Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

A novel insecticidal gene crylAh was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active CrylAh toxin has a toxicity level similar to that of the full-length CrylAh toxin. In this study, plant expression vector pMhGM harboring truncated crylAh gene was transformed into maize （Zea mays L.） immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions （madMARs） were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer （Ostriniafurnacalis）. These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the crylAh gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene crylAh was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T l-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.

Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China

Ninety-two Bacillus thuringiensis isolates were screened from 683 soil samples collected from tropical and semitropical primeval forests in Yunnan and Hainan provinces of China. Several shapes of crystals, including bipyramidal, square, ovoid, spherical, and amorphous, were observed in the B. thuringiensis isolates. Twenty-six pairs of primers were used to identify 31 holotype cry genes at primary rank of the B. thuringiensis cry gene nomenclature system. The cry gene-types of 92 B. thuringiensis isolates and 33 B. thuringiensis isolates screened from Northeast region of China were identified by PCR-RFLP and SDS-PAGE methods. Fifty-eight isolates harbored cry1 genes, 32 isolates cry2 genes, 12 isolates cry8 genes, 3 isolates cry9 genes, 12 isolates cry11 genes, and 13 isolates cry30 genes. Of the tested isolates, 42 produced no reaction product with 26 pairs of primers and also exhibited no toxicity against 8 insect species tested. The isolate Z2-34 harbored a novel cry30 gene, exhibited insecticidal activity against Aedes albopictus of Dipterans. The accession number of the novel genes in this study is AY916046. Isolation and identification of B. thuringiensis and cry gene are important for investigating the diversity of B. thuringiensis resources and cloning new cry gene.

Developing transgenic maize(Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

Utilizing modified ubi1 introns to enhance exogenous gene expression in maize(Zea mays L.) and rice(Oryza sativa L.)

supported by the National Natural Science Fund of China （30970231）;the Genetically Modified Organisms Breeding Major Project of China （2014ZX08003001）