温度对转基因741杨抗虫性的影响

在不同温度下测定转基因741杨的抗虫性,发现转抗虫基因741杨的抗虫性与温度密切相关．随着温度的升高,舞毒蛾幼龄幼虫的总死亡率、累计死亡率均明显升高,且随着昆虫龄级的增加,幼虫对转基因株系和温度的敏感性逐渐降低．温度对取食对照株系的昆虫生长发育有促进作用．温度高,舞毒蛾幼虫的生长发育明显加快,历期缩短,各龄期体重、排粪量增长快,而以转基因杨饲喂的昆虫发育进度随温度变化差异不显著．对1998～2000年温度资料和相应的杨扇舟蛾死亡率建立了回归方程,各高抗和中抗株系的温度-昆虫死亡率曲线均表现为明显的回归关系．     

转双抗虫基因三倍体毛白杨外源基因表达及性状相关性分析

用部分改造的BtCry1Ac基因与慈菇蛋白酶抑制剂（API）基因构建的双抗虫基因表达载体,通过农杆菌介导法转化了三倍体毛白杨Populus tomentosa Carr.,获得一批转双抗虫基因株系。对转基因株系的抗虫毒蛋白表达进行了ELISA和Western Blot检测,同时用转基因株系叶片对杨扇舟蛾Clostera anachoreta Fabricius和舞毒蛾Lymantria dispar L.幼虫进行室内饲虫试验,并对各项外源基因表达指标进行了相关分析。结果表明：在检测的28个转基因株系中,对杨扇舟蛾高抗株系占总参试系号的41％,中抗系号占35.0％,低抗系号占24％,对舞毒蛾高抗株系占参试系号的70％。转基因植株可明显抑制存活幼虫的生长发育,且不同转基因株系饲养的幼虫发育存在显著差异。连续两年相关分析表明,不同转基因株系幼虫死亡率间存在极显著相关。转基因植株对舞毒蛾和杨扇舟蛾均表达出抗虫性,并存在极显著相关。ELISA检测结果表明,不同转基因株系Bt毒蛋白表达量存在差异,变化在0.0011％～0.0161％。转基因植株对害虫的杀虫效果与Bt杀虫蛋白的表达量存在显著相关,表明Bt毒蛋白在抗虫效果中占有重要地位。转基因植株对卡那霉素表现出一定的抗性,但与抗虫程度相关不明显,单纯用卡那霉素作为筛选手段并不能完全反映植株的真实抗虫效果。     

转新型双价抗虫基因杨树对光肩星天牛及美国白蛾的抗性鉴定

为明确双价基因chitinase-BmkIT对鞘翅目害虫光肩星天牛及鳞翅目害虫美国白蛾的抗虫效果, 分别以转该双价基因的杨树茎干和叶片为材料,采用室内茎干接虫和叶片接虫方法鉴定其对光肩星天牛Anoplophora glabripennis和美国白蛾Hyphantria cunea幼虫的抗性。结果表明：转基因株系132-2,144-1,123-1对美国白蛾幼虫的致死作用和生长发育影响显著高于取食未转化叶片的处理（P＜0.05）,且对其后代产生了影响,表现为幼虫不能化蛹或化蛹后不能羽化或羽化不正常;但供试的8个转基因株系对光肩星天牛幼虫无明显的致死和生长抑制作用,表现为幼虫死亡率和体重均与取食非转化茎干幼虫无显著差异（P＞0.05）。结果提示,双价基因chitinase-BmkIT可作为杨树鳞翅目害虫的抗虫基因源,并且对延缓害虫抗性的产生具有一定的意义。     

昆虫病原线虫对腰果细蛾的致病力测定

室内测定了Steinernema carpocapsae All、S.carpocapsae Biosys、S.glaseri NC52、S.longicaudum X-7、Heterorhabditis bacteriophora H06和H.indica LN2共6个昆虫病原线虫品系对腰果细蛾Acrocercops syngramma Meyrick幼虫的致病力。结果表明,线虫S.carpocapsae All、Biosys品系与线虫S.glaseri NC52对腰果细蛾幼虫的致死效果显著高于其它线虫,但供试的6个线虫品系均未能在腰果细蛾幼虫体内繁殖下一代,说明供试线虫在腰果细蛾幼虫体内难以繁殖。选择小卷蛾斯氏线虫S.carpocapsae All进一步研究其对腰果细蛾的致病力,结果发现,All线虫对腰果细蛾3龄幼虫处理24 h后的致死中浓度LC50为19.88 IJs/虫。腰果细蛾各龄幼虫和蛹对小卷蛾斯氏线虫S.carpocapsae All均表现出高度的敏感性,但不同幼虫龄期和处理时间敏感程度不同。小卷蛾斯氏线虫S.carpocapsae All对不同龄期腰果细蛾幼虫和蛹均表现出较强的致病力。     

椰子织蛾幼虫龄数及取食量的雌雄差异

为了明确椰子织蛾幼虫的龄数、取食量及龄期。在室温25℃±3℃,寄主食料椰子叶饲养条件下,测量了雌、雄幼虫头壳宽、取食量并记录了各龄幼虫的发育历期。结果表明,椰子织蛾雌性幼虫有9-10个龄数,雄性幼虫有8-10个龄数。1-10龄雌虫的头壳宽约为0.2300,0.3250,0.4300,0.5267,0.7700,0.9633,1.3775,1.5850,1.8200,2.1929 mm。1-10龄雄虫的头壳宽约为0.2233,0.3214,0.4125,0.5300,0.6529,0.8675,1.1267,1.3375,1.4950,1.8925 mm。9-10龄雌虫的头壳宽显著大于雄虫的头壳宽。头壳宽与龄数具有很强的相关性。椰子织蛾幼虫的取食量随龄数的增大而增加。椰子织蛾1代雌性幼虫平均取食椰子叶的面积(3607.23±146.83 mm~2)显著高于1代雄性幼虫平均取食椰子叶的面积(1991.25±143.92 mm~2)。1-5龄幼虫的取食量最小,小于50 mm~2。8-10龄为暴食期。1-10龄幼虫的发育历期分别为4.55±0.16,5.69±0.24,5.73±0.37,5.22±0.15,5.11±0.46,4.61±0.46,5.12±0.68,6.00±0.43,6.86±0.40和8.75±1.55 d。对于雌、雄成虫个体差异较大的昆虫,对其幼虫头壳宽值和取食量的测定应雌、雄幼虫分别测定。椰子织蛾的防治适期应在未造成严重为害的1-5龄幼虫高蜂期进行。以幼虫头壳宽为主,同时结合取食面积判定幼虫所处龄数,可为准确掌握防治时期提供科学依据。     

转单、双Bt基因741杨外源基因表达和抗虫性比较

【目的】研究联合使用两种或两种以上的抗虫基因的抗虫效果, 同时鉴定并筛选出转双Bt基因741杨对鳞翅目和鞘翅目害虫有较强抗性的株系。【方法】选取转三基因（Cry3Aa+Cry1Ac+API）741杨8个株系、 转双基因（Cry1Ac+API）741杨1个株系和转单基因（Cry3Aa）741杨3个株系为试材, 从外源基因PCR检测、 毒蛋白表达和抗虫性三方面对转基因株系进行对比分析。【结果】经PCR扩增后各转基因株系出现了预期的电泳条带。ELISA蛋白检测显示转基因株系中都有与所含基因相应的Bt杀虫蛋白表达。用转基因株系新鲜叶片进行柳蓝叶甲Plagiodera versicolora和美国白蛾Hyphantria cunea室内饲虫实验表明： 转入不同抗虫基因的杨树对昆虫的抗性具有选择性, 对非靶标昆虫没有毒杀作用。转双Bt基因741杨具有双抗性, 不同转基因株系表现出高中低的抗性水平： 在对柳蓝叶甲的抗性上, 筛选出的其中5个高抗株系（pCCA1, pCCA2, pCCA5, pCCA6和pCCA9）的抗性水平明显比含Cry3Aa单Bt基因的3个高抗株系（pCC11, pCC53和pCC84）高; 在对美国白蛾的抗性上, 有7个株系（pCCA2~pCCA7和pCCA9）的抗性水平与含Cry1Ac单Bt基因株系（pB29）表现一致, 只有1个株系（pCCA1）对美国白蛾表现出了极低的抗性。【结论】多个抗虫基因在741杨上的联合使用, 不仅扩大了抗虫谱, 其中的高抗株系还具有了更高的抗虫能力, 有效地发挥了基因的叠加效应。     

转蜘蛛杀虫肽与Bt毒蛋白C肽基因小黑杨对杨扇舟蛾的抗性

为评价转蜘蛛杀虫肽与Bt毒蛋白C肽融合蛋白基因小黑杨对杨扇舟蛾Clostera anachoreta (Fabricius)的抗性,采用苗木套笼饲喂法和石蜡切片法,对取食转基因小黑杨的杨扇舟蛾幼虫的发育、死亡情况和中肠结构进行了研究。结果表明,杨扇舟蛾幼虫分别取食TT3（转基因无性系3）、TT1（转基因无性系1）和CK（非转基因对照）小黑杨后 总历期依次为35.63天、30.39天和28.74天,幼虫化蛹率依次为12.1%、29.3%和44.3%,平均蛹重依次为0.1077 g、0.1714 g和0.1893 g。转基因小黑杨能明显延长杨扇舟蛾幼虫的发育历期,降低化蛹率和蛹重。同时,转基因小黑杨有抑制幼虫蜕皮、增加其死亡率和致蛹畸形的作用,且能破坏幼虫中肠,使中肠细胞排列松散、肠腔食物减少、中肠变形,其破坏作用随时间延长而加剧。一般来讲,TT3对杨扇舟蛾的各种影响作用均大于TT1。     

湿度对三角新小卷蛾实验种群生长的影响

在相对湿度(RH)为55%、65%、75%、85%和95%条件下,观察湿度对三角新小卷蛾Olethreutes leucaspis Meyrick各虫态生长、发育及存活的影响,组建三角新小卷蛾实验种群生命表。结果表明:在RH为55%-95%条件下,三角新小卷蛾卵、幼虫、蛹和世代的发育历期在RH为85%时最短,发育历期分别为1.67、12.46、7.67和25.42 d。高湿条件下卵、幼虫和蛹的存活率较高。三角新小卷蛾各虫态发育历期、存活率与相对湿度呈抛物线关系。在RH为55%-95%条件下该虫内禀增长力(rm)均大于0,种群趋势指数(I)均大于1,该虫种群呈增长趋势。三角新小卷蛾发育适宜的RH为75%。     

虫害诱导黑杨对分月扇舟蛾生长发育的影响

在实验室条件下,通过人工饲养研究了虫害处理的69杨和895杨对分月扇舟蛾Clostera anastomosis幼虫、蛹及成虫的生长发育的影响。结果表明:虫害处理的叶片对分月扇舟蛾幼虫表现为驱避作用;取食虫害处理的叶片,使得幼虫的发育历期延长,死亡率增加,对食物的利用和转化效率下降;蛹重减轻,蛹期延长;成虫的羽化率降低,产卵量减少。说明昆虫取食危害能够诱导杨树对分月扇舟蛾产生抗虫性。     

A method to quantitative determine the stomach poison on stored grain pests—mixed drug sheet method

本实验制定一种定量测定储粮害虫胃毒作用的方法,命名为混药薄片法.通过试验验证饲料薄片对烟草甲Lasioderma serricorre(Fabricius)的幼虫成活率、发育历期、虫体重量、产卵量等生长发育无影响.在排除烯虫酯对烟草甲的触杀作用的前提下,将混有烯虫酯溶液的混药薄片作为饲料饲养烟草甲3龄幼虫,得到烯虫酯对烟草甲3龄幼虫的准确致死剂量为(0.46±0.03) μg.     

我国抗虫转基因杨树生态安全性研究进展

转基因树木与农作物相比, 人们更关注其长时间种植可能导致转基因扩散到周围野生近缘种。由于生长周期长, 转基因树木会增加转基因不稳定性, 对非靶标生物的影响, 靶标害虫对转基因植物产生抗性, 增加树木入侵性(杂草化), 以及由于基因漂移或基因逃逸对环境产生的负面影响或新的环境风险。过去十几年, 针对我国抗食叶害虫的两个商业化转Bt基因欧洲黑杨(Populus nigra)和转双抗虫基因741杨[P. alba× (P. davidiana + P. simonii) × P. tomentosa], 已开展了有关生态安全性方面的多项研究。本文围绕抗虫转基因树木生态安全性研究进展进行了综述。抗虫转基因杨树对节肢动物种群和群落结构产生了一定影响, 使昆虫的多样性提高, 但对土壤微生物区系未见明显影响。转基因欧洲黑杨通过花粉和种子发生的基因漂移几率很低。转基因杨树通过内生菌发生的水平转移可能会对环境造成的潜在危险也进行了评价。文章最后指出对抗虫转基因杨树农林复合生态系统开展生物安全研究的必要性。     

向小黑杨转化蜘蛛杀虫毒素基因（英文）

近年来,危害小黑杨Populus simonii×Ｐ.nigra的害虫发生严重,给林业生产造成很大损失。为了提高小黑杨的抗虫能力,避免使用杀虫剂带来的污染,用农杆菌介导法将澳大利亚漏斗蛛Atrax robustus的毒蛋白基因和苏云金芽孢杆菌CryⅠA（b）基因C末端的融合基因BGT转化入小黑杨。PCR 和Southern印记分析转基因植株,结果表明,BGT杀虫基因已经整合在小黑杨基因组上。活性实验表明,取食转基因杨树6天和9天后,舞毒蛾Lymantria dispar 2龄幼虫的校正死亡率分别是37.0%和92.6%。方差分析表明取食转基因和对照杨树的舞毒蛾幼虫体重差异显著。这些结果显示转基因杨树上的舞毒蛾的发育速率受到影响。     

Overexpression and oral immunogenicity of a dengue antigen transiently expressed in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

To understand the genetic and expression stability of transgenic insect-resistant poplar 741, this study compared the experimental plantations of transgenic insect-resistant poplar 741 lines (pb1, pb6, pb11, pb17, and pb29) with non-transgenic poplar 741, P. tomentosa Carr.f.yixianensis (poplar 84 K) and transgenic hybrid progeny lines cultured from immature embryos. The insect resistance and growth stability of transgenic poplar 741 were investigated by detecting exogenous genes by polymerase chain reaction (PCR), measuring the diameter at breast height (DBH) and volume growth, and performing insect-resistance tests against Clostera anachoreta and Hyphantria cunea. The inheritance and expression of the exogenous gene was also examined in transgenic hybrid progeny lines. The results revealed that the exogenous gene was stable, remaining stable in 8–10-year-old transgenic poplar 741 trees. No significant difference was found between the height of 10-year-old transgenic poplar 741 and non-transgenic poplar 741 in the experimental plantations in Baoding, China. The DBH and volume growth of pb17 was significantly greater than that of pb29 and pb11. The 8-year-old transgenic poplar 741 pb29 grown in Zhuozhou showed no significant difference from poplar 741 in terms of height growth, DBH, and volume. From 1999 to 2013, pb29-fed larvae (C. anachoreta larvae and H. cunea) exhibited stable mortality rates >79%. Likewise, pb11-fed larvae showed stable mortality rates (C. anachoreta larvae had mortality rates >75%, and H. cunea larvae exhibited rates >80%). pb17 conferred low insect-resistant stability, showing mortality rates that varied from 28.2 to 99.27% in C. anachoreta and H. cunea larvae. Among the hybrid progeny lines acquired by hybridization of pb1, pb29, and pb11 with 84 K poplar, the ratios of PCR-positive to PCR-negative lines for the BtCry1Ac gene were 1.31, 1.15, and 0.86, respectively. X ² tests showed that the ratio was consistent with the Mendelian law of 1:1 segregation controlled by an allele pair. The hybrid progeny of pb6?×?84 K had a segregation ratio of 3:1. The nptII gene followed the same segregation rule as Cry1Ac. The transgenic hybrid progeny that contained Cry1Ac gene exhibited the same insect resistance as the parent plants.     

转基因抗虫作物对非靶标昆虫的影响

转基因抗虫作物自 1996年被批准商业化种植以来 ,它的抗虫性和经济效益已得到了普遍肯定 ,同时 ,转基因抗虫作物对非靶标生物的影响 ,如转基因抗虫作物的长期种植 ,是否会导致次要害虫上升为主要害虫 ,是否会影响有益昆虫 ,包括重要经济昆虫、捕食性和寄生性天敌以及重要蝶类的种类及种群数量 ,已成为转基因抗虫作物生态风险评估的重要内容。一些研究结果表明 ,转基因抗虫作物在对靶标害虫有效控制的同时 ,一些对杀虫蛋白不敏感的非靶标害虫有加重危害的趋势 ,由于种植转基因抗虫作物 ,减少了化学农药的使用 ,客观上也使非靶标害虫种群数量上升 ,这对转基因抗虫作物害虫综合治理提出了新的要求。靶标害虫数量的减少直接影响了害虫天敌种群数量 ,靶标害虫取食转基因抗虫作物后发育迟缓 ,也间接影响了天敌昆虫的生长发育 ,转基因抗虫作物的花粉或花蜜是一些重要经济昆虫如蜜蜂、熊蜂和一些寄生蜂 ,甚至捕食性天敌的食物来源 ,或花粉飘落到一些鳞翅目昆虫如家蚕或重要蝶类昆虫的寄主植物上 ,直接或间接对这些昆虫造成一定影响。目前大多数研究表明转基因抗虫作物对非靶标昆虫 ,特别是对有益昆虫没有明显的不利影响 ,也有研究报道认为对某些有益昆虫有一定的不良影响。这为深入开展转基因抗虫作物的生态安全     

Relative resistance of transgenic tomato tissues expressing high levels of tobacco anionic peroxidase to different insect species.

Different parts of genetically transformed tomato (Lycopersicon esculentum L.) plants that express the tobacco anionic peroxidase were compared for insect resistance with corresponding wild type plants. Leaf feeding by first instar Helicoverpa zea and Manduca sexta was often significantly reduced on intact transgenic plants and/or leaf disks compared to wild type plants, but the effect could depend on leaf age. Leaves of transgenic plants were generally as susceptible to feeding damage by third instar Helicoverpa zea (Boddie) and Manduca sexta (L.) as wild type plants. Green fruit was equally susceptible to third instar larvae of H. zea in both type plants, but fruit of transgenic plants were more resistant to first instar larvae as indicated by significantly greater mortality. Basal stem sections were more resistant to neonate larvae of H. zea and adults of Carpophilus lugubris Murray compared to wild type plants as indicated by significantly greater mortality and/or reduced feeding damage. Thus, tobacco anionic peroxidase activity can increase plant resistance to insects in tomato, a plant species closely related to the original source plant species, when expressed at sufficiently high levels. However, the degree of resistance is dependent on the size of insect and plant tissue involved.     

Limited fitness advantages of crop-weed hybrid progeny containing insect-resistant transgenes (Bt/CpTI) in transgenic rice field

Background

The spread of insect-resistance transgenes from genetically engineered (GE) rice to its coexisting weedy rice (O. sativa f. spontanea) populations via gene flow creates a major concern for commercial GE rice cultivation. Transgene flow to weedy rice seems unavoidable. Therefore, characterization of potential fitness effect brought by the transgenes is essential to assess environmental consequences caused by crop-weed transgene flow.

Methodology/Principal Findings

Field performance of fitness-related traits was assessed in advanced hybrid progeny of F₄ generation derived from a cross between an insect-resistant transgenic (Bt/CpTI) rice line and a weedy strain. The performance of transgene-positive hybrid progeny was compared with the transgene-negative progeny and weedy parent in pure and mixed planting of transgenic and nontransgenic plants under environmental conditions with natural vs. low insect pressure. Results showed that under natural insect pressure the insect-resistant transgenes could effectively suppress target insects and bring significantly increased fitness to transgenic plants in pure planting, compared with nontransgenic plants (including weedy parent). In contrast, no significant differences in fitness were detected under low insect pressure. However, such increase in fitness was not detected in the mixed planting of transgenic and nontransgenic plants due to significantly reduced insect pressure.

Conclusions/Significance

Insect-resistance transgenes may have limited fitness advantages to hybrid progeny resulted from crop-weed transgene flow owning to the significantly reduced ambient target insect pressure when an insect-resistant GE crop is grown. Given that the extensive cultivation of an insect-resistant GE crop will ultimately reduce the target insect pressure, the rapid spread of insect-resistance transgenes in weedy populations in commercial GE crop fields may be not likely to happen.     

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

2000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（SGK321, 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Helicoverpa armigera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321(SGK321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321,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321和Bt棉,但其食物利用率显著高于石远321和Bt棉。