brassica oleracea var.botrytis) mediated by Agrobacterium.Thirty regenerated plants were obtained,18 of them were positive by PCR detection,which demonstrated that cry1 Ⅰ a gene was integrated into the cauliflower genome.The PCR positive rate is 60%.Moreover,RT-PCR and Western blot analysis showed that cry1 Ⅰ a gene were transcripted and translated,respectively in transgenic cauliflower.Bioassay results showed that the transgenic cauliflowers harboring cry1 Ⅰ a gene had the high toxicity to diamondback moth and the mortality of insect larvae was accounted for 100%.The transgenic cauliflower can be used for further insect resistant cauliflower breeding.
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