转cry1Ac玉米对亚洲玉米螟的抗性评价

目标性状的有效性评价是转Bt基因抗虫玉米研发和安全性评价研究的重要内容之一。采用酶联免疫吸附剂测定（ELISA）、室内生测和田间人工接虫鉴定研究了转cry1Ac玉米纯合群体BT-X和分离群体BT-38、BT-181、BT-43、BT-105等转化事件Cry1Ac蛋白的表达量以及对亚洲玉米螟Ostrinia furnacalis（Guenée）的室内杀虫和田间抗虫效果。转cry1Ac玉米鲜组织中Cry1Ac蛋白含量在44.07～438.00 ng/g之间,不同转化事件及不同组织之间差异显著,其中BT-X鲜花丝中Bt蛋白表达量显著低于未展开心叶。室内生测试验中除BT-43与对照没有显著差异外,取食其它转基因玉米心叶的亚洲玉米螟初孵幼虫6或7天的存活率为0～15.3%,显著低于非转基因对照;取食夏播BT-X鲜花丝6天的幼虫存活率为62.5%,显著低于Bt11及非转基因对照。田间心叶期接虫鉴定显示BT-X高抗亚洲玉米螟。说明转cry1Ac玉米BT-X、BT-181、BT-38和BT-105对亚洲玉米螟有很高的杀虫作用,BT-X田间抗虫效果良好。     

应用农杆菌介导法获得转大麦黄矮病毒GPV株系复制酶基因ORF1的小麦植株

 以生产用4种基因型小麦为材料,利用农杆菌介导法将大麦黄矮病毒GPV株系复制酶基因ORF1分别转化愈龄40d的幼胚愈伤组织,经过G418筛选后,共再生出15株抗性植株,拓宽了小麦农杆菌转化的受体基因型。PCR及Southern杂交分析证实大麦黄矮病毒复制酶基因ORF1已经整合到小麦基因组中,共获得14株转基因植株。抗病性初步检测结果显示,转基因植株对GPV株系具有抗性。研究结果还表明,对于不同的基因型小麦进行转化时,需选择适合本基因型的菌株。     

转cry1Ac+cry2Ab基因棉对棉田节肢动物群落的影响

以Bt棉和常规棉为对照,研究了转cry1Ac+cry2Ab棉对棉田节肢动物群落的影响。研究结果表明,转cry1Ac+cry2Ab棉田、转cry1Ac棉田和常规棉田节肢动物群落、害虫亚群落和天敌亚群落的结构与组成无明显差异;转cry1Ac+cry2Ab棉田刺吸类害虫、蛀食类害虫和食叶类害的数量均低于转cry1Ac棉田和常规棉田;转cry1Ac+cry2Ab棉田蜘蛛类、瓢虫类和捕食类天敌的数量均低于转cry1Ac棉田和常规棉田;转cry1Ac+cry2Ab棉田节肢动物群落、害虫亚群落和天敌亚群落的多样性指数与转cry1Ac棉田和常规棉田相比均无明显差异,说明转cry1Ac+cry2Ab棉田节肢动物群落、害虫亚群落和天敌亚群落的稳定性和转cry1Ac棉和常规棉相比均无明显差异。     

转cry1Ab+cry1C双价抗虫水稻对二化螟的抗性评价

为明确转cry1Ab+cry1C双价抗虫水稻对二化螟的抗性及其杀虫蛋白的时空表达,本研究采用离体水稻组织生测法系统评价了cry1Ab、cry1C、正交cry1Ab+cry1C和反交cry1C+cry1Ab 4种抗虫水稻品系对二化螟的杀虫效果,并用ELISA方法测定了Cry1Ab、Cry1C蛋白在各个抗虫水稻品系中的表达情况。结果显示,二化螟在不同生育期的4种转基因抗虫水稻上取食的叶面积显著低于非转基因对照亲本明恢63。4个转基因抗虫水稻品系在生长前期（苗期、分蘖和拔节期）对二化螟表现极高的杀虫效果,生长后期（孕穗期和成熟期）杀虫效果有所下降。两双价抗虫水稻杀虫效果最好,其次为转cry1Ab和cry1C水稻。Bt蛋白表达水平随着抗虫水稻生育期的变化而变化,且差异显著;Cry1Ab的蛋白表达量在水稻整个生长期均显著高于Cry1C。Cry1Ab蛋白在单价抗虫水稻叶片和茎杆中均表现出随生育期先升高后下降的趋势,但在双价抗虫水稻中表现出随生育期逐渐下降的趋势。Cry1C蛋白在单、双价抗虫水稻的叶片组织中均表现出逐渐升高的趋势。与单价抗虫水稻相比,双价抗虫水稻中的Cry1Ab、Cry1C的蛋白表达水平并没有表现出显著的降低。因此,双价抗虫水稻不仅能高效防治害虫,还能延缓害虫抗性,在生产上表现出良好的应用前景。该研究结果可望为转Bt基因抗虫作物的环境安全评价提供科学数据和理论依据。     

转苏云金杆菌毒蛋白基因水稻抗螟性的离体叶片测定法

提出了转苏云金杆菌ｃｒｙｌＡｂ基因水稻抗螟性的快速离体叶片测定法,并用该方法评价了转ｃｒｙ１／Ａｂ基因水稻对二化螟、三化螟和大螟的抗性程度。结果表明,３种螟虫蚁螟取食纯合株系ＰＲ１６及分离株系ＰＲ１８、ＰＲ５７或ＰＲ５８中抗性植株中片后,食叶量与对照和分离株系中的非抗性植株相比极显著下降,接虫后第４ｄ的幼虫死亡率高达１００％。该方法评价转ｃｒｙ１／Ａｂ基因水稻对３种螟虫的抗性结果与活化成株测定法评     

Resistance enhancement of transgenic tomato to bacterial pathogens by the heterologous expression of sweet pepper ferredoxin-I protein

ABSTRACT Expression of a foreign gene to enhance plant disease resistance to bacterial pathogens is a favorable strategy. It has been demonstrated that expressing sweet pepper ferredoxin-I protein (PFLP) in transgenic plants can enhance disease resistance to bacterial pathogens that infect leaf tissue. In this study, PFLP was applied to protect tomato (Lycopersicon esculentum cv. cherry Cln1558a) from the root-infecting pathogen, Ralstonia solanacearum. Independent R. solanacearum resistant T(1) lines were selected and bred to produce homozygous T(2) generations. Selected T(2) transgenic lines 24-18-7 and 26-2-1a, which showed high expression levels of PFLP in root tissue, were resistant to disease caused by R. solanacearum. In contrast, the transgenic line 23-17-1b and nontransgenic tomato, which showed low expression levels of PFLP in root tissue, were not resistant to R. solanacearum infection. The expansion of R. solanacearum populations in stem tissue of transgenic tomato line 24-18-7 was limited compared with the nontransgenic tomato Cln1558a. Using a detached leaf assay, transgenic line 24-18-7 was also resistant to maceration caused by E. carotovora subsp. carotovora; however, resistance to E. carotovora subsp. carotovora was less apparent in transgenic lines 26-2-1a and 23-17-1b. These results demonstrate that PFLP is able to enhance disease resistance at different levels to bacterial pathogens in individual tissue of transgenic tomato.     

Bt蛋白在水稻不同遗传背景下的含量及对抗虫性的影响

为明确Bt基因在水稻杂交转育过程中抗虫作用的稳定性,以分别转入cry1C和cry2A基因的抗虫水稻品系T1C-19和T2A-1为供体亲本,与10个恢复系杂交并回交,筛选BC1F3代转基因纯合株系,用ELISA法测定水稻叶片和茎秆中Bt蛋白含量。结果表明,4个转cry1C株系和5个转cry2A株系叶片和茎秆的蛋白含量显著低于亲本,且降低了16.62%~62.03%;相同遗传背景下,叶片和茎秆中的Bt蛋白含量之间呈显著正相关;在室内转cry1C株系和转cry2A株系上二化螟幼虫的校正死亡率分别为62.50%~95.83%和37.50%~83.33%,其中在cry2A株系上的校正死亡率与茎秆中的Cry2A蛋白含量呈极显著正相关;在田间自然发虫条件下,纯合株系的单株卷叶数和枯心率最高仅为3.97和9.37%,受害程度与相应组织中Bt蛋白含量之间存在相关性。表明Bt蛋白含量会受到水稻遗传背景的影响,但杂交后代株系在田间仍能表现出很好的抗性。     

氯虫苯甲酰胺在甘蔗及土壤中的残留消解动态

建立了氯虫苯甲酰胺在甘蔗和土壤中残留的高效液相色谱检测方法,应用该方法对氯虫苯甲酰胺在甘蔗和土壤中的残留消解规律进行了研究。样品用乙腈提取,经Florisil固相萃取柱及N-丙基乙二胺(PSA)净化,紫外检测器检测,外标法定量。结果表明:当添加水平为0.05~2 mg/kg时,氯虫苯甲酰胺在甘蔗植株地上部分(包括茎秆和叶片)、茎秆(去除叶片后的地上至肥厚带部位)和土壤中的平均回收率为77%~97%,相对标准偏差(RSD)为3.4%~11.6%。氯虫苯甲酰胺在3种基质中的最小检出量(LOD)均为0.2 ng,最低检测浓度(LOQ)均为0.05mg/kg。两年两地的田间残留试验结果表明:0.4%氯虫苯甲酰胺颗粒剂在甘蔗下种时一次沟施后覆土,以有效成分用量120和180 g/hm2施药时,在土壤中的原始沉积量在1.18~3.57 mg/kg之间,半衰期为12.4~18.2 d;成熟时采收,甘蔗茎秆中氯虫苯甲酰胺的残留量低于0.05 mg/kg。参照我国制定的甘蔗上氯虫苯甲酰胺的临时最大残留限量(TMRL)值0.05 mg/kg,按照推荐剂量及推荐剂量的1.5倍分别施药1次,成熟时采收甘蔗是安全的。     

Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes

BACKGROUND: Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis Berliner (Bt) were first commercialised in 1996. The risk that pests have the potential to evolve resistance to Bt toxins is one of the most serious challenges to this technology. Gene stacking, pyramiding two Bt genes into one variety, is considered to be an effective insect resistance management (IRM) strategy. In this study, insect‐resistant rice expressing two Bt genes was developed by sexual crossing, and then characterised. RESULTS: Homozygous rice lines of two pyramided Bt genes were obtained in the F₃ generation. Quantification of Bt toxin showed that protein concentrations of Cry1Ab, Cry1Ac and Cry2A in the two‐gene lines were comparable with their single‐gene parents, while the expression of cry1C gene decreased after gene stacking. Four two‐gene lines showed higher activity to striped stem borer (Chilo suppressalis Walker) than parental lines in the laboratory bioassay. All pyramided lines and their hybrids exhibited excellent efficacy against stemborers and leaffolders in field evaluation, while most pyramided lines had no significant differences from original variety in yield under spraying of insecticide. CONCLUSION: These results demonstrate that the two‐gene lines have commercial potential and could serve as a valuable IRM strategy. Copyright © 2011 Society of Chemical Industry     

应用农杆菌介导法获得转大麦黄矮病毒GPV株系复制酶基因ORF2小麦植株

以生产用5种基因型小麦为材料,利用农杆菌介导法将大麦黄矮病毒GPV株系复制酶基因ORF2转化愈龄40d的幼胚愈伤组织,经过G418筛选后,共再生出9株抗性植株,拓宽了小麦农杆菌转化的受体基因型。PCR及Southern杂交分析证实大麦黄矮病毒复制酶基因ORF2已经整合到小麦基因组中。初步抗病性检测结果显示转基因植株对GPV株系具有抗性。研究结果还表明,受体基因型、外植体的生理状态对农杆菌侵染起着至关重要的作用。     

转cry2Ah-vp基因玉米的抗虫性鉴定

为获得新型抗虫转基因玉米,将通过群体筛选获得的具有抗虫性的转cry2Ah-vp基因玉米VP1-5采用PCR、Southern blot、实时荧光定量PCR(qPCR)、酶联免疫吸附测定(ELISA)等方法进行阳性植株鉴定、拷贝数分析、转录水平和翻译水平分析,同时通过室内和田间生物活性测定鉴定转基因玉米VP1-5对东方黏虫Mythimna separata和亚洲玉米螟Ostrinia furnacalis的抗性。结果表明,在转基因玉米VP1-5中cry2Ah-vp基因已整合到玉米基因组,以单拷贝的形式插入;cry2Ah-vp基因在转基因玉米VP1-5不同部位组织中均可以正常转录,在灌浆期叶片中的mRNA表达量最高,相对表达量为32.67,在灌浆期穗轴中的mRNA表达量最低,相对表达量为3.74;Cry2Ah-vp蛋白在转基因玉米VP1-5的6叶期各组织中表达量均较高,其中在叶片中的表达量达到2 155.18 ng/g FW,在抽雄期花丝中的表达量最高,达到2 165.86 ng/g FW;且转基因玉米VP1-5对东方黏虫有很高的杀虫活性,接虫3 d后幼虫死亡率达到100.00%;对亚洲玉米螟幼虫也有明显的生长抑制作用。表明转基因玉米VP1-5可作为玉米抗虫育种和害虫防治的种质资源。     

甘蔗优良新品种(系)抗褐锈病基因Bru1的分子检测及自然抗性评价

甘蔗褐锈病是目前中国蔗区发生最普遍、危害最严重的病害之一,选育和种植抗病品种是防治该病最为经济有效的措施。为明确近年国家甘蔗体系育成的50个新品种(系)对甘蔗褐锈病的抗性,确定其应用潜力,本研究通过在甘蔗褐锈病高发蔗区云南德宏、保山2个区域化试验站,采用田间自然抗性调查与分子标记辅助鉴定抗性基因的方法,于2014年和2015年对中国近年选育的50个优良新品种(系)及2个主栽品种进行抗褐锈病基因Bru1的分子检测及自然抗性评价。田间自然发病调查结果表明,50个优良新品种(系)及2个主栽品种中,高抗至中抗的有34个,占65.38%。其中15个材料表现高抗,占28.85%,16个材料表现抗病,占30.77%,3个材料表现中抗,占5.77%;分子检测结果显示,共29个抗病材料含有抗褐锈病基因Bru1,出现频率为55.77%,表明中国近年选育的优良新品种(系)中抗褐锈病性主要由Bru1控制;其余5个抗病材料均不含抗褐锈病基因Bru1,暗示除了Bru1外,可能还有其他抗褐锈病基因存在。不同系列品种田间感病品种的频率和含抗褐锈病基因Bru1的频率不同,粤糖系列品种感病品种的频率最高达到60%,含Bru1的频率最低,只有30%,抗性最弱;云蔗系列品种感锈病品种的频率最低只有12.5%,含Bru1的频率最高,达到81.25%,抗性最好。本研究结果为深入开展甘蔗抗褐锈病育种,选育和推广优良抗病品种,有效防控甘蔗褐锈病提供了科学依据和优良抗性新品种(系)。     

转cry1Ab/cry2Aj玉米对亚洲玉米螟的抗性评价

为探讨转cry1Ab/cry2Aj玉米对亚洲玉米螟Ostrinia furnacalis(Guenée)的抗性,采用室内生测和田间人工接虫鉴定方法评价了6个转cry1Ab/cry2Aj玉米品系对亚洲玉米螟的杀虫效果,并利用酶联免疫吸附法(ELISA)测定了Cry1Ab蛋白在各品系主要组织的表达水平.结果显示,亚洲玉米螟幼虫取食转基因玉米各品系雄穗、苞叶、花丝和雌穗48 h后死亡率在90％左右,取食心叶的死亡率在70％左右,96h后大部分幼虫死亡,而取食非转基因玉米的死亡率为3.3％～8.7％;春播和夏播田转基因玉米叶片、茎秆、雌穗基本没有被害,而对照玉米叶片、茎秆、雌穗被害严重,春播和夏播玉米平均每株分别有2.9、2.3头幼虫和蛹,2.8、3.4个蛀孔,隧道长度为7.5、14.2 cm,雌穗被害级别为5.3和5.1;6个转基因玉米品系中Cry1Ab蛋白在心叶、雄穗、花丝、苞叶和雌穗中稳定表达.综合评价认为,转基因玉米各品系在整个生育期内对亚洲玉米螟有很好的抗性,其中N50品系抗虫效果最佳,可以作为抗虫转多基因玉米育种的备选材料.     

V-ATPase H表达量下调增强棉铃虫对Cry1Ac的抗性

棉铃虫Helicoverpa armigera是世界性重要农业害虫。目前防治棉铃虫的主要手段是种植转苏云金芽胞杆菌Bacillus thuringiensis(Bt)杀虫蛋白的转基因作物。本文旨在研究棉铃虫V-ATPase H在Cry1Ac蛋白毒力和抗性中的作用。利用实时荧光定量qRT-PCR技术分析V-ATPase H在Cry1Ac抗、感品系棉铃虫幼虫中肠及敏感品系棉铃虫幼虫受Cry1Ac诱导后的表达情况;在昆虫Sf9细胞中过表达V-ATPase H对其进行细胞定位,通过细胞毒力试验验证其对Cry1Ac毒力的影响。结果发现棉铃虫V-ATPase H基因在抗性品系中低表达,并且V-ATPase H在受到Cry1Ac诱导时也低表达;在Sf9细胞内表达V-ATPase H蛋白发现其在整个细胞中都有分布,过表达该蛋白后增强了细胞对Cry1Ac蛋白的敏感性。结果表明V-ATPase H参与Cry1Ac蛋白的毒力。     

Herbicide resistance in transgenic plants with mammalian P450 monooxygenase genes

Transgenic potato and rice plants were generated by the introduction of human P450 species, CYP1A1, CYP2B6, CYP2C9 and CYP2C19, which metabolized a number of herbicides, insecticides and industrial chemicals. The transgenic potato plant T1977 co-expressing CYP1A1, CYP2B6 and CYP2C19 genes showed remarkable cross-resistance to several herbicides with different structures and modes of action due to metabolism of these herbicides by the P450 species expressed. The transgenic rice plant 2C9-57R2 expressing CYP2C9 gene showed resistance to sulfonylureas, and the transgenic rice plant 2C19-12R1 expressing CYP2C19 gene showed cross-resistance to certain herbicides with different structures and modes of action. These transgenic plants appear to be useful for herbicide resistance as well as phytoremediation of environmental contaminants.     

对绿盲蝽具有杀虫活性Bt菌株的筛选及Cry15Aa蛋白活性的研究

Bt棉有效控制了棉田主要害虫棉铃虫(Helicoverpa armigera),然而原来处于次要地位的刺吸式口器害虫盲蝽(Heteroptera:Miridae)为害逐年加重,目前对绿盲蝽(Apolygus lucorum Meyer-Dür)有效的抗虫基因未见报道。本研究以本实验室保存的Bt杀虫基因和菌株为材料,对绿盲蝽进行杀虫活性筛选。利用本实验室先前克隆的Mtx类杀虫基因cry15Aa表达产物进行绿盲蝽杀虫活性测定,研究结果显示Cry15Aa蛋白对绿盲蝽具有一定的杀虫活性。通过杀虫活性测定,获得对绿盲蝽有效的Bt菌株20株;对这些菌株表达蛋白进行质谱鉴定,LC-MS/MS质谱鉴定结果显示,这些菌株可能含有Cry1Aa、Cry1Ab、Cry1Ac、Cry1Ae、Cry1Af、Cry1Ag、Cry1Ah、Cry1Ba、Cry1Be、Cry8Ha等十余种对鳞翅目、鞘翅目害虫有杀虫活性的蛋白片段,并且有4株Bt菌株样品中检出了Mtx类杀虫蛋白Cry15Aa的片段。但是进一步基因鉴定结果表明,这4株菌株并不含有cry15Aa全长基因,推测这些菌株中含有Cry15类的新基因。上述研究结果表明这些菌株中可能存在对盲蝽有效的新型杀虫蛋白。本研究在发现了cry15Aa基因所表达的蛋白对绿盲蝽具有杀虫活性的同时,获得了对绿盲蝽具有杀虫活性的Bt新菌株,对绿盲蝽的生物防治具有重要的意义。     

从配合力分析探讨甘蔗家系抗黑穗病的育种潜力

采用5×4不完全双列杂交设计选配的第1次无性种茎为试验材料,在人工浸渍接种黑穗病菌条件下,估算了9个亲本及其组合在新植蔗和宿根蔗的配合力效应。结果表明:①20个家系抗性差异较大,以CP67/412、ROCl为母本的半同胞家系表现较强抗病性,而以CP72/1210、CP65/357为母本的半同胞家系表现一定感病性;②甘蔗抗黑穗病遗传是由加性效应基因和非加性效应基因共同控制;③发现CP67/412、ROClgca效应值较高,CP57/614次之,均具有可作为抗源亲本的育种潜力,④根据配合力总效应值评价组合,认为CP67/412×崖84/153、ROCl×崖71/374、CP67/412×崖71/374、ROCl×崖84/153、CP67/412×崖73/512、CP57/614×崖84/153是抗黑穗病较强的组合,可用于今后抗黑穗病育种计划。     

转Bt 基因抗虫玉米对棉铃虫杀虫活性的初步评价

国内外在转 Bt基因抗虫作物的研究和应用上已取得重大进展 ,其中 Bt抗虫棉已在美国、澳大利亚、中国等进入商品化应用阶段 ,近年来种植面积增长很快 ,并取得了显著的经济、生态和社会效益。 1 997年下半年 ,我国农业部分别批准了孟山都公司申报的保铃棉○R在河北省商业化生产和 Bt玉米在我国多省份的田间种植。其中保铃棉 ○R已由河北省定名为新棉 33B,1 998年在全省实际种植了近 1 0万 hm2 ,1 999年的种植面积已达 1 5万 hm2 。我国自主研制的 Bt抗虫棉也于 1 998年开始商品化应用 ,当年种植面积即达 1 0万 hm2以上 ,1 999年推广总面积…     

Development of insect‐resistant transgenic rice with Cry1C*‐free endosperm

BACKGROUND: Yellow stem borer (Tryporyza incertulas Walker), striped stem borer (Chilo suppressalis Walker) and leaf folder (Cnaphalocrocis medinalis Guenec) are three lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, novel insect‐resistant transgenic rice was developed in which Bt protein expression was nearly absent in the endosperm. The resistant gene, cry1C*, driven by the rice rbcS promoter (small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase), was introduced into Zhonghua 11 (Oryza sativa L. ssp. japonica) by Agrobacterium‐mediated transformation. RESULTS: A total of 83 independent transformants were obtained, 19 of which were characterised as single‐copy foreign gene insertion. After preliminary screening of the T₁ families of these 19 transformants in the field, six highly insect‐resistant homozygous lines were selected. These six homozygous transgenic lines were field tested for resistance to leaf folders and stem borers, and for their agronomic performance. The Cry1C* protein levels in leaves and endosperm were measured by ELISA. Subsequently, the elite transgenic line RJ5 was selected; this line not only possessed high resistance to leaf folders and stem borers, normal agronomic performance, but also Cry1C* expression was only 2.6 ng g^?1 in the endosperm. CONCLUSION: These results indicated that RJ5 has the potential for widespread utility in rice production. Copyright © 2009 Society of Chemical Industry     

转cry1Ie基因抗虫玉米对田间节肢动物群落多样性的影响

为评价转cry1Ie基因（IE09S034转化体）抗虫玉米对田间节肢动物群落的影响,采用直接观察、地面陷阱、吸虫器和空中水盆诱捕4种方法,连续2年调查了转cry1Ie基因抗虫玉米田和非转基因对照玉米田在自然条件下的节肢动物物种数和个体数,比较分析了节肢动物群落各特征参数。结果显示,2012年依次采用此4种方法调查转cry1Ie基因抗虫玉米与非转基因对照玉米田间的节肢动物群落物种数、Simpson优势集中性指数、Shannon-Wiener多样性指数、Pielou均匀度指数分别为29.1、0.7、2.6、0.5,12.6、0.6、2.2、0.6,13.7、0.9、3.1、0.9和6.7、0.4、1.3、0.5,各参数之间均无显著差异。2013年调查结果与2012年一致,也显示2种玉米田的节肢动物群落各特征参数之间均无显著差异。表明转cry1Ie抗虫玉米对田间节肢动物群落多样性无显著影响。