The phenomenon of intron-mediated enhancement (IME) was discovered in 1990 based on the observation that plant introns can stimulate gene expression, particularly in monocots. However, the intrinsic mechanism of IME remains unclear because many studies have yielded various results depending on the promoter, reporter gene, flanking sequences of the intron, and target cell or tissue. In this study, the effect of the first intron of the maize ubiquitin gene (ubi1 intron) was investigated by changing insertion sites, deleting specific regions and mutating individual motifs in maize (Zea mays L.) and rice (Oryza sativa L.) using ubi1 intron-containing GUS (β-glucuronidase) constructs. In maize callus, the integration of the full-length ubi1 intron into the GUS coding sequence at the +13, +115 and +513 positions by particle bombardment increased GUS activity approximately five-, four- and two-fold, respectively. Eight truncated ubi1 introns in the pSG(13i)N construct significantly influenced GUS gene expression to different degrees in transient assays. Notably, the 3´ region deletions significantly reduced the IME effect, whereas a 142-nt deletion, pSG(13i-P5)N, in the 5´ region caused a 1.5-fold enhancement relative to pSG(13i)N. Furthermore, four site-directed mutageneses were performed in pSG(13i-P5)N; these constructs resulted in the up-regulation of GUS gene expression to different levels. The most effective modified ubi1 intron, pSG(13i-M4)N, was further evaluated and proved in rice using transient experiments. In addition, the sequences flanking the GUS insertion significantly influenced the IME effect of the vectors that were constructed. The modified ubi1 intron had the potential application on crop genetic engineering.

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 cry1Ah gene with the 2mG2-epsps gene and combined the wide-used manA gene as a selective marker to construct one coordinated expression vector called p2EPUHLAGN. 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.

A novel insecticidal gene cry1Ah was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active Cry1Ah toxin has a toxicity level similar to that of the full-length Cry1Ah toxin. In this study, plant expression vector pMhGM harboring truncated cry1Ah 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 (Ostrinia furnacalis). These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the cry1Ah gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene cry1Ah was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T1-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.

Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana, was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear cry1Ah gene (800 ng mL-1) and supercoiled plasmid DNA (800 ng mL-1) under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannon’s index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic cry1Ah maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.

Insect pest and weeds are two major problems for forage and turf grasses. In this study, scarab larvae- and herbicideresistant transgenic perennial ryegrass (Lolium perenne L.) was obtained by transforming it with cry and bar genes simultaneously via the Agrobacterium-mediated method. To optimize the callus induction and plant regeneration conditions, various concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine were assayed. The transformation efficiencies of different Agrobacterium suspension media, used during Agrobacterium-mediated transformation, were compared. Then, plasmids of pCAMBIA3301 containing cry gene (cry8Ca2 or cry8Ga) and bar gene, driven by ubiquitin promoter, were transformed into perennial ryegrass. The transformants were generated and confirmed by both Southern hybridization analysis and Western hybridization analysis. Further, the resistance of transgenic perennial ryegrass plants to scarab larvae and herbicide were analyzed. After 30 d of co-cultivation with scarab larvae, the damage to the root system of transgenic plants was less than that of non-transgenic control plants. Additionally, the leaves of transgenic plants were resistant to Basta®, while leaves of the wild plants wilted after Basta® spraying. These results show that cry gene and bar gene were successfully transferred into perennial ryegrass by the Agrobactgerium-mediated method, and convey resistance to scarab larvae and herbicide in transgenic perennial ryegrass plants.