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.

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

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.

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.

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 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.

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.

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.

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.

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.

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。

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

转Bt基因棉田节肢动物群落结构特征研究

比较研究了转Bt基因棉———保铃棉、中棉 30和常规棉常规防治、常规棉不防治 4种类型棉田的节肢动物群落构成及结构 .结果表明 ,保铃棉、中棉 30的物种丰富度有一定降低 ( 2 4%～ 16 3% ) ,个体总量显著减小 ( 71 0 %～ 78 3% ) .其中植食性亚群落的优势种组成及次序改变 ,其个体总量降低最多 ,捕食性和寄生性亚群落的个体总量明显增加 .中棉 30、保铃棉与常规棉不防治处理群落的相似系数依次最高 ( 0 82 43)、次高( 0 732 0 ) ,保铃棉与常规棉常规防治居第三 ( 0 3380 ) ,常规棉常规防治与常规棉不防治居第四 ( 0 312 8) ,中棉30与常规棉常规防治最小 ( 0 2 665 ) .各处理多样性指数和均匀度值的高低次序为中棉 30 (分别为 2 3712和0 64 2 8) >常规棉不防治 ( 2 365 4和 0 62 5 1) >保铃棉 ( 2 1364和 0 5 791) >常规棉常规防治 ( 1 0 877和0 2 949) ,优势度次序为常规棉常规防治 ( 0 872 6) >保铃棉 ( 0 35 2 8) >常规棉不防治 ( 0 1178) >中棉 30( 0 10 48) .转Bt基因棉的害虫防治宜采取与常规棉不同的IPM策略和方法 .

转基因棉Bt毒性表达的时空动态及对棉铃虫生存、繁殖的影响

转Ｂｔ基因棉３１０品系在不同发育阶段，对棉铃虫的毒性表达测定表明，（１）棉株各空间组织在不同发育阶段均能强力阻止棉铃虫取食，表达出较高的抗虫效果。（２）棉株不同空间组织在同一时期，毒性表达存在差异，以营养组织，中、小蕾高于大蕾和铃。（３）同一组织毒性表达随着棉株生长、发育、成熟、衰老逐渐呈动态下降。（４）前后各阶段，同一组织饲喂棉铃虫，死亡所需要天数呈动态增加，但最终生存率降低到一个较低水平。存活老熟幼虫重量、蛹重及蛹羽化率比对照棉铃虫下降３０％～５０％，羽化的蛾子进行自交或异交均能产下受精率、孵化率很高的卵。取食Ｂｔ转基因棉的雌蛾产卵量比对照减少５０％～７０％。

渍涝与干旱对不同转Bt基因抗虫棉的影响

对 3个转 Bt基因棉花品种的研究表明 ,花铃期田间渍涝显著降低皮棉产量和棉株内的 Bt蛋白含量。积水后棉花主茎功能叶和幼蕾的 Bt蛋白含量平均分别比对照降低 2 7.7%和 2 9.9% ,主茎下部老叶的 Bt蛋白含量仅降低 6 .6 % ,说明渍涝对生长旺盛器官 Bt蛋白合成的影响远远大于衰老器官。干旱对棉株内 Bt蛋白合成的抑制更强 ,当花铃期干旱使棉叶致萎时 ,主茎下部老叶、幼蕾和主茎功能叶的 Bt蛋白含量分别降低了52 .9%、3 7.0 %和 3 0 .0 %。为充分发挥和利用转Bt基因棉花品种的产量与抗虫潜力 ,棉花生长发育的关键时期应尽量避免缺水干旱 ,及时排除田间积水 。

转Bt基因棉杂种优势及性状配合力研究

采用 NC 设计方法 ,选用我国构建的 Bt( Bacillus thuringiensis)基因棉 GK3和美国构建的 Bt基因棉新棉 33B等 Bt基因棉为父本 ,配制杂交组合 ,分析了含 Bt基因杂交棉组合的性状优势表现及配合力效应。性状优势分析表明 ,组合的中亲优势和竞争优势明显 ,皮棉产量中亲优势率达 1 0 0 %,竞争优势率达 86.7%,多数品质性状较对照为优 ,表明转 Bt基因棉品系与常规品种间杂种优势利用潜力较大 ;通径分析表明 ,在增产因素中 ,铃重和铃数所起贡献较大 ;配合力分析表明 ,父本间和母本间的 GCA均方值均达显著或极显著水平 ,组合的 SCA均方值除子、皮棉产量性状外 ,其余均不显著 ,在 F1性状的遗传中 ,各性状的加性效应起主导作用 ;F1的性状表型值与父、母本的 GCA呈高度正相关 ,并达显著水平 ,表明选配含 Bt基因杂交组合时 。

转Bt基因棉Bt毒蛋白的ELISA检测方法的研究(英文)

利用纯化的Ｂｔ毒素蛋白（６７ｋＤａ）作为标准蛋白和免疫抗原，建立了检测转基因抗虫棉植株内Ｂｔ毒蛋白含量的抗体夹心ＥＬＩＳＡ方法。检测极限１μｇ·Ｌ－ １，线性范围１－ １５μｇ·Ｌ－ １，板内误差Ｃ．Ｖ．＜ ５％ ，板间误差Ｃ．Ｖ．＜ ５％ ，平均样品回收率９４．９４％ 。经稀释度试验等质量控制试验，表明所建立的ＥＬＩＳＡ方法是可靠的，可用于转基因棉花植株中Ｂｔ毒素蛋白含量的测定。