Zhixing Wang

Gene-splitting technology: a novel approach for the containment of transgene flow in Nicotiana tabacum.

The potential impact of transgene escape on the environment and food safety is a major concern to the scientists and public. This work aimed to assess the effect of intein-mediated gene splitting on containment of transgene flow. Two fusion genes, EPSPSn-In and Ic-EPSPSc, were constructed and integrated into N. tabacum, using Agrobacterium tumefaciens-mediated transformation. EPSPSn-In encodes the first 295 aa of the herbicide resistance gene 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) fused with the first 123 aa of the Ssp DnaE intein (In), whereas Ic-EPSPSc encodes the 36 C-terminal aa of the Ssp DnaE intein (Ic) fused to the rest of EPSPS C terminus peptide sequences. Both EPSPSn-In and Ic-EPSPSc constructs were introduced into the same N. tabacum genome by genetic crossing. Hybrids displayed resistance to the herbicide N-(phosphonomethyl)-glycine (glyphosate). Western blot analysis of protein extracts from hybrid plants identified full-length EPSPS. Furthermore, all hybrid seeds germinated and grew normally on glyphosate selective medium. The 6-8 leaf hybrid plants showed tolerance of 2000 ppm glyphosate in field spraying. These results indicated that functional EPSPS protein was reassembled in vivo by intein-mediated trans-splicing in 100% of plants. In order to evaluate the effect of the gene splitting technique for containment of transgene flow, backcrossing experiments were carried out between hybrids, in which the foreign genes EPSPSn-In and Ic-EPSPSc were inserted into different chromosomes, and non-transgenic plants NC89. Among the 2812 backcrossing progeny, about 25% (664 plantlets) displayed glyphosate resistance. These data indicated that transgene flow could be reduced by 75%. Overall, our findings provide a new and highly effective approach for biological containment of transgene flow.

Construction of a standard reference plasmid containing seven target genes for the detection of transgenic cotton.

Insect resistance and herbicide tolerance are the dominant traits of commercialized transgenic cotton. In this study, we constructed a general standard reference plasmid for transgenic cotton detection. Target genes, including the cowpea trypsin gene cptI, the insect resistance gene cry1Ab/1Ac, the herbicide tolerance gene cp4-epsps, the Agrobacterium tumefaciens nopaline synthase (Nos) terminator that exists in transgenic cotton and part of the endogenous cotton SadI gene were amplified from plasmids pCPT1, pBT, pCP4 and pBI121 and from DNA of the nontransgenic cotton line K312, respectively. The genes cry1Ab/1Ac and cptI, as well as cp4-epsps and the Nos terminator gene, were ligated together to form the fusion genes cptI-Bt and cp4-Nos, respectively, by overlapping PCR. We checked the validity of genes Sad1, cptI-Bt and cp4-Nos by DNA sequencing. Then, positive clones of cptI-Bt, cp4-Nos and Sad1 were digested with the corresponding restriction enzymes and ligated sequentially into vector pCamBIA2300, which contains the CAMV 35S promoter and nptII gene, to form the reference plasmid pMCS. Qualitative detection showed that pMCS is a good positive control for transgenic cotton detection. Real-time PCR detection efficiencies with pMCS as a calibrator ranged from 94.35% to 98.67% for the standard curves of the target genes (R(2)⩾0.998). The relative standard deviation of the mean value for the known sample was 11.95%. These results indicate that the strategy of using the pMCS plasmid as a reference material is feasible and reliable for the detection of transgenic cotton. Therefore, this plasmid can serve as a useful reference tool for qualitative and quantitative detection of single or stacked trait transgenic cotton, thus paving the way for the identification of various products containing components of transgenic cotton.

Development and Event-specific Detection of Transgenic Glyphosate-resistant Rice Expressing the G2-EPSPS Gene

Glyphosate is a widely used herbicide, due to its broad spectrum, low cost, low toxicity, high efficiency, and non-selective characteristics. Rice farmers rarely use glyphosate as a herbicide, because the crop is sensitive to this chemical. The development of transgenic glyphosate-tolerant rice could greatly improve the economics of rice production. Here, we transformed the Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene G2-EPSPS, which conferred tolerance to glyphosate herbicide into a widely used japonica rice cultivar, Zhonghua 11 (ZH11), to develop two highly glyphosate-tolerant transgenic rice lines, G2-6 and G2-7, with one exogenous gene integration. Seed germination tests and glyphosate-tolerance assays of plants grown in a greenhouse showed that the two transgenic lines could greatly improve glyphosate-tolerance compared with the wild-type; The glyphosate-tolerance field test indicated that both transgenic lines could grow at concentrations of 20,000 ppm glyphosate, which is more than 20-times the recommended concentration in the field. Isolation of the flanking sequence of transgenic rice G2-6 indicated that the 5′-terminal of T-DNA was inserted into chromosome 8 of the rice genome. An event-specific PCR test system was established and the limit of detection of the primers reached five copies. Overall, the G2-EPSPS gene significantly improved glyphosate-tolerance in transgenic rice; furthermore, it is a useful candidate gene for the future development of commercial transgenic rice.

Effect on transcriptome and metabolome of stacked transgenic maize containing insecticidal cry and glyphosate tolerance epsps genes

Gene stacking is a developing trend in agricultural biotechnology. Unintended effects in stacked transgenic plants are safety issues considered by the public and researchers. Omics techniques provide useful tools to assess unintended effects. In this paper, stacked transgenic maize 12-5×IE034 that contained insecticidal cry and glyphosate tolerance G10-epsps genes was obtained by crossing of transgenic maize varieties 12-5 and IE034. Transcriptome and metabolome analyses were performed for different maize varieties, including 12-5×IE034, 12-5, IE034, and conventional varieties collected from different provinces in China. The transcriptome results were as follows. The nine maize varieties had obvious differences in gene expression. There were 3561-5538 differentially expressed genes between 12-5×IE034 and its parents and transgenic receptor, which were far fewer than the number of differentially expressed genes in different traditional maize varieties. Cluster analysis indicated that there were close relationships between 12-5×IE034 and its parents. The metabolome results were as follows. For the nine detected maize varieties, the number of different metabolites ranged from 0 to 240. Compared with its parents, 12-5 and IE034, the hybrid variety 12-5×IE034 had 15 and 112 different metabolites, respectively. Hierarchical cluster analysis with Pearsons correlation analysis showed that the differences between 12-5×IE034 and its parents were fewer than those between other maize varieties. Shikimate pathway-related genes and metabolites analysis results showed that the effects of hybrid stacking are less than those from transformation and differing genotypes. Thus, the differences due to breeding stack were fewer than those due to natural variation among maize varieties. This paper provides scientific data for assessing unintended effects in stacked transgenic plants.

Structural and Functional Analysis of a Bidirectional Promoter from Gossypium hirsutum in Arabidopsis

Stacked traits have become an important trend in the current development of genomically modified crops. The bidirectional promoter can not only prevent the co-suppression of multigene expression, but also increase the efficiency of the cultivation of transgenic plants with multigenes. In Gossypium hirsutum, Ghrack1 and Ghuhrf1 are head-to-head gene pairs located on chromosome D09. We cloned the 1429-bp intergenic region between the Ghrack1 and Ghuhrf1 genes from Gossypium hirsutum. The cloned DNA fragment GhZU had the characteristics of a bidirectional promoter, with 38.7% G+C content, three CpG islands and no TATA-box. Using gfp and gus as reporter genes, a series of expression vectors were constructed into young leaves of tobacco. The histochemical GUS (Beta-glucuronidase) assay and GFP (green fluorescence protein) detection results indicated that GhZU could drive the expression of the reporter genes gus and gfp simultaneously in both orientations. Furthermore, we transformed the expression vectors into Arabidopsis and found that GUS was concentrated at vigorous growth sites, such as the leaf tip, the base of the leaves and pod, and the stigma. GFP was also mainly expressed in the epidermis of young leaves. In summary, we determined that the intergenic region GhZU was an orientation-dependent bidirectional promoter, and this is the first report on the bidirectional promoter from Gossypium hirsutum. Our findings in this study are likely to enhance understanding on the regulatory mechanisms of plant bidirectional promoters.

Development of glyphosate-tolerant transgenic cotton plants harboring the G2-aroA gene

Abstract Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural production in China, the development of glyphosate-resistant cotton holds great promise. Using an Agrobacterium-mediated transformation method, a new G2-aroA gene that encodes 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was transformed into cotton cultivar K312. The transgenic cotton plants were regenerated from a callus tissue culture via kanamycin selection. Ten regenerated cotton plants were obtained and allowed to flower normally to produce fruit. The results from polymerase chain reaction (PCR) and Southern and Western blot analyses indicated that the target gene was integrated into the cotton chromosome and was expressed effectively at the protein level. The glyphosate tolerance analysis showed that the transgenic cotton had a high resistance to glyphosate. Further, even cotton treated with 45.0 mmol L−1 of glyphosate was able to slowly grow, bloom and seed. The transgenic cotton may be used for cotton breeding research of glyphosate-tolerant cotton.

Function Deletion Analysis of Light-Induced Gacab Promoter from Gossypium arboreum in Transgenic Tobacco: Function Deletion Analysis of Light-Induced Gacab Promoter from Gossypium arboreum in Transgenic Tobacco

分离了金华中棉（Gossypiun arboreum var. jinhua）光诱导基因cab 5＇上游的调控序列1 009 bp，并对其功能进行了分析，证明获得的这一DNA片段具有驱动光诱导表达的功能。为了进一步分离具有最大转录活性的最小光诱导启动子，根据光诱导表达调控元件所在的位置，构建了Gacab P和197 bp、504 bp、779 bp的5＇端缺失体，并将这些缺失体分别与gus （uid A）基因融合，构建植物表达载体。用农杆菌介导法转化烟草，获得转基因烟草。GUS组织化学分析表明，转基因烟草的T1代种子在光下培养时，只有Gacab P驱动gus基因在转基因烟草的叶片表达，其他3个启动子驱动gus基因在转基因烟草的整个植株中均有表达；当转基因烟草的T1代种子在暗中萌发及培养时，Gacab P驱动gus基因在转基因烟草中无表达，其他3个启动子驱动gus基因在转基因烟草的整个植株中均有表达。GUS定量分析表明，–504 - –1 bp的启动子缺失体启动活性最高，比CaMV35S启动子高0.6倍。上述结果表明只有全长的Gacab启动子具有光诱导和绿色组织特异表达特性，且–504 - –1 bp的启动子缺失体启动活性最高。

Function Deletion Analysis of Light-induced Gacab Promoter from Gossypium arboreum in Transgenic Tobacco

The 1009 bp promoter sequence of a light-induced gene, cab, has been cloned from the Gossypium arboreum in an earlier study. This gene encodes the chlorophyll a/b binding protein, which belongs to a class of light-inducible proteins. For designing a minimal length light-inducible Gacab promoter, the full-length Gacab P and the 5′ truncations of 197, 504, and 779 bp in length were fused with gus (uid A) gene and ligated into plant expression vectors. All the constructs were transformed into Nicotiana tabacum variety NC89 using Agrobacterium-mediated transformation method. Seeds from the T1 generation of transgenic tobacco were germinated and grown in either light or dark condition. A GUS histochemical assay showed that only the full-length Gacab promoter was light inducible and expressed in a tissue-specific manner. Promoter activities were quantified by GUS quantity analysis, and the promoter fragment from −504 to −1 bp had the highest activity, which was 0.6-fold higher than that of the CaMV35S promoter.