Research on Frequency of Exogenous Gene Flow from Marber-free Insect-resistant Transgenic Rice to Conventional Rice Varieties

[Objective] This study aimed to investigate the frequency of exogenous gene flow to non-transgenic conventional rice cultivars and assess the potential risks of marker-free of insect-resistant transgenic rice to agricultural ecological environment. [Method] Insect-resistant transgenic rice variety HUAHUI No.1 was planted as the experimental material and surrounded by several non-transgenic conventional rice cultivars. F1 non-transgenic rice seeds were collected according to different distances and identified by using PCR technology, the frequency of exogenous gene flow from insect-resistant transgenic rice to non-transgenic conventional rice cultivars was counted and analyzed. [Result] The average frequency of exogenous Bt gene flow to P13381 and CHUNJIANG063 was 0. Transgene flow occurred to varying degrees from insect-resistant transgenic rice HUAHUI No.1 to several non-transgenic rice lines including HEX122-2, TIANXlANG, MINGHUI63 and Pl157, with the maximum average gene flow frequency of 0.875%. The frequency of gene flow was gradually reduced with the increase of distance, and the average transgene flow frequency de- creased to 0 in all the sampling points 7 m away from transgenic rice material. [Conclusion] This study revealed that the exogenous gene flow frequency of insect-re- sistant transgenic rice variety HUAHUI No.1 was very low, leading to very small risk to the eco-environment. Rational distribution in the field for physical isolation, keeping the appropriate distance and scientific farming arrangement to avoid the synchronization of flowering can effectively control the exogenous gene flow from transgenic rice and reduce he ecological risks caused by transgene escape.

The Effect of Dietary Supplementation with Phytase Transgenic Corn on Growth Performance,Phosphorus Utilization and Excretion in Growing Pigs (Sus scrofa)

Two experiments were conducted to investigate the effect of dietary supplementation with phytase transgenic corn （PTC） on growth performance,phosphorus （P） utilization and excretion in growing pigs.In Exp.1,180 pigs （Large White × Landrace,BW=37.7 kg） were randomly allotted to 4 treatments with 5 replicates of 9 pigs each in order to evaluate the effect of PTC supplementation in low-P diets on growth performance.Four corn soybean meal-based diets consisted of a positive control （PC） diet,a diet containing 500 units （U） of exogenous phytase kg-1 （EP） on the basis of low-P （inorganic P reduced by 0.05% from PC diet） and the low-P＋500 （PTC1） or 750 （PTC2） phytase U of PTC kg-1.In Exp.2,20 barrows （Large White×Landrace,BW=31 kg,4 treatments with 5 replicates of 1 pig each） were randomly selected to evaluate the effect of PTC in low-P diets on serum parameters and nutrient utilization.Diets in Exp.2 were similar to those in Exp.1 except that the EP group was replaced by a low-P diet without exogenous phytase supplementation as a negative control （NC） group.The results from Exp.1 showed that the average daily gain （ADG） in the PTC2 group was significantly higher （P〈0.05） than that in the EP group over all periods.On the other hand,the feed：gain （F：G） ratio of the EP group was significantly higher （P〈0.05） than that of the PTC2 group during 1-21 and 1-42 d,respectively.There were no differences in average daily feed intake （ADFI） among all treatments （P〉0.05）.The results from Exp.2 showed that the concentration of serum Ca in the NC group was the highest （P〈0.05）,while the concentration of serum P in the PTC2 group was the highest （P〈0.05） among all treatments.There was a significant decrease （P〈0.05） in the P apparent digestibility of the NC group compared with the other groups,and that of PTC2 group was the best.Furthermore,fecal P excretion was reduced （P〈0.05） from 1.80 g d-1 in the PC group to 1.28 g d-1 in the PTC2 group.In conclusion,dietary supplementation with PTC could reduce the application of inorganic P,decrease fecal P excretion,and improve the growth performance of growing pigs.

Field Evaluation of the Asian Corn Borer Control in Hybrid of Transgenic Maize Event MON 810

In this study, a transgenic Bt maize hybrid (event MON 810 from Monsanto Company) expressing Cry1Ab protein derived from Bacillus thuringiensis (Bt) and its negative isoline hybrid were evaluated for control of the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), in a field trial. Maize plants were artificially infested with neonate larvae of Asian corn borer at the mid-whorl (first-generation), pre-tassel (first- and/or second-generation), and silk (second-generation) growth stages. The transgenic Bt maize hybrid sustained significantly less leaf feeding damage (rating 1.0±0.0) than its negative isoline control (rating 7.3±0.1). With the Bt maize, 1.36.8% of plants were damaged by corn borer tunneling with <0.5 cm tunneling per stalk under different levels of infestation, compared with 100% of plants damaged with 9.325.0 cm tunneling per stalk for the negative isoline control. On average, transgenic Bt maize hybrids had only 0.010.05 tunnels per stalk and no stems were broken. In contrast, the negative isoline control had 3.118.36 tunnels per stalk and 31.273.9% of stems broken. Yields were significantly higher in transgenic Bt maize than in the control. These results demonstrate that transgenic Bt maize can significantly minimize yield losses caused by the Asian corn borer through resistance to the first- and second-generation larvae.

Effect of Transgenic Corn Cultivation and Sampling Location on Feeding Habits of Collembola

Transgenic corn was modified to combat lepidopteran pests, herbicide and other deteriorate environmental factors. However, fewer studies concerned the effects of transgenic corn cultivation on the soil Collembola in black soil region in China. In Jilin black soil region, a six treatments study including three corn varieties （transgenic corn, control and local variety） and two sampling locations （in rizhosphere and in between plants） were deployed to study the effect of transgenic corn cultivation and sampling location on densities, species richness and community structure of Collembola. We found that root biomass was marginally decreased in transgenic corn and control variety when compared to local variety. Collembola significantly affected by sampling location treatments. Densities and species richness of collembolans significantly decreased in ＂between plants＂ than in ＂rizhosphere＂. Densities of Collembola were impacted by corn varieties. In the whole growing season, densities of Collembola significantly decreased in control than in local variety. Collembolans in transgenic variety only slightly decreased in ＂jointing stage＂ than local variety. Collembolans adversely affected in ＂between plants＂ when compared to ＂in rhizosphere＂ in ＂seeding stage＂ but not other stages. These variations of collembolans were in lines with the trend of corn root biomass. It is indicated that Collembola variations could be caused by food resources （root biomass） but not corn varieties （transgenic, control and local）.

Evaluation of Impact of Pollen Grains from Bt,Bt/CpTITransgenic Cotton and Bt Corn Plants on the Growth andDevelopment of the Mulberry Silkworm,Bombyx moriLinnaeus (Lepidoptera:Bombycidae)

The S-endotoxin genes of Bacillus thuringiensis (Bt) and proteinase inhibitor (PI) genes are two kinds of genes popularly used for developing transgenic plants resistant to insect pests. To clarify whether there is any risk concerning the effects of pollens from these transgenic crops on non-target insects with economic importance, such as the effects on the growth and development as well as cocoon quality of the silkworm, Bombyx mori Linnaeus, a series of feeding experiments were conducted, using pollens from transgenic cotton or corn containing cry 1Ac, cry1A+CpTI or crylAb genes, compared with pollens from non-transgenic normal cotton and corn as well as the non-pollen treatment. In contrast to the latter ones, pollens from transgenic plants showed no significant adverse effects on larval mortality, cocoon weight, pupa weight, cocoon shell weight, pupation rate, emergence rate and fecundity of the silkworm after neonates were fed with the pollens for 72 h. In addition, no dosage effects of pollens were found. Though the duration of 1st instar larvae was prolonged in the case of feeding with transgenic pollens as compared with those of the non-pollen treatment , but they were not significantly different from those fed with pollens from non-transgenic cotton or corn. Meanwhile, the body weight of the 3rd instar molters fed with transgenic pollens was obviously different from those for non-pollen treatment, and was all significantly heavier than that of the controls. Consequently, it is considered that the adverse effect of pollens from transgenic insect-resistant cotton and corn on the growth and development of the silkworm is negligible.

Temporal and spatial changes in Bt toxin expression in Bt-transgenic poplar and insect resistance in field tests

Extensive planting of Bacillus thuringiensis （Bt）-transgenic plants economically benefits society; how-ever, the potential risk they pose is receiving increasing attention. This study used enzyme-linked immunosorbent assay and fluorescence quantitative PCR （RT-PCR） to monitor the temporal and spatial dynamics of the expression of Bt toxic protein in a forest of 6- to 8-year-old trees of transgenic insect-resistant poplar 741 for three consec- utive years. The enrichment, distribution, and degradation of Bt toxic protein and the influence of transgenic poplars on the targeted insect population, Hyphantria cunea, were investigated. The content of CrylAc toxic protein dynamically changed in transgenic poplar. During the annual growth cycle, the content initially increased, then decreased in the long and the short branches of the crown and in the root system, peaking in August. During the study, the protein did not accumulate overtime. The mRNA transcription of gene CrylAc was almost consistent with the level of the protein, but transcription peaked in July. In the transgenic and control forestland, microscale levels of the CrylAc toxic protein were detected from the soil, but increased accumulation was not observed with the planting year of transgenic poplar. Meanwhile, Bt was isolated and detected molecularly from the soil in the experimental forestland. A systematic investigation of the density of H. cunea in the experimental transgenic poplar forest indi- cated that transgenic Pb29 poplar could resist insects to a certain degree. At peak occurrence of the targeted insects, the density of H. cunea in the experimental forest was significantly lower than in the nontransgenic poplar forest.

生产加工过程食用玉米淀粉基因降解情况

含有转基因成分的玉米在生产加工过程中核酸成分受到不同程度破坏,增加出口过程中转基因成分检验难度。针对转基因食用玉米淀粉在加工过程中主要环节进行样本收集,使用荧光定量聚合酶链式反应(polymerase chain reaction,PCR)对加工过程中样品内、外源基因不同扩增片段进行定量研究。研究发现,浸泡后的浆料湿磨及精磨分离阶段的样品DNA降解严重。随着检测转基因成分方法的不断创新和提高及数字PCR在检测领域的开发和应用,利用数字PCR方法检验痕量转基因成分,不仅可以定性检测,并且可以对转基因成分含量进行定量检测。

Performance of Hybrids between Weedy Rice and Insect-resistant Transgenic Rice under Field Experiments: Implication for Environmental Biosafety Assessment

Transgene escape from genetically modified （GM） rice into weedy rice via gene flow may cause undesired environmental consequences. Estimating the field performance of crop-weed hybrids will facilitate our understanding of potential introgression of crop genes （including transgenes） into weedy rice populations, allowing for effective biosafety assessment. Comparative studies of three weedy rice strains and their hybrids with two GM rice lines containing different insect- resistance transgenes （CpTI or Bt/CpTI） indicated an enhanced relative performance of the crop-weed hybrids, with taller plants, more tillers, panicles, and spikelets per plant, as well as higher 1000-seed weight, compared with the weedy rice parents, although the hybrids produced less filled seeds per plant than their weedy parents. Seeds from the F1 hybrids had higher germination rates and produced more seedlings than the weedy parents, which correlated positively with 1000-seed weight. The crop-weed hybrids demonstrated a generally enhanced relative performance than their weedy rice parents in our field experiments. These findings indicate that transgenes from GM rice can persist to and introgress into weedy rice populations through recurrent crop-to-weed gene flow with the aid of slightly increased relative fitness in F1 hybrids.

Expression of two insect-resistant genes cryIA (b&c)/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesized Bacillus thuringiensis insecticidal protein gene crylA(b&c) and the synthesized gene GNA, (the mannose specific lectin from snowdrop ( Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes of Nico-tiana tabacum var. K326 have been transformed with Agrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants

Commercial production of transgenic Bt insect-resistant cotton varieties and the resistance management for bollworm (Helicoverpa armigera Hubner)

There are currently three kinds of transgenic Bt insect-resistant cotton germplasm lines, Shanxi 94-24, Zhongxin 94 and R19, in China. They showed high resistance to the neonate larvae of bollworm (Helicoverpa armigera). Transgenic Bt insect-resistant cotton varieties or hybrids have been bred using the three kinds of germplasm lines as parents. Our researches reveal that there exist different expressions in resistant level at different developmental stages in the three categories of germplasm lines. When neonate larvae are fed with leaves of cotton plant at the seeding stage with less than 10 leaves on the main stem, the mortality of the neonate larvae is 100%, but the resistance level will decline at later season. When Bt gene has been transferred to the cotton genome, it can be steadily transferred to the progeny, the level of resistance to bollworm keeps fundamentally uniform. Such insects as tobacco budworm (Heliothis virencens) in laboratory directive selection are very apt to produce resistance

Integration and inheritance stability of foreign Bt toxin gene in the bivalent insect-resistant transgenic cotton plants

Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R3, R4 and R5 generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of R3, R4 and R5 generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.

Safety Evaluation of Stacked Genetically Modified Corn Event (MON89034 ×MON88017) Using Zebrafish as an Animal Model

This study assessed the effect of feeding stacked genetically modified (GM) corn event (MON89034 × MON88017) on embryonic development, adult tissue histology and assessed the possibility of transgenic DNA transfer, using zebrafish as an animal model. Adult zebrafish were separated into male and female groups. After 3 weeks of feeding with experimental diets, males and females were paired once a week and fertilized embryos were collected, then the male and female fish were re-turned to their sex-specific groups. This fertilization and embryo collection process were repeated 3 times. Embryonic morphological measurements were obtained on developing embryos up to 96 hours after fertilization, and then the embryos were fixed in 4% phosphate-buffered paraformal-dehyde for morphologic assessment. At the end of the feeding study, various adult zebrafish tissues were histologically examined for abnormalities. Both zebrafish tissues and embryos were analyzed for presence of plant specific genes and transgenic sequences. No changes or abnormalities were observed in embryonic morphology nor in any of the tissues examined histologically. In addition, no plant DNA reference genes or transgenic DNA were found in any of the analyzed samples. These results demonstrate that the safety and nutrition of MON89034 × MON88017 corn are similar to non-GM corn.

Identification and Quantification of Corn, Soybean and Cotton Genetically Modified by Real Time PCR

In order to obtain a cheaper method for quantification of transgenic events in corn, soybeans and cotton, primers for real time PCR have been developed and optimized, with fluorescent BRYT Green system. The DNA was extracted from grains, with and without event, by CTAB method. The following events have been studied for corn: MON810, Bt11, MON89034, GA21, TC1507, NK603, MIR162, PRO3;Soybean: GTS-40-3-2, MON87701;MON89788;for cotton: MON1445, MON531, LLCotton25, 281-24-236;3006-210-23, GHB614, T304-40;GHB119, MON15985, MON88913, besides the respective primers for the endogenous genes of corn, soybean and cotton. The sensitivity was 0.057%, the coefficient of linearity R2 ranged from 0.98 to 0.99 and the efficiency of PCR 0.9 to 1.1. The quantification of events ranged from 92 to 115, with a relative error (RE) from 2 to 18%, and a variance of 0.33 to 3.0. The precision acceptance criterion was observed for all analyses, as well the repeatability and reproducibility. As it was found that the measurement of accuracy and reproducibility were within the international acceptance criterion, it may infer the robustness of the methodology. Therefore, the results from replicates with two different technicians, and validation of results by comparison with those obtained by Eurofins Brazil, showed the possibility of specific and quantitative analysis of transgenic events with a cheaper method with sensitivity, repeatability and robustness.

Interaction Between Heficoverpa zea Damage with Corncob Diseases on Genetically Modified Corn in Sinaloa, México

GM （genetically modified） corn with the CrylAb, mCry3A and Vip3Aa20 protein of Bt （Bacillus thuringiensis） and its respective isoline as control with and without chemical control for corn earworm were used in this research. Fusarium corncob damage was lower on GM corn with （12.5%） and without （25.7%） insecticide treatment as compared with the isoline that had 48.3% and 83.1% of damaged corncobs with and without chemical control, respectively. Corn smut cob damage was also lower on GM corn with （3.2%） and without （6.3%） insect control compared with 15.5% and 49.7% damage with and without insecticide treatment, respectively. Fusarium sp. corncob rot was also lower on GM corn with 5.7% and 9.5% whereas a 24.6% and 63% rot was observed on the isoline with and without insecticide control, respectively. Ustilago maydis severity was also lower on Bt corn finding 0.07% and 0.25% damage on treatments with and without insect control as compared with the isoline that showed an 11.6% and a 41.4% smut rot with and without insecticide treatment, respectively. The authors conclude that GM corn resistant to Helicoverpa zea prevents damage by the pest, eliminating the entrance pathway for Fusarium sp. and Ustilago maydis.

Cotton Plants Transformed with the Activated Chimeric Cry1Ac and API-B Genes

A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.

转抗虫基因棉花和玉米花粉对家蚕生长发育影响的评价

苏云金杆菌 (Bacillusthuringiensis,Bt)杀虫蛋白基因和蛋白酶抑制剂基因是培育转抗虫基因植物的两大类基因。为了正确评价转抗虫基因作物花粉对非靶标重要经济昆虫的安全性 ,本试验以家蚕为对象 ,研究转cry1Ac基因棉花、转cry1A +CpTI双价抗虫棉花以及转cry1Ab基因玉米花粉对家蚕生长发育的影响。结果表明 ,与非转基因常规棉花或玉米花粉以及无花粉对照相比 ,转抗虫基因棉花或转抗虫基因玉米花粉对家蚕各龄期的死亡率、蛹重、茧重、茧层重、化蛹率、羽化率和产卵量均无多大影响 ,且无明显的剂量效应。虽然取食转抗虫基因花粉后家蚕一龄历期有所延长 ,但与非转基因的花粉处理相比 ,则无显著差异 ;同样 ,家蚕三龄眠蚕体重虽与无花粉对照相比表现出较大差异 ,但其体重均大于对照。可见 ,转抗虫基因棉花和玉米花粉对家蚕的历期和体重亦无多大影响。综上所述 ,转抗虫基因棉花和玉米花粉对家蚕的生长发育不会产生明显的负面影响。

转Bt基因玉米幼苗残体中Cry1Ab杀虫蛋白田间降解动态

【目的】研究间苗后留在田间地表的转Bt基因玉米幼苗残体中Cry1Ab杀虫蛋白的降解规律,比较两种Bt玉米幼苗残体中Cry1Ab杀虫蛋白的降解速度。【方法】以两种表达Cry1Ab杀虫蛋白的转Bt基因抗虫玉米MON810和Bt11为材料,采用ELISA方法测定各取样时期中幼苗残体中Cry1Ab杀虫蛋白残留量。【结果】转Bt基因玉米幼苗残体中杀虫蛋白降解是逐渐的,且降解速度较快,到50d时幼苗残体已经完全腐烂,Bt11幼苗残体中的杀虫蛋白已经完全降解,在MON810中还能检测到微量的杀虫蛋白。两种转基因玉米幼苗残体中的Bt杀虫蛋白的初始含量差异不显著,但在同一时间段的Bt杀虫蛋白降解速度存在差异均显著,在30d前MON810幼苗残体中Bt杀虫蛋白降解速度比Bt11降解的快,30d后,则降解趋势相反,到50d取样结束时MON810和Bt11分别降解了初始含量的99.81%和100%。【结论】两种转Bt基因玉米间苗后留在田间的幼苗残体中的Cry1Ab杀虫蛋白降解速度不同,在50d完全腐烂时,其中的杀虫蛋白完全降解或仅有微量残留。

转基因玉米外源基因通过花粉漂移的频率和距离

基因漂移是转基因生物可能引起生态环境安全性问题的主要风险之一,选用美国孟山都公司的转基因抗除草剂玉米GA21进行了外源基因向周边环境遗传漂移的距离和频率的研究,结果表明:转基因玉米的外源基因可以向周边玉米品种进行漂移,其最大漂移频率为45.10%,150m处仍能检测到外源基因的漂移,防止转基因玉米基因向外扩散的最佳途径为设置隔离带,隔离距离以200m以上为好。

转Bt基因抗虫玉米对亚洲玉米螟幼虫几种主要酶系活性的影响

研究了表达Cry1Ab杀虫蛋白的转Bt基因抗虫玉米对亚洲玉米螟Ostriniafurnacalis(Guen啨e)幼虫解毒酶、保护酶和中肠蛋白酶活性的影响,测定比较了取食转Bt基因玉米后幼虫体内α乙酸萘酯酶、乙酰胆碱酯酶、谷胱甘肽S转移酶、过氧化氢酶、超氧化物歧化酶、中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶的活力。结果表明,取食转Bt基因玉米48h后亚洲玉米螟幼虫体内的α乙酸萘酯酶、谷胱甘肽S转移酶活力明显低于对照;而乙酰胆碱酯酶活力显著高于对照,在取食48h、60h和72h的活力分别是对照的2.00、1.50和2.50倍。保护酶系、中肠总蛋白酶、弱碱性类胰蛋白酶和类胰凝乳蛋白酶的活性在取食48h后明显受到抑制;但强碱性类胰蛋白酶的活性显著高于对照,取食48h、60h和72h的活力分别是对照的4.00、1.67和1.33倍。乙酰胆碱酯酶和强碱性类胰蛋白酶可能与亚洲玉米螟对Bt的抗性有关。

转基因抗虫玉米Bt蛋白表达量的研究

应用酶联免疫吸附测定法(ELISA)定量检测转基因抗虫玉米不同生长期不同部位的Bt蛋白表达量。结果显示:Bt毒蛋白在玉米乳熟末期的第15叶中表达量(0.225%)最高,在花粉中表达量(0.011%)最低。叶、茎、髓、花丝、穗轴及粒的Bt蛋白含量及比例随着作物的生长呈上升的趋势。Bt毒蛋白最低检出量为0.5ng/mL。