Effect of Transgenic Bt plus CpTI Cotton on Carboxylesterase and Acetylcholinesterase of Cotton Aphid Aphis gossypii

[Objective] The research aimed to assess the effect of transgenic Bt plus CpTI cotton variety SGK321 on carboxylesterase and acetylcholinesterase of cotton aphid Aphis gossypii and provide theoretical basis for studying the biosafety of transgenic cotton.[Method] Cotton aphids were fed with SGK321 and Shiyuan321（normal parental varieties） for over 40 generations.Enzyme activities were compared between cotton aphids feeding on SGK321 for 1,2,3,41,42 and 43 generations with those on Shiyuan321.[Result] The carboxylesterase activity of cotton aphids feeding on SGK321 for 1 generation was significantly higher than those feeding on Shiyuan321.Acetylcholinesterase activity of cotton aphids feeding on SGK321 for 1,2 and 3 generations were significantly higher than those feeding on Shiyuan321 in the same generation.But there was no significant difference of enzyme activity between cotton aphids feeding on SGK321 for a long term and those feeding on parental cotton.[Conclusion] The cotton aphid that feeding on transgenic Bt plus CpTI cotton SGK321 for a long time has adaptivity to SGK321 by regulating the detoxifying enzyme.

Effects of Transgenic Bt+CpTI Cotton Cultivation on Functional Diversity of Microbial Communities in Rhizosphere Soils

[Objective] This study aimed to investigation the effects of tranagenic Bt ＋ CpTI cotton cultivation on functional diversity of microbial communities in rhizospbere soils. E Method] By using the Biolog method, a comparative study was conducted on the utilization level of single carbon source by microbes in the rhi- zosphere soils of transgenic Bt ＋ CpTI cotton sGK321 and its parental conventional cotton ＇ Shiyuan 321＇ at different growth stages. [ Result ] The results showed that, compared with the parental conventional cotton, the average well-color development （AWCD） value of micmhial communities in rhizospbere soils of transgenie Bt ＋ CpTI cotton were significantly higher （P 〈 O. 05） at seedling stage and budding stage while significantly lower at flower and boll stage and bell opening stage. Shannon-Wiener diversity index （H） and Simpson dominance index （D） of microbial communities in rhlzesphere soils of transgenic cotton and conventional cotton varied with the different growth stages, whereas the Shannon-Wiener evenness index （E） showed no significant difference between transgenie cotton and convention- al cotton at four growth stages. Principal component analysis indicated that the patterns of carbon source utilization by microbial communities in rhizospbere soils were similar among transgenic cotton at seeding stage and flower and boll stage and parental conventional cotton at seeding stage and budding stage, which were also similar between tranagenic cotton at budding stage and parental conventional cotton at flower and boll stage. [ Conclusion] Analysis of different carbon sources indi- cated that the main carbon sources utilized by soil microbes were carbohydrates, amino acids, carboxylie acids and polymers.

Impact of Transgenic Bt+CpTI Cotton on Soil Enzyme Activities and Soil Microorganisms

Due to its strong and effective insecticidal properties, transgenic Bt＋CpTI cotton has witnessed an expanding planting area in recent years, and the impact of its cultivation on soil ecosystem becomes an important part of environmental risk assessment. Using transgenic Bt＋CpTI cotton sGK321 and its parental homologous conventional cotton Shiyuan 321 as the study objects, a comparative analysis was conducted on the changes in enzyme activities （urease, alkaline phosphatase, and catalase） of the rhizosphere soil and changes in the number of culturable microor-ganisms （bacteria, fungi, and actinomycetes） at different growth stages （seedling stage, budding stage, flower and bol stage, and bol opening stage） of sGK321 and Shiyuan 321 under the condition of 13 years field plantings. The results showed that, the populations of bacteria, fungi, and actinomycete and the soil enzyme activi-ties of urease, alkaline phosphatase and catalase had a similar variation trend along with the cotton growing process for transgenic cotton and conventional cotton. Some occasional and inconsistent effects on soil enzyme activities and soil fungi composi-tion in the rhizosphere soil of transgenic Bt＋CpTI cotton were found at the seedling stage, budding stage, flower and bol stage as compared with that of conventional cotton. The amount of bacteria and actinomycetes were not significantly different during a certain stage; however, the activities of urease, catalase, alkaline phos-phatase, also with the number of fungi were significantly different, e.g. the urease activities at seedling stage, the alkaline phosphatase at seedling and budding stages, and the soil culturable fungi at flower and bol stage were less than that of conven-tional cotton, while the soil alkaline phosphatase activities at flower and bol stage were higher. Cluster analysis showed that soil enzyme activities and microbial popu-lation changed mainly along the growth processes, suffering little from the planting of transgenic Bt＋CpTI cotton.

Effects of Transgenic Bt+CpTI Cotton on Field Abundance of Non-Target Pests and Predators in Xinjiang, China

Transgenic insect-resistant cotton is being increasingly planted in Xinjiang cotton-planting regions, where geographical climate conditions and species composition of pests and natural enemies are greatly unique in China. Limited studies have been conducted on the ecological impacts of transgenic insect-resistant cotton, especially for transgenic double genes （Bt＋CpTI） cotton, in this region. In this study, the potential effects of transgenic Bt＋CpTI cotton on the seasonal abundance of non-target pests and predators were assessed from 2009 to 2011 in Korla, Xinjiang. The results showed that species composition and seasonal abundance of 5 groups of pests and 5 groups of predators were not significantly different between transgenic Bt＋CpTI cotton and non-transgenic cotton every year. It suggests that transgenic Bt＋CpTI cotton per se does not affect the population dynamics of non-target pests and predators on this crop in Xinjiang.

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.

Study on Spatial-temporal Dynamics of Bt Toxic Protein Expression in Insect-resistant Transgenic Cotton and Its Degradation in Soil

[Objective] This study aimed to investigate the spatial-temporal dynamics of Bt toxic protein expression in insect-resistant transgenic cotton and its degradation in soil. [Method] Btcry1Ac toxic protein expression in roots, stems and leaves of transgenic cotton Guoshen GK45 at different developmental stages and the annual average content of BtCry1Ac toxin protein in the topsoil, rhizosphere soil and following cotton-growing area were explored and analyzed by using enzyme linked immuno sorbed assay (ELISA). [Result] The content of exogenous BtCry1Ac toxin protein decreased during the growth process of insect-resistant transgenic cotton; to be specific, the content of BtCry1Ac toxin protein in cotton stems and leaves decreased more slowly and always maintained a high level, while that in roots decreased rapidly and reached a minimum level to the following plant growth and development stage. BtCry1Ac toxin protein was detected in topsoil of both non-transgenic and transgenic cotton-growing areas, and the content of BtCry1Ac toxin protein increased in topsoil of following cotton-growing area, which was very low in rhizosphere soil. [Conclusion] Determination of Btcry1Ac toxic protein provides scientific basis for the risk assessment of the cultivation of genetically modified crops and the safety evaluation of soil ecosystem.

NaCl-Induced Changes of Ion Fluxes in Roots of Transgenic Bacillus thuringiensis (Bt) Cotton (Gossypium hirsutum L.)

Bacillus thuringiensis （Bt） cotton is grown worldwide, including in saline soils, but the effect of salinity on ion fluxes of Bt cotton remains unknown. Responses of two transgenic Bt cotton genotypes （SGK321 and 29317） and their corresponding receptors, Shiyuan 321 （SY321） and Jihe 321 （J321）, to 150 mmol L-1 NaCl stress were studied in a growth chamber. The root dry weight of SGK321 and 29317 under NaCl treatment was decreased by 30 and 31%, respectively. However, their corresponding receptor cultivars SY321 and J321 were less affected （19 and 24%, respectively）. The root length and surface area of the Bt cultivars were significantly decreased relative to their receptors under salt stress. NaCl treatment significantly increased CrylAc mRNA transcript levels in SGK321 and 29317 but did not affect Bt protein content in leaves or roots of either cultivar at 1 and 7 d after NaCl treatment. Fluxes of Na^＋, K^＋, and H^＋ in roots were investigated using the scanning ion-selective electrode technique. Both mean K^＋ efflux rate and transient K^＋ efflux of the Bt cultivars increased four-fold compared to their corresponding receptors when exposed to salinity stress. There were no significant differences in Na^＋ efflux between Bt and non-Bt cottons. Furthermore, the Na^＋ contents in roots and leaves of all genotypes dramatically increased under salt stress, whereas K^＋ contents decreased. Our results suggested that Bt cotton cultivars are more sensitive to salt stress than their receptor genotypes.

Combining Ability and Heterosis Between High Strength Lines and Transgenic Bt (Bacillus thuringiensis) Bollworm-Resistant Lines in Upland Cotton (Gossypium hirsutum L.)

To analyse the combining ability and heterosis between high-strength lines and transgenic Bt bollworm-resistant lines in upland cotton, 5 high-strength lines were crossed as female lines with 12 transgenic Bt bollworm-resistant lines according to NCII design. It was demonstrated that the compositions of variance in various traits were quite different. For seed cotton yield, lint yield, boll numbers per plant and boll weight, the dominant (special combining ability) effects were the major effects, accounting for 87.38, 84. 40, 80. 04 and 64. 46% of the total phenotypic variances, respectively, while for fibre strength and micronaire value, the additive (general combining ability) effects had the major effects, with a ratio of additive variance to phenotypic variance of 78.85 and 43.80%. As for lint percent and 2.5% span length, the dominant and additive variances had similar effects, in phenotypic variances (54.94 and 40.11% for lint percent, 45.76 and 42.49% for 2. 5% span length, respectively). The mid-parent heterosis (Hpm), surpassing parent heterosis (Hpb) and competitive heterosis (Hck) for seed cotton yield and lint yield were both extremely significant. For fibre properties, the Hck and Hpm of 2.5 % fibre span length were extremely significant, the Hck of fibre strength was significant, and the favorable negative Hck of micronaire was also extremely significant. The increments of hybrid over common variety were 17% for lint yield and fibre strength, 7% for fibre span length, and 4 % for fineness.

Effects of External Chemical Regulation on Bt Transgenic Cotton Plants under Combined Stress of High Temperature and Water Deficit

[Objective] The study aimed to find a possible way to combat or alleviate the negative effects caused by high temperature and water deficit at the growth stage of peak boll-setting.[Method] With Bt transgenic cotton GK22 as the test cultivar,a potted experiment was carried out to investigate the effects of the regulation of external substances(the water solutions of pix,urea and their mixture) on the physiological parameters,insecticidal protein content,yield and yield component of cotton plants in artificial climate chambers treated with high temperature and water deficit.[Result] The application of external pix,urea or their mixture was effective in stabilizing the physiological parameters of cotton plants,insecticidal protein content,yield and yield components.Compared with the exclusive application of pix and urea,the mixture of pix and urea played the most effective role in stabilizing the content of chlorophyll,soluble sugar and insecticidal protein,alleviating the increase of the content of free amino acids and proline,and increasing boll number per plant,boll weight and seed cotton yield.[Conclusion] The water solutions of pix,urea or their mixtures can be used to combat or alleviate the stress of high temperature and water deficit if they are sprayed onto cotton plants prior to stress occurrence.

Differential Expression of Bt Protein in Transgenic Bt Cotton

[Objective] The paper was to study the temporal and spatial dynamics of Bt protein expression in transgenic Bt cotton and to determine the inner relationship of Bt protein expression and transgenic Bt cotton. [Method] With transgenic cotton cultivar（ GK19） as the test material,Bt protein contents in different organs,main stem functional leaves at different growth stages and different positions of main stem leaves at different growth stages were studied by enzyme-linked immunosorbent assay. [Result] There were differences in Bt protein content among different organs of transgenic Bt cotton; the Bt protein content of leaves at seedling stage was the highest,followed by flowers,bubs and bolls,and those of roots and stems were relatively low. The Bt protein content of main stem function leaves gradually decreased with the progressing development. There were great differences in Bt protein content among different positions of main stem leaves at different growth stages; the Bt protein content of the 1^（st）-7^（th） top leaves at seedling stage and full budding stage gradually decreased,while those at full flowering stage and full bolling stage first slowly increased then gradually stabilized. [Conclusion] Bt protein expression was found in all organs of transgenic cotton at all growth stages,and the expression level presented temporal and spatial dynamics.

Effects of Transgenic Cry1Ac plus CpTI Cotton on the Bioecology of Main Parasitoids in Laboratory Studies

Effects of transgenic Cry1Ac plus CpTI cotton(double genes cotton) on development of main parasitoids were studied in the laboratory.Compared with the traditional cotton,the differences of

90份转BT基因抗虫棉品种(系)在新疆早熟棉区的适应性评价

【目的】评价转BT基因抗虫棉品种(系)的适应性与应用价值,为棉花新品种的选育提供适合的亲本。【方法】以90份转BT基因抗虫棉为材料,研究其农艺性状和品质性状的变异情况及相关性,并以主要农艺性状和品质性状对90份转BT基因抗虫棉进行主成分和聚类分析。【结果】供试90份转BT基因抗虫棉品种(系)纤维品质、农艺性状间差异显著,其中44份转BT基因抗虫棉为早熟材料,46份转BT基因抗虫棉为中早熟材料;前5个主成分特征值均大于1,累计贡献率达71.62%,第1主成分与产量有关,第2主成分与纤维品质有关,第3主成分与植株性状有关。90份转BT基因抗虫棉划分为4个类群,第Ⅰ类群籽指性状表现好;第Ⅱ类群断裂比强度、伸长率较好;第Ⅲ类群单株铃数、铃重、衣分较高;第Ⅳ类群纤维品质较好。【结论】筛选出了G21-2、G21-14、G21-1、G21-77等综合性状优异、铃重均在5.00 g以上、衣分在42%以上的转BT基因抗虫棉品种(系)18份。

双价基因(Bt+CpTI)抗虫棉对棉铃虫的杀虫活性及抑制生长作用

比较了转 Bt+ Cp TI双价基因抗虫棉 (双价棉 )与转 Bt单基因棉 ( Bt棉 )对棉铃虫不同Cry1 Ac抗性种群杀虫活性的时间动态和对不同龄期幼虫的抑制生长作用。结果表明 ,双价棉叶对棉铃虫的杀虫活性于 6月底最高 ,7月底和 8月下旬逐渐下降 ,高于同期测定的 Bt棉的杀虫活性。双价棉对棉铃虫抗性种群 2～ 5龄幼虫的死亡率、存活幼虫体重、化蛹率和成虫羽化率等生长发育的影响 ,均显著高于 Bt棉。用 Bt棉叶连续饲养不同龄期抗性棉铃虫 ,2龄幼虫就可部分化蛹和羽化 ,而用双价棉饲养 ,5龄以下幼虫不能化蛹和羽化 ,表明双价棉对抗性棉铃虫具有较强的抗虫性。

转双价(Bt＋CpTI)棉种植对土壤速效养分和酶活性的影响

本研究利用三室根箱对棉花根部土壤进行分区采集,以转双价棉SGK321及其亲本常规棉石远321为研究对象,对3个生长时期(播种后40、50 d和60 d)不同根区(S1、S2和S3)土壤速效养分(硝态氮、铵态氮和速效磷)含量及酶(脲酶、碱性磷酸酶和过氧化氢酶)活性变化进行了对比分析。研究结果表明,与常规棉相比,转双价棉的种植促进了S2根区土壤中磷素向有效态的转化,使S2和S3根区土壤硝态氮含量下降,而对各根区土壤脲酶、碱性磷酸酶和过氧化氢酶活性及土壤铵态氮无显著影响。主成分分析结果表明,土壤速效养分和酶活性变化主要受生长时期的影响,转双价棉种植对其影响是非常有限的。

利用根箱法解析转双价(Bt+CpTI)基因棉花对土壤微生物数量及细菌多样性的影响

利用根箱法对棉花根部土壤进行分区采集,并采用传统平板培养和DGGE克隆测序技术相结合的方法,对转双价基因棉和常规棉3个生长时期(播种后40 d、50 d和60 d)不同根区土壤细菌、放线菌和真菌数量及细菌多样性进行对比分析。平板培养结果表明,与常规棉相比,转双价基因棉S1根区土壤细菌、放线菌的数量显著降低,而S2根区土壤细菌、放线菌、真菌和S3根区土壤细菌、真菌的数量显著增加(P<0.05);DGGE图谱分析表明,在棉花生长的3个时期内,转双价基因棉和常规棉根区土壤细菌多样性指数、均匀度和条带数均无显著差异(P>0.05),2种棉花处理间有着很强的相似性,不受转双价基因棉种植的影响。转双价基因棉种植仅改变了根区土壤可培养微生物数量,对细菌多样性无显著影响。

种植转双价基因(Bt＋CpTI)棉对主要土壤养分和酶活性的影响

采用灭菌盆栽试验,以转双价棉品种sGK321及其亲本常规棉品种石远321为研究对象,对4个生长时期(苗期、蕾期、花铃期、吐絮期)根际土壤养分(全氮、全磷、速效磷和有机质)含量及某些酶(脲酶、碱性磷酸酶和过氧化氢酶)活性变化进行了对比分析。结果表明,在观测期间,与同源常规棉相比,转双价棉根际土壤全磷含量和速效磷含量在同一时期均无显著差异,不同生育期差异明显,根际土壤全氮含量、有机质含量、酶活性在一些时期则明显不同,其变化随棉花品种、生育期不同而不同。转双价棉蕾期根际土壤全氮含量和苗期根际土壤过氧化氢酶活性以及苗期、蕾期的碱性磷酸酶活性显著高于常规棉(P<0.05),而蕾期根际土壤有机质含量、根际土壤过氧化氢酶活性和苗期、花铃期根际土壤脲酶活性以及苗期碱性磷酸酶活性则显著低于常规棉(P<0.05)。聚类分析表明,土壤养分和酶活性变化主要受生长时期的影响,转双价棉种植对其影响是非常有限的。

转CpTI-Bt基因棉和转Bt基因棉对棉铃虫幼虫存活、生长及营养利用的影响

20 0 0年 7月中旬和 8月中旬 ,分别测定了采自田间的转CpTI Bt基因双价抗虫棉 (SGK32 1,以下简称CpTI Bt棉 )和转Bt基因抗虫棉 (中 30 ,以下简称Bt棉 )对棉铃虫 Helicoverpaarmigera幼虫存活、生长的影响。结果表明 :7月中旬两种转基因抗虫棉抗虫效果均较好 ,尤其是CpTI Bt棉棉叶和花瓣对 4龄幼虫 3天内致死率为 92 %以上 ;8月中旬两种转基因棉的抗虫活性均明显降低 ,且Bt棉的杀虫活性显著低于CpTI Bt棉 ,其幼虫死亡率与对照受体棉中 16的死亡率之间无显著差异 ,仅显著抑制了幼虫的生长 ;石远 32 1(SGK32 1受体品系 )的花瓣具有一定的抗虫活性 ,可显著降低取食幼虫的体重 ,甚至造成部分幼虫死亡 ;CpTI Bt棉中 ,花瓣和棉叶的抗虫性明显高于蕾和铃心。对 5龄幼虫取食棉铃 1日后的营养指标测定结果显示 :两种转基因抗虫棉处理的幼虫相对生长率和相对取食量均显著低于石远 32 1,但两者之间无显著差异 ;CpTI Bt棉处理的幼虫近似消化率显著低于石远 32 1和Bt棉 ,但其食物利用率显著高于石远 32

美洲黑杨杂种优良无性系转抗虫基因(Bt和CpTI)的研究

以美洲黑杨杂种优良无性系南林895杨(Populus×euramericanac‘v.Nanlin895’)为转基因受体材料,以嫩芽或腋芽为外植体材料组织培养再生植株,利用农杆菌介导法转化Bt基因和CpTI基因。结果显示较合适的组培再生与遗传转化系统为:叶分化培养基为MS+6-BA0.5mg/L+TDZ0.002mg/L,芽伸长培养基为MS+6-BA0.2mg/L+TDZ0.001mg/L+Km10mg/L+Carb500mg/L,生根培养基为1/2MS;预培养3d,菌液浓度OD6001.0～1.3左右,侵染时间20min,共培养4d,叶盘转化频率可达28.7%。对Kmr植株经PCR分析,筛选获得了18株整合有Bt基因和1株整合有CpTI基因的转基因植株。部分转基因植株的初步饲虫实验表明,饲喂转基因杨树叶片可明显抑制杨小舟蛾的生长发育。

表达CpTI或CpTI+Bt基因水稻对二化螟的杀虫活性

明恢86是目前我国应用最为广泛的优良籼稻恢复系,明恢86CpTI和明恢86CpTI+Bt分别为表达豇豆胰蛋白酶抑制剂(CpTI)基因(单基因)和CpTI+Bt蛋白基因(双基因)的明恢86.以明恢86为对照,系统测定了明恢86CpTI和明恢86CpTI+Bt对二化螟的杀虫活性.结果表明,明恢86CpTI和明恢86CpTI+Bt对二化螟都表现出很强的杀虫活性.取食分孽期明恢86CpTI和明恢86CpTI+Bt7d后,二化螟1龄幼虫的校正死亡率高达88.6%和92.1%,对二化螟均表现高抗.该转基因水稻还对存活二化螟幼虫的生长发育产生了明显的影响,取食明恢86、明恢86CpTI和明恢86CpTI+Bt后存活的二化螟幼虫历期分别为(36±1.9)、(47±2.9)和(46±2.0)d,饲喂该转基因水稻后二化螟的幼虫历期明显延长.此外,二化螟老熟幼虫取食该转基因水稻后,鲜重平均减少了8-11mg,蛹重明显减轻,繁殖能力明显下降.

转Bt-CpTI-GNA基因棉花的研究(英文)

采用农杆菌介导法将外源三价抗虫基因(Bt-CpTI-GNA)导入常规棉花品种中,获得转基因再生株,分子检测表明外源基因已在棉花体内表达,并遗传给后代材料。PCR分子检测与转化的标记基因和外源目的基因抗性三者极有规律性。其所携带的基因在转基因棉花中有分离现象,这可能是外源基因整合到受体棉株体内“基因沉默”而引起所致。