转双抗虫基因741杨的抗虫效应及其规律研究

通过室内饲虫试验 ,应用昆虫的总死亡率、各龄期死亡率、各龄历期及发育速率、昆虫体重增长速率等指标 ,测定了转双抗虫基因 74 1杨的抗虫性。研究结果表明 ,转基因抗虫杨不同株系对昆虫的致死效应有显著差异 ;不同昆虫种类和同一昆虫幼虫的不同龄级对转基因杨株系的敏感性有明显差异 ,4种测试昆虫的敏感性强弱依次为 :杨小舟蛾 >美国白蛾 >杨扇舟蛾 >舞毒蛾 ,随着昆虫幼虫龄级的增加 ,昆虫对转基因杨株系的敏感性逐渐降低 ;高抗虫株系主要表现为对低龄幼虫的强致死作用 ,中等抗虫株系表现为对各龄幼虫稍低的但是持续的致死效应 ;各转基因杨株系对测试昆虫的生长和发育都有明显抑制作用 ,可导致昆虫的发育历期延长、发育速率和体重增长速率降低。     

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。     

转基因741杨节肢动物群落主要害虫及天敌的动态变化

在时间序列过程中,转基因741杨对目标害虫鳞翅目食叶害虫表现出持续的抗性,其数量明显减少,高抗和中抗741杨之间差异不大.研究表明,转基因741杨对目标昆虫和非目的植食性害虫存在负效应,而对天敌和中性节肢动物组成和发生无明显负作用.因而在制定害虫综合治理策略和途径上宜采取与对照741杨不同的措施,抗性株系应以生态调控为主.     

温度胁迫对舞毒蛾营养成分和总抗氧化力的影响

【目的】为了探究温度胁迫对舞毒蛾Lymantira dispar幼虫营养成分和总抗氧化力的影响。【方法】测定了不同温度(15、20、25、30、35、40℃)下处理3 h的舞毒蛾幼虫可溶性蛋白、碳水化合物、海藻糖、脂质含量以及总抗氧化力,以饲养温度25℃的舞毒蛾幼虫为对照。【结果】舞毒蛾幼虫在不同温度下可溶性蛋白含量变化无显著差异;碳水化合物与海藻糖含量均随着温度的升高,呈现逐渐下降趋势;脂质含量无论处理温度低于还是高于对照温度均会增加;而舞毒蛾幼虫总抗氧化力在经不同温度处理后均显著降低。【结论】温度变化可以显著影响舞毒蛾幼虫碳水化合物、海藻糖、脂质3种营养成分含量以及舞毒蛾幼虫的总抗氧化力的能力,而对舞毒蛾幼虫可溶性蛋白含量无显著影响。本研究对舞毒蛾幼虫的耐热性及其在不同温度下的防治具有指导意义。     

转不同rol基因741杨株系相关性状比较分析

对已转入rolB、rolC基因741杨株系进行分子检测,并对其根系生长状况、苗高、叶绿素a和b、叶绿素荧光以及内源激素等指标进行测定。结果表明,转rolB、rolC基因741杨各株系发根率、发根量均高于未转基因对照。转rolB基因741杨株系苗高与对照无显著差异,叶绿素a、b含量上升,Fv/Fm、PI值下降,IAA、ZT含量上升,ABA含量下降。转rolC基因741杨各株系顶端优势减弱,植株矮化,叶绿素a、b含量降低,Fv/Fm、PI值下降,IAA、ZT、ABA含量上升。总体比较,转rolB基因741杨株系生根率,叶绿素a、b含量,PI值、ZT和IAA含量均高于转rolC基因741杨株系。     

不同光照强度下兴安落叶松对舞毒蛾幼虫生长发育及防御酶的影响

为了探讨舞毒蛾对光照环境变化引起的落叶松抗性的变化是如何响应的,分析了不同光照强度处理的兴安落叶松对舞毒蛾幼虫生长发育状况和生理指标变化的影响。研究发现,取食50%和25%光照强度下生长的落叶松后,舞毒蛾幼虫平均体重、蛹重、化蛹率和羽化率与对照组相比均显著下降（P<0.05）。幼虫体内保护酶SOD、POD、CAT和解毒酶ACP、AKP、CarE、GSTs、MFO活性与对照组(100% 光照)相比均显著降低（P<0.05）且除CAT外在50%光照强度下降低最为显著（P<0.05）;4龄和5龄幼虫取食同一光强处理的落叶松后,SOD、POD、CarE、GSTs和MFO活性5龄显著低于4龄（P<0.05）,ACP和AKP活性5龄却显著高于4龄（P<0.05）,表明昆虫在不同的发育阶段启用不同的排毒酶系。光照差异对取食人工饲料的舞毒蛾生长发育和排毒代谢酶活性均没有显著影响,说明舞毒蛾生长发育和酶活性的变化是因寄主植物抗性的影响而非光照强度的作用。本研究结果表明,采取适当的营林措施调节林分内的光照条件,可以提高落叶松的自主抗虫性,增强其抵御害虫危害的能力,有效控制害虫危害,减少化学农药的施用量。     

Cd胁迫对舞毒蛾幼虫食物利用的影响及其Cd的外泌机制

为探明植食性昆虫对受重金属胁迫的寄主植物的生理生态响应机制,本研究用Cd胁迫下银中杨的叶片饲养舞毒蛾幼虫,分析舞毒蛾幼虫对食物的利用情况以及其对Cd的排毒代谢机制.结果表明: 取食Cd胁迫下银中杨的叶片后,舞毒蛾3～6龄幼虫体内的Cd浓度和Cd含量均显著高于对照,但随着幼虫龄期增长,其体内Cd浓度显著降低,而Cd含量有不同程度的提高;舞毒蛾幼虫粪便和虫蜕中的Cd浓度均显著高于对照;舞毒蛾3～5龄幼虫的食物消耗率显著高于对照,而转化率显著低于对照;3～4龄幼虫的食物利用率均与对照差异不显著,但在5龄时显著低于对照.说明在Cd胁迫下,舞毒蛾幼虫能通过有效的排毒代谢途径将体内富集的部分Cd排出体外,且高龄幼虫的排毒代谢能力强于低龄幼虫;舞毒蛾幼虫体质量的增加会对体内的Cd浓度形成一种稀释效应;舞毒蛾幼虫能通过调整食物消耗率和转化率之间的比例,来维持其正常生长发育所需的食物利用率,但超过一定限度后仍会造成食物利用率降低.     

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。     

小黑杨转双价抗虫基因的研究

用根癌农杆菌介导的叶盘法,将蜘蛛杀虫肽与Bt基因C肽序列的融合基因导入小黑杨(Populus simonii×P.nigra),所获得的3株卡那霉素抗性再生植株进行PCR和PCR-Southern杂交检测的结果均为阳性,Southern杂交检测的结果2株为阳性.抗虫试验结果显示,3个转基因株系均具有明显的抗虫效果,能显著抑制舞毒蛾幼虫的生长发育,表现出强烈的抗虫作用.     

靶标昆虫杨扇舟蛾对转Bt基因741杨胁迫效应的SSR分析

应用SSR分子标记技术,研究了以不同抗性转基因741杨为食物的杨扇舟蛾实验种群的遗传分化,探讨以转Bt基因杨树作为食物对靶标昆虫的胁迫效应.利用筛选出的10对引物,分别对经取食转基因741杨高抗品系‘Pb29’、中抗品系‘Pb17’及未转基因杨(对照)筛选出的杨扇舟蛾实验种群进行遗传多样性和遗传分化研究.结果表明: 10对引物共检测到76条等位基因,平均等位基因7.6个,平均有效等位基因为2.2,平均观测杂合度为0.5167,平均期望杂合度为0.5167,平均多态位点百分率达96.7%.经取食转基因741杨筛选出的杨扇舟蛾实验种群的遗传多样性水平显著高于未转基因种群,且取食转基因741杨的杨扇舟蛾与CK样本间的遗传相似性最低,表明经取食转Bt基因杨树的杨扇舟蛾实验种群遗传多样性有增高趋势.基于SSR分析,转基因741杨对杨扇舟蛾实验种群的胁迫筛选效应明显.     

靶标昆虫杨扇舟蛾对转Bt基因741杨胁迫效应的SSR分析

应用SSR分子标记技术,研究了以不同抗性转基因741杨为食物的杨扇舟蛾实验种群的遗传分化,探讨以转Bt基因杨树作为食物对靶标昆虫的胁迫效应.利用筛选出的10对引物,分别对经取食转基因741杨高抗品系‘Pb29’、中抗品系‘Pb17’及未转基因杨(对照)筛选出的杨扇舟蛾实验种群进行遗传多样性和遗传分化研究.结果表明: 10对引物共检测到76条等位基因,平均等位基因7.6个,平均有效等位基因为2.2,平均观测杂合度为0.5167,平均期望杂合度为0.5167,平均多态位点百分率达96.7%.经取食转基因741杨筛选出的杨扇舟蛾实验种群的遗传多样性水平显著高于未转基因种群,且取食转基因741杨的杨扇舟蛾与CK样本间的遗传相似性最低,表明经取食转Bt基因杨树的杨扇舟蛾实验种群遗传多样性有增高趋势.基于SSR分析,转基因741杨对杨扇舟蛾实验种群的胁迫筛选效应明显.     

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.     

转双抗虫基因741杨节肢动物群落营养结构及生态位变化

转双抗虫基因741杨（简称转基因741杨）节肢动物群落中,基位物种的植食性昆虫丰富度显著降低,但中性节肢动物丰富度却明显增加.高抗和中抗的节肢动物群落中位物种和顶位物种较之对照有所增多.转基因741杨节肢动物群落的害虫功能团,其优势状况,与对照相比有所改变：天敌优势度高于对照,中性节肢动物丰富度增加,并在天敌-害虫的营养链中起着重要的调控作用.鳞翅目害虫的空间生态位宽度最小,其它各功能类群的生态位宽度较大;捕食性和寄生性天敌与鳞翅目害虫的生态位重叠均较小,而与腐生和游逛种类的生态位重叠较大;各类害虫之间、捕食性天敌与寄生性天敌之间亦存在激烈竞争.转基因741杨对寄生性天敌和捕食性天敌在利用时间资源上有正作用.各种功能类群的时-空二维生态位宽度和生态位重叠均不如单维生态位宽度和生态位重叠值大,但抗性株系天敌类群对环境的适应性优于对照.     

Bacillus thuringiensis protein transfer between rootstock and scion of grafted poplar

Bacillus thuringiensis (Bt) Cry1Ac protein is a toxin against different leaf‐eating lepidopteran insects that attack poplar trees. In the present study, the mode of migration of the Bt‐Cry1Ac protein within poplar grafts was investigated. Grafting was done using Pb29 (transgenic poplar 741 with cry1Ac genes), CC71 (transgenic poplar 741 with cry3A genes), non‐transgenic poplar 741 and non‐transgenic Populus tomentosa, either as scion or as rootstock. In order to detect migration of Bt‐Cry1Ac protein from one portion of the graft union to different tissues in the grafted plant, ELISA analysis was employed to assess the content of Bt‐Cry1Ac protein in the phloem, xylem, pith and leaves of the grafted poplar. To further verify migration of Bt‐Cry1Ac protein, Clostera anachoreta larvae, which are susceptible to Bt‐Cry1Ac protein, were fed leaves from the control graft (i.e., graft portion that originally did not contain Bt‐Cry1Ac protein). The results showed that Bt‐Cry1Ac protein was transported between rootstock and scion mainly through the phloem. Migration of Bt‐Cry1Ac protein in the grafted union was also evidenced in that the leaves of the control graft did have a lethal effect on C. anachoreta larvae in laboratory feeding experiments.     

Expression of Bt-Cry3A in transgenic <Emphasis Type="Italic">Populus alba</Emphasis> × <Emphasis Type="Italic">P. glandulosa</Emphasis> and its effects on target and non-target pests and the arthropod community

During the growing seasons of 2006–2008, feeding tests and field studies were conducted in Beijing, China, to investigate the effects of transgenic Bacillus thuringiensis (Bt) poplar (BGA-5) expressing the Cry3A protein (0.0264–0.0326% of the total soluble protein) on target and non-target pests and the arthropod community. The effects of BGA-5 on the target pest Plagiodera versicolora (Coleoptera, Chrysomelidae) and a non-target pest Clostera anachoreta (Lepidoptera, Notodontidae), were assessed under laboratory conditions. Total mortality of P. versicolora larvae fed with BGA-5 leaves was significantly higher than that of the control (P < 0.05). The exuviation index of P. versicolora larvae fed with BGA-5 tended to be higher than that of CK, but it was not significantly different. The pupation rate and eclosion rate of the survived larvae fed with BGA-5 were lower than that of CK, but it was also not significantly different. Additionally, no significant differences were detected in the mortality, exuviations index, pupation rate, or eclosion rate of C. anachoreta fed with leaves of transgenic and non-transgenic poplars. Furthermore, the arthropod communities in the Bt poplar and CK field stands were similar, as indicated by four diversity indices (Berge-Parker index, Shannon-Wiener indices, evenness index, and Simpson’s inverted index) and the Bray-Curtis index. Therefore, the Bt-Cry3A poplar decreased damage by the target pest (P. versicolora), had no effects on a non-target pest (C. anachoreta), and generally did not have any significant negative effect on the poplar arthropod community.     

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae)

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.