外源基因导入对玉米叶片物理性状的影响

【目的】探讨外源基因导入对玉米叶片物理性状的影响,为转基因玉米的安全性评价提供基础资料,也为转基因玉米的科学、有效利用提供依据。【方法】在扬州大学实验农牧场种植大北农转基因(转Cry Ab和epsps基因)和大北农(对照)、IE09S034转基因(转Cry IE基因)和IE09S034(对照)、808-双抗-12-5转基因(转Cry Ab/cry2Aj和Gloevo-epsps基因)和808瑞丰-1(对照)3对玉米品种,室内测定了不同时期(苗期、穂期和花粒期)各品种叶片的蜡质含量、叶绿素含量、茸毛密度、维管束埋深及Si、K、Ca、S、P和Cl含量。【结果】转基因玉米的叶片中蜡质含量、叶绿素含量和维管束埋深较对应的常规亲本品种大,而叶片茸毛密度则较对应的常规亲本品种小。其中,穗期的大北农、IE09S034和808-双抗-12-5转基因品种叶片蜡质含量分别较对照高17.95%、48.30%和39.31%;IE09S034和808-双抗-12-5转基因品种穗期叶片的维管束埋深分别较对照高13.70%和9.21%,花粒期分别高10.81%和14.47%;IE09S034和808-双抗-12-5转基因品种穗期叶片的叶绿素含量分别较对照高18.11%和13.13%,花粒期分别高16.62%和14.61%;大北农、IE09S034和808-双抗-12-5转基因品种花粒期叶片茸毛密度分别较对照低17.70%、17.43%和17.78%。3个品种的转基因玉米叶表面元素含量均大于相应的对照。其中,与常规亲本相比,穗期大北农转基因品种叶片中Ca和S含量分别高64.71%和61.18%,IE09S034转基因品种叶片中Si、Ca、S、P和Cl含量分别高110.26%、16.67%、44.44%、46.32%和20.00%,808-双抗-12-5转基因品种叶片中Si、Ca、S和P含量分别高34.78%、50.52%、115.47%和20.41%。【结论】外源基因的导入会诱导玉米叶片相关物理性状的改变。     

Dissection of genotype-phenotype associations in rice grains using metabolome quantitative trait loci analysis

A comprehensive and large‐scale metabolome quantitative trait loci (mQTL) analysis was performed to investigate the genetic backgrounds associated with metabolic phenotypes in rice grains. The metabolome dataset consisted of 759 metabolite signals obtained from the grains of 85 lines of rice (Oryza sativa, Sasanishiki × Habataki back‐crossed inbred lines). Metabolome analysis was performed using four mass spectrometry pipelines to enhance detection of different classes of metabolites. This mQTL analysis of a wide range of metabolites highlighted an uneven distribution of 802 mQTLs on the rice genome, as well as different modes of metabolic trait (m‐trait) control among various types of metabolites. The levels of most metabolites within rice grains were highly sensitive to environmental factors, but only weakly associated with mQTLs. Coordinated control was observed for several groups of metabolites, such as amino acids linked to the mQTL hotspot on chromosome 3. For flavonoids, m‐trait variation among the experimental lines was tightly governed by genetic factors that alter the glycosylation of flavones. Many loci affecting levels of metabolites were detected by QTL analysis, and plausible gene candidates were evaluated by in silico analysis. Several mQTLs profoundly influenced metabolite levels, providing insight into the control of rice metabolism. The genomic region and genes potentially responsible for the biosynthesis of apigenin‐6,8‐di‐C‐α‐l‐ arabinoside are presented as an example of a critical mQTL identified by the analysis.     

Impacts of Bt maize inoculated with rhizobacteria on development and food utilization of Mythimna separata

The current trend of increasing proportion of cultivation of transgenic Bt crops is pushing towards dramatic destabilization of the agroecosystem, thus raising severe concerns about the sustainability of transgenic Bt crops as an effective management tool for the control of target insect pests in the future. Rhizobacteria is the key biological regulator to ameliorate soil‐nitrogen utilization efficiency of crop plants, especially transgenic Bt crops. A laboratory study quantified the impacts of transgenic Bt maize (Line IE09S034 with Cry1Ie vs. non‐Bt maize cv. Xianyu335) inoculated with Azospirillum brasilense (AB) and Azotobacter chroococcum (AC) on the growth, development and food utilization of a target lepidopteran insect, Mythimna separata. The results showed that the inoculation of rhizobacteria significantly prolonged the larval lifespan and pupal duration, increased RCR and AD, reduced pupal weight, pupation rate, fecundity, RGR, ECD and ECI, and shortened adult longevity of M. separata fed on transgenic Bt maize, while exact opposite trends were found in these measured indexes of growth, development and food utilization for M. separata fed on non‐Bt maize inoculated with AB and AC compared with the buffer control in both years. Thus, the results clearly depicted that the inoculation of rhizobacteria had opposite influences on the growth, development and food utilization of M. separata fed on transgenic Bt maize. Presumably, rhizobacteria inoculation can be used to stimulate plant–soil‐nitrogen uptake and promote plant growth for transgenic Bt maize and non‐Bt maize, simultaneously increasing Bt toxin production and enhancing resistance efficiency against target lepidopteran pests for transgenic Bt maize.     

Persistence of transgenes in wild rice populations depends on the interaction between genetic background of recipients and environmental conditions

The persistence of transgenes in wild populations may cause unintended ecological consequences, and the possibility of transgenes' persistence and introgression is dependent on fitness performance of transgenic crop–wild hybrids. To investigate the effects of transgene and genotype × environment on the fitness of crop–wild rice hybrids, a total of 11 cross‐combination progenies between insect‐resistant transgene (CpTI and Bt/CpTI) rice lines and wild rice (Oryza rufipogon) were evaluated at different sites with contrasting insect treatments. The results showed that fitness performance varied between transgenic hybrids having different wild parents and under different environmental conditions, indicating that fitness effects of transgenes on hybrid progenies depend heavily on the genetic background of recipient plants and growing environment. Significant fitness advantages conferred by transgenes were found only in some hybrids under high insect pressure condition, demonstrating that the level of target insects in the field environment influences the persistence and spread of insect‐resistant transgenes in wild rice populations. These findings suggest that evolutionary fate of escaped transgenes is different in wild populations with diverse genetic backgrounds under various environmental conditions.     

转Bt基因抗虫作物培育现状及Bt蛋白的改造和聚合策略的利用

Bt基因是目前世界上应用最为广泛的抗虫基因,已经被转入多种作物中。其中,棉花、玉米、马铃薯等转Bt基因抗虫作物已经商品化生产,创造了可观的经济效益。结合作者的资料收集和研究结果,综述了Bt基因以及转Bt基因抗虫作物的培育现状,同时对提高Bt蛋白杀虫活力的方法和Bt基因聚合策略的利用进行了深入的探讨。     

Using Metabolomics To Estimate Unintended Effects in Transgenic Crop Plants: Problems, Promises, and Opportunities

Transgenic crops are widespread in some countries and sectors of the agro-economy, but are also highly contentious. Proponents of transgenic crop improvement often cite the “substantial equivalence” of transgenic crops to the their nontransgenic parents and sibling varieties. Opponents of transgenic crop improvement dismiss the substantial equivalence standard as being without statistical basis and emphasize the possible unintended effects to food quality and composition due to genetic transformation. Systems biology approaches should help consumers, regulators, and other stakeholders make better decisions regarding transgenic crop improvement by characterizing the composition of conventional and transgenically improved crop species and products. In particular, metabolomic profiling via mass spectrometry and nuclear magnetic resonance can make broad and deep assessments of food quality and content. The metabolome observed in a transgenic variety can then be assessed relative to the consumer and regulator accepted phenotypic range observed among conventional varieties. I briefly discuss both targeted (closed architecture) and nontargeted (open architecture) metabolomics with respect to the transgenic crop debate and highlight several challenges to the field. While most experimental examples come from tomato (Solanum lycoperiscum), analytical methods from all of systems biology are discussed.     

The influence of planting date,transgenic Bt maize and hybrid relative maturity on European corn borer Ostrinia nubilalis (Lepidoptera: Crambidae) ovipositional patterns

A three year study was carried out at Hoytville and at Wooster, Ohio, USA from 2006 to 2008 to investigate the influence of planting date, transgenic maize and hybrid maturity on Ostrinia nubilalis (Hubner) population dynamics and oviposition patterns. Maize plants were planted in late April or early May, mid‐May and early June during each year. The moth flight pattern showed bivoltine generations during the three years. The first moth flight peaked in June, with the populations declining during July. The second moth flight peaked in August and declined towards the end of September or early October. Egg mass density did not differ significantly between transgenic and non‐transgenic maize of different maturities. Significant differences were observed, however, among planting dates, sampling dates, and sampling date × planting date interactions. Generally higher numbers of egg masses from second generation moths were deposited on late planted maize than middle and early plantings.     

Freestanding stacked mesh‐like hydrogel sheets enable the creation of complex macroscale cellular scaffolds

Hydrogel‐based bottom‐up tissue engineering depends on assembly of cell‐laden modules for complex three‐dimensional tissue reconstruction. Though sheet‐like hydrogel modules enable rapid and controllable assembly, they have limitations in generating spatial microenvironments and mass transport. Here, we describe a simple method for forming large‐scale cell‐hydrogel assemblies via stacking cell‐embedded mesh‐like hydrogel sheets to create complex macroscale cellular scaffolds. Freestanding stacked hydrogel sheets were fabricated for long‐term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets (8.0 mm × 8.7 mm) were aligned using a polydimethylsiloxane drainage well. The stacked hydrogel sheets were precisely aligned so that the openings could facilitate mass transport through the stacked sheets. Despite the relatively large height of the stacked structure (400–700 μm), which is larger than the diffusion limit thickness of 150–200 μm, the freestanding cell‐ydrogel assemblies maintained cell viability and exhibited enhanced cellular function compared with single hydrogel sheets. Furthermore, a three‐dimensional co‐culture system was constructed simply by stacking different cell‐containing hydrogel sheets. These results show that stacked hydrogel sheets have significant potential as a macroscale cell‐culture and assay platform with complex microenvironments for biologically relevant in vitro tissue‐level drug assays and physiological studies.     

Stacking three late blight resistance genes from wild species directly into African highland potato varieties confers complete field resistance to local blight races

Considered responsible for one million deaths in Ireland and widespread famine in the European continent during the 1840s, late blight, caused by Phytophthora infestans, remains the most devastating disease of potato (Solanum tuberosum L.) with about 15%–30% annual yield loss in sub‐Saharan Africa, affecting mainly smallholder farmers. We show here that the transfer of three resistance (R) genes from wild relatives [RB, Rpi‐blb2 from Solanum bulbocastanum and Rpi‐vnt1.1 from S. venturii] into potato provided complete resistance in the field over several seasons. We observed that the stacking of the three R genes produced a high frequency of transgenic events with resistance to late blight. In the field, 13 resistant transgenic events with the 3R‐gene stack from the potato varieties ‘Desiree’ and ‘Victoria’ grew normally without showing pathogen damage and without any fungicide spray, whereas their non‐transgenic equivalent varieties were rapidly killed. Characteristics of the local pathogen population suggest that the resistance to late blight may be long‐lasting because it has low diversity, and essentially consists of the single lineage, 2_A1, which expresses the cognate avirulence effector genes. Yields of two transgenic events from ‘Desiree’ and ‘Victoria’ grown without fungicide to reflect small‐scale farm holders were estimated to be 29 and 45 t/ha respectively. This represents a three to four‐fold increase over the national average. Thus, these late blight resistant potato varieties, which are the farmers’ preferred varieties, could be rapidly adopted and bring significant income to smallholder farmers in sub‐Saharan Africa.     

Impact of genetic structures on haploid genome-based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications

Real-time Polymerase Chain Reaction (PCR) based assays are widely used to estimate the content of genetically modified (GM) materials in food, feed and seed. It has been known that the genetic structures of the analyte can significantly influence the GM content expressed by the haploid genome (HG) % estimated using real-time PCR assays; this kind of influence is also understood as the impact of biological factors. The influence was first simulated at theoretical level using maize as a model. We then experimentally assessed the impact of biological factors on quantitative results, analysing by quantitative real-time PCR six maize MON 810 hybrid kernels with different genetic structures: (1) hemizygous from transgenic male parent, (2) hemizygous from transgenic female parent and (3) homozygous at the transgenic locus. The results obtained in the present study showed clear influences of biological factors on GM DNA quantification: 1% of GM materials by weight (wt) for the three genetic structures contained 0.39, 0.55 and 1.0% of GM DNA by HG respectively, from quantitative real-time PCR analyses. The relationships between GM wt% and GM HG% can be empirically established as: (1) in the case of the presence of a single GM trait: GM HG% = GM wt% × (0.5 ± 0.167Y), where Y is the endosperm DNA content (%) in the total DNA of a maize kernel, (2) in the case of the presence of multiple GM traits: GM HG% = N × GM wt% × (0.5 ± 0.167Y), where N is the number of GM traits (stacked or not) present in an unknown sample. This finding can be used by stakeholders related to GMO for empirical prediction from one unit of expression to another in the monitoring of seed and grain production chains. Practical equations have also been suggested for haploid copy number calculations, using hemizygous GM materials for calibration curves.     

RNA‐directed DNA methylation and seed development: an unexpected difference between Arabidopsis thaliana and Brassica rapa

Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer‐DNA (T‐DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site‐specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5‐kbp T‐DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T‐DNA left border, and only 8% contained sequence from outside the T‐DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high‐quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering.     

The examinations of grain yield and yield components in new white maize varieties using line × tester analysis method

Line × tester set was obtained by crossing 10 inbred lines with 2 testers in maize. 20 F1`s along with 12 parents (10 S6 lines and 2 testers) and two standard white checks (TWC321 and TWC324) were evaluated randomized complete block Design (RCBD) with three replications during summer season 2016 to detect the best new white crosses for the potential comercial crosses. Data were recorded on cob length, ear diameter row no. per ear, no. of kernels per row, 100-kernel weight and grain yield. The results of mean squares showed highly significant differences in the all examined traits. For mean grain yield of the crosses P5 × SC24, P6 × SC21, P6 × SC24, P7 × SC21, P7 × SC24 and P10 × SC21 had significant in all traits and their combined data was higher in case of TWC321 and TWC324.Hence, it could be concluded that these crosses may be useful for improving grain yield of maize. Crosses P1 × SC21, P1 × SC24, P3 × SC24, P5 × SC24, P6 × SC21, P6 × SC24, P7 × SC21 and P7 × SC24 had highest significant and positively higher percentages over check varieties TWC321 and TWC324 for grain yield and some of yield components. These lines were having promising performance which could be used in future maize breeding programs for hybrid production. Based on the overall performance of the hybrids and parental lines, some of the lines could be used as parents of hybrids of maize with high grain yield potential. Thus, these crosses could be commercially exploited after critical evaluation for its superiority in performance as maize hybrids.     

Stability of the MON 810 transgene in maize

We analysed the DNA variability of the transgene insert and its flanking regions in maize MON 810 commercial varieties. Southern analysis demonstrates that breeding, since the initial transformation event more than 10 years ago, has not resulted in any rearrangements. A detailed analysis on the DNA variability at the nucleotide level, using DNA mismatch endonuclease assays, showed the lack of polymorphisms in the transgene insert. We conclude that the mutation rate of the transgene is not significantly different from that observed in the maize endogenous genes. Six SNPs were observed in the 5′flanking region, corresponding to a Zeon1 retrotransposon long terminal repeat. All six SNPs are more than 500 bp upstream of the point of insertion of the transgene and do not affect the reliability of the established PCR-based transgene detection and quantification methods. The mutation rate of the flanking region is similar to that expected for a maize repetitive sequence. We detected low levels of cytosine methylation in leaves of different transgenic varieties, with no significant differences on comparing different transgenic varieties, and minor differences in cytosine methylation when comparing leaves at different developmental stages. There was also a reduction in cryIAb mRNA accumulation during leaf development.     

Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

Effects of transgenic <Emphasis Type="Italic">cry1Ie</Emphasis> maize on non-lepidopteran pest abundance,diversity and community composition

Non-lepidopteran pests are exposed to, and may be influenced by, Bt toxins when feeding on Bt maize that express insecticidal Cry proteins derived from Bacillus thuringiensis (Bt). In order to assess the potential effects of transgenic cry1Ie maize on non-lepidopteran pest species and ecological communities, a 2-year field study was conducted to compare the non-lepidopteran pest abundance, diversity and community composition between transgenic cry1Ie maize (Event IE09S034, Bt maize) and its near isoline (Zong 31, non-Bt maize) by whole plant inspections. Results showed that Bt maize had no effects on non-lepidopteran pest abundance and diversity (Shannon–Wiener diversity index, Simpson’s diversity index, species richness, and Pielou’s index). There was a significant effect of year and sampling time on those indices analyzed. Redundancy analysis indicated maize type, sampling time and year totally explained 20.43 % of the variance in the non-lepidopteran pest community composition, but no association was presented between maize type (Bt maize and non-Bt maize) and the variance. Nonmetric multidimensional scaling analysis showed that sampling time and year, rather than maize type had close relationship with the non-lepidopteran pest community composition. These results corroborated the hypothesis that, at least in the short-term, the transgenic cry1Ie maize had negligible effects on the non-lepidopteran pest abundance, diversity and community composition.