转Bt cry1Ah基因抗虫玉米的分子检测及农艺性状分析

利用农杆菌介导法将具有自主知识产权的Bt cry1Ah基因转入玉米自交系综31中,在获得的1 764株转基因植株中筛选获得1株对玉米螟有显著抗性的转基因植株。对该抗性植株进行了室内和田间抗虫性分析及外源基因表达分析,并研究了其杂交后代的农艺性状。结果表明,该转基因植株对亚洲玉米螟田间抗虫性达到高抗水平;外源基因能够正常高效表达,在玉米抽穗期的叶片中Cry1Ah蛋白表达量最高,达到1μg/g鲜重;株高、穗位高、穗长、穗粗等与非转基因对照植株相比差异不大,秃尖长明显短于对照,千粒重及单株产量明显高于对照,这种差异在人工模拟虫害爆发情况下达到极显著水平。     

基因枪法转化水稻E32后代非目标农艺性状变异的研究

采用基因枪法将4个抗真菌性病害基因(RAC22、RCH10、β-1,3-Glu和B-Rip)导入超级稻(Oryza sativa L.)恢复系E32中,经选择获得10个T4代转基因株系。分蘖盛期纹枯病抗性鉴定表明外源基因已在转基因株系中得到表达并获得抗性,这些转基因株系除了产生抗病性等目标性状的变异外,还发生生育期、植株形态、谷粒外观及产量性状等非目标农艺性状变异。对这些非目标性状进行遗传增益和稳定性分析表明,不同转化株系间或性状间的遗传增益均存在较大差异,其中遗传增益最高的性状是单株穗数、着粒密度和单株产量;各性状中以株高、穗长和谷粒外观性状的变异程度最低,单株穗数和千粒重的变异系数最大。通过选择可获得产量与品质性状均有提高的转基因水稻。     

转(Bt Cry1Ac+CP_4EPSPS)基因抗虫抗草甘膦棉花对草甘膦的耐受性研究

本研究比较了转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花与常规棉花在新疆棉区对草甘膦的耐受性差异。两年的研究结果表明,转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花对草甘膦有较好的耐受性,而对照棉花中棉所49对草甘膦耐受性较差。苗期喷施草甘膦后转基因抗棉铃虫抗草甘膦棉花生长发育没有受到影响,而对照棉花中棉所49喷施草甘膦后生长发育受到了明显的影响,个别植株甚至死亡。转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花株高、真叶数、蕾数、产量等指标与对照相比差异显著。转Bt Cry1Ac+CP_4EPSPS基因棉花对草甘膦的耐受程度显著高于非转基因棉花。草甘膦对转基因抗草甘膦棉花无负面影响。     

Bt水稻田重要非靶标节肢动物暴露于Cry2Aa蛋白的程度分析

根据风险=危险×暴露的原理,在实验室条件下评价转基因作物对非靶标节肢动物影响时,所选择的代表性非靶标生物通常是在农田系统中较高地暴露于转基因外源杀虫蛋白的节肢动物种.为了弄清Bt稻田主要节肢动物暴露于Cry蛋白的程度,选择合适的非靶标节肢动物,用于转基因抗虫水稻的风险评价,本文采用酶联免疫技术检测了水稻不同生长期从转cry2Aa基因水稻田采集的不同节肢动物体内Cry2Aa蛋白的含量.结果表明: 不同节肢动物种体内的Cry蛋白含量差异显著.一些节肢动物体内不含Cry蛋白,而一些节肢动物体内含有较高的Cry蛋白;相对于花期后采集的节肢动物,在Bt水稻花期采集的节肢动物,特别是捕食性节肢动物体内的Cry蛋白含量较高;寄生性节肢动物体内未检测到Cry蛋白.这为在实验室条件下评价转基因水稻对农田非靶标节肢动物的影响奠定了基础.     

转Bt基因及其亲本水稻上褐飞虱对三唑磷和溴氰菊酯的反应

以转Bt基因抗虫水稻T1C-19(含cry1C基因)和T2A-1(含cry2A基因)及其亲本水稻MH63为材料,用20和40 mg·L^-1的三唑磷以及1、3和6 mg·L^-1的溴氰菊酯喷雾分别处理稻株上的3龄褐飞虱若虫,研究了两种农药对转Bt基因抗虫水稻上褐飞虱再猖獗的影响.结果表明： 三唑磷处理对褐飞虱的若虫历期无显著性影响,溴氰菊酯则能显著降低若虫历期,但随着两种药剂处理浓度的升高,若虫的存活率降低、成虫的产卵量增加.在同一浓度农药药剂处理下,3个水稻品种上的褐飞虱若虫发育历期、若虫存活率、初羽雌成虫体质量、产卵量和卵孵化率等生态学参数均没有显著差异.表明褐飞虱在两种转Bt水稻上对三唑磷和溴氰菊酯诱导再猖獗的反应能力与其亲本水稻MH63没有差异.     

转拟南芥NPR1基因桂99 T3代植株的抗病性及农艺性状表现

以转拟南芥AtNPR1基因的恢复系品种桂99T3代纯合株系为材料,考查其农艺性状及其抗病性,并比较转基因植株与桂99侵染水稻白叶枯病菌后的农艺性状。结果表明:转基因植株表现出对水稻白叶枯病的抗性显著增强77%以上;穗长、剑叶长、有效穗数、一次枝梗数、每穗实粒数、单株产量和谷粒宽等农艺性状与未转基因桂99无显著差别。在受到水稻白叶枯病菌侵染后,转基因植株的一次枝梗数、每穗粒数、每穗实粒数和单株产量等方面均比对照桂99高出13%～78%。说明AtNPR1基因增强了水稻的抗病能力,从而降低了病害引起的产量损失。转基因植株的恢复力不受影响,稻米品质比桂99更加优良。本工作为转基因水稻抗病育种的研究奠定了基础。     

广西兴安转Bt水稻大田两迁害虫发生动态

以转Bt水稻华恢1号(Cry1Ac/ CryAb融合基因型,简称HH1)及其对照亲本明恢63(简称MH63)稻田两迁害虫稻纵卷叶螟和白背飞虱为研究对象,系统研究转基因抗虫水稻种植下两迁害虫的发生规律及其致害力差异.结果表明:转Bt水稻及其对照亲本上稻纵卷叶螟的落卵量和幼虫发生量无显著差异,但转Bt水稻的卷叶株率和卷叶率都显著低于对照亲本.表明转Bt水稻对靶标害虫稻纵卷叶螟具有较高抗性.转Bt水稻及对照亲本上白背飞虱若虫、成虫及整个种群的发生动态差异不显著,且转Bt水稻对长翅型和短翅型成虫的种群发生影响也不显著.白背飞虱发生高峰期,转Bt水稻上若虫及短翅型成虫发生量均明显高于对照亲本;相反,转Bt水稻上长翅型成虫发生量明显低于对照亲本,且水稻生育后期长翅型成虫雌性比明显低于对照亲本.转Bt水稻大面积商业化种植下其非靶标害虫白背飞虱的发生危害变得更为复杂.     

转Bt基因抗虫玉米HGK60的农艺性状分析

为了确定转Bt cry1Ah抗虫玉米HGK60的自交系及其杂交后代外源基因的遗传表达稳定性和农艺性状,通过实时荧光定量PCR和ELISA分析外源基因的遗传表达稳定性,通过室内外生测和田间性状考量分析农艺性状。荧光定量PCR结果表明Bt cry1Ah基因在玉米的不同组织中可以正常转录,但RNA表达水平存在一定的差异;ELISA结果表明在转基因植株的不同发育时期、不同器官中Cry1Ah的蛋白表达量顺序:雄穗叶片苞叶籽粒花丝穗轴。两地连续三代的田间及室内抗虫性检测结果表明HGK60抗虫玉米对亚洲玉米螟均表现出很好的抗性。性状考量结果表明HGK60抗虫玉米与受体材料对照比较,种子发芽率、生育期、穗行数、穗长、千粒重等农艺性状均无显著差异。通过多年多点田间试验和分子检测结果证明HGK60转基因抗虫玉米中外源基因稳定的遗传和表达,对亚洲玉米螟有很好的抗性,农艺性状与对照材料无显著差异。HGK60转基因抗虫玉米对亚洲玉米螟的良好抗性使其具有很好的产业化应用前景。     

转Bt抗虫基因水稻的研究进展和生物安全性及其对策

转基因抗虫水稻的商品化种植已成为国内外广泛关注的热点问题.综述了转Bt(Bacillus thuringiensis)抗虫基因水稻的研究进展、食品安全性、生态安全性以及对转Bt基因水稻本身的性状变化的影响,并提出了防范策略,以期为转Bt基因水稻的生物安全性评价和商品化种植提供科学参考.     

NaCl胁迫对转基因杨树外源基因表达的影响

以具高抗虫性的转抗虫基因‘741杨’及在此基础上转入了发根农杆菌Ri质粒T-DNA株系的组培苗为材料,研究了转基因株系BtCrylAc抗虫基因和发根基因的表达及其对NaCl胁迫的反应。结果表明,转入Ri质粒T- DNA上的rol基因后,导致苗木根系数目增加,根系长度减小,IAA和GA含量显著提高,抗虫BtCrylAc基因编码的毒蛋白的表达量降低;随着NaCl胁迫强度的增加,苗高、根系数量、叶绿素含量及IAA、GA含量逐步降低,而根系的长度加大,Bt毒蛋白含量显著提高,表明NaCl胁迫使转基因杨外源Bt毒蛋白基因的表达增强,而发根农杆菌Ri质粒T-DNA的表达下降。     

Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae)

Ten transgenic Bacillus thuringiensis Bt rice, Oryza sativa L., lines with different Bt genes (two Cry1Ac lines, three Cry2A lines, and five Cry9C lines) derived from the same variety Minghui 63 were evaluated in both the laboratory and the field. Bioassays were conducted by using the first instars of two main rice lepidopteran insect species: yellow stem borer, Scirpophaga incertulas (Walker) and Asiatic rice borer, Chilo suppressalis (Walker). All transgenic lines exhibited high toxicity to these two rice borers. Field evaluation results also showed that all transgenic lines were highly insect resistant with both natural infestation and manual infestation of the neonate larvae of S. incertulas compared with the nontransformed Minghui63. Bt protein concentrations in leaves of 10 transgenic rice lines were estimated by the sandwich enzyme-linked immunosorbent assay. The cry9C gene had the highest expression level, next was cry2A gene, and the cry1Ac gene expressed at the lowest level. The feeding behavior of 7-d-old Asiatic rice borer to three classes of Bt transgenic rice lines also was detected by using rice culm cuttings. The results showed that 7-d-old larvae of Asiatic rice borer have the capacity to distinguish Bt and non-Bt culm cuttings and preferentially fed on non-Bt cuttings. When only Bt culm cuttings with three classes of different Bt proteins (CrylAc, Cry2A, and Cry9C) were fed, significant distribution difference of 7-d-old Asiatic rice borer in culm cuttings of different Bt proteins also was found. In the current study, we evaluate different Bt genes in the same rice variety in both the laboratory and the field, and also tested feeding behavior of rice insect to these Bt rice. These data are valuable for the further development of two-toxin Bt rice and establishment of appropriate insect resistance management in the future.     

Field performance of transgenic elite commercial hybrid rice expressing bacillus thuringiensis delta-endotoxin

Here we describe development of transgenic elite rice lines expressing a Bt fusion gene derived from cryIA(b) and cryIA(c) under the control of rice actinI promoter. The lines used in the study were indica CMS restorer line of Minghui 63 and its derived hybrid rice Shanyou 63. The level of Bt fusion protein CryIA(b)/CryIA(c) detected in Minghui 63 (T51-1) plants was 20 ng/mg soluble protein. The Bt Shanyou 63 was field-tested in natural and repeated heavy manual infestation of two lepidopteran insects, leaffolder and yellow stem borer. The transgenic hybrid plants showed high protection against both insect pests without reduced yield.     

Tolerance and compensatory response of rice to sugarcane borer (Lepidoptera: Crambidae) injury

A 3-yr field experiment was conducted to evaluate the tolerance and compensatory response of rice (Oryza sativa L.) to injury caused by sugarcane borer, Diatraea saccharalis (F.), as affected by cultivar (Cocodrie, Francis, and Jefferson), stage of crop growth during which the injury occurred (third tiller stage, panicle differentiation stage, and heading stage), and sugarcane borer density. The proportion of rice tillers with sugarcane borer injury (leaf and leaf sheath injury and/or stem injury) was lower when injury occurred at the third tiller stage (0.05) than at panicle differentiation (0.19) and heading (0.18). When injury occurred at the two latter stages, both the proportion of tillers with injury and the proportion of tillers with stem injury were negatively correlated with rainfall. Rainfall resulted in dislodgement and mortality of sugarcane borer eggs and larvae before the larvae entered the stems. Rice plant density in this study (111.1 plants/m2) was higher than recorded for previous research on rice compensation using potted rice or conducted in low-density hill production systems (26.7-51.3 plants/m2). Two mechanisms of within-plant tolerance/compensation were observed. Stem injured plants produced approximately 0.69 more tillers than uninjured plants, whereas tillers with leaf and leaf sheath injury produced larger panicles, up to 39.5 and 21.0% heavier than uninjured tillers, when injury occurred at third tiller stage and at panicle differentiation, respectively. Rice yield was not reduced with up to 23% injured tiller and up to 10% injured stems at the third tiller stage, 42% injured tillers and 17% injured stems at panicle differentiation, and 28% injured tillers and 14% injured stems at heading. Significant between-plant compensation was not detected, suggesting competition between adjacent plants is not significantly reduced by injury. Our results suggest that rice can tolerate and/or compensate for a level of stem borer injury previously considered to be economically damaging.     

Overview on current status of biotechnological interventions on yellow stem borer Scirpophaga incertulas (Lepidoptera: Crambidae) resistance in rice

Yellow stem borer (YSB), Scirpophaga incertulas (Lepidoptera: Crambidae), a monophagous pest of paddy is considered as most important pest of rain fed low land and flood prone rice eco-systems. Breeding of yellow stem borer resistance in rice is difficult owing to the complex genetics of the trait, inherent difficulties in screening and poor understanding of the genetics of resistance. On the other hand, a good level of resistance against the widespread yellow stem borer has been rare in the rice germplasm. Resistance to insects has been demonstrated in transgenic plants expressing genes for δ-endotoxins from Bacillus thuringiensis (Bt), protease inhibitors, enzymes and plant lectins. The performance of insect resistant GM rice in trials in China has been quite impressive. The present review is an attempt to assess the current state of development in biotechnological intervention for yellow stem borer resistance in rice.     

长江三角洲主要超级稻CH4排放特征及其与植株生长特性的关系

采用盆栽试验研究了长江三角洲14个主要超级稻品种(6个粳型超级稻和8个籼型杂交超级稻)CH₄排放特征及其与植株生长特性之间的关系.结果表明: 粳型和籼型超级稻全生育期CH₄排放均呈双峰模式,排放峰值分别出现在分蘖盛期和孕穗期.粳型超级稻的平均CH₄排放总量比籼型超级稻高37.6%(P<0.01),品种间排放差异主要出现在生长后期.虽然两种类型超级稻的CH₄排放总量均与最大叶面积呈显著正相关,但CH₄排放与其他生长特性的关系因品种类型而异.在株高上,粳型超级稻CH₄排放总量与株高呈显著正相关,而籼型超级稻的相关不显著.在生产力上,籼型超级稻CH₄排放总量与其总生物量、籽粒产量和收获指数呈显著负相关,而粳型超级稻的相关不显著.籼型超级稻CH₄排放量低主要是由于其根系生物量显著高于粳型超级稻.     

高温胁迫对Bt棉铃壳中Bt蛋白含量及氮代谢的影响

以Bt基因来源于中国的棉花品种泗抗1 号(常规种)、泗抗3 号(杂交种)和来源于美国的棉花品种99B(常规棉)、岱杂1 号(杂交棉)为材料,研究了不同高温水平下Bt 棉盛铃期铃壳中Bt 蛋白含量变化及氮代谢生理特征.结果表明: 铃壳中Bt 蛋白含量随温度升高而降低,与对照相比(32 ℃),常规棉品种在38 ℃、杂交棉品种在40 ℃以上时,铃壳中Bt 蛋白含量大幅度下降.其中,常规种泗抗1号和99B在38 ℃时分别下降53.0%和69.5%;杂交种泗抗3号和岱杂1号在40 ℃时下降64.8%和54.1%.铃壳Bt 杀虫蛋白含量下降显著时,其可溶性蛋白含量明显下降,游离氨基酸含量明显提高,GPT活性显著下降,蛋白酶活性显著增加.高温影响铃壳的氮代谢引起Bt蛋白的分解加剧,合成减弱,从而造成Bt蛋白含量减少,抗虫性下降.     

南方菟丝子寄生对加拿大一枝黄花生长、繁殖及防御的影响

为了阐明南方菟丝子寄生对加拿大一枝黄花生长和繁殖的影响及机制,分析了南方菟丝子寄生对加拿大一枝黄花形态、生长与繁殖指标的影响,并分析了其生物量与加拿大一枝黄花次生代谢产物含量的相关性.结果表明： 南方菟丝子寄生显著降低加拿大一枝黄花株高、基径、主根长度和根直径,显著降低根、茎、叶和总生物量以及花序数、花序主轴长和花序分枝数;尤其是株高、花序数、茎生物量3个指标,被南方菟丝子寄生的植株仅为无寄生植株的1/2、1/5和1/8.南方菟丝子寄生强度对加拿大一枝黄花的株高、根长、茎生物量、总生物量等指标没有显著影响,但高强度寄生组的基径、根体积、叶生物量、根生物量、花序数、花序主轴长、花序分枝数等指标均显著低于低强度寄生组.南方菟丝子寄生显著增加加拿大一枝黄花根鞣质含量和茎黄酮含量.加拿大一枝黄花的生物量与根鞣质含量及茎黄酮含量存在显著负相关.这表明南方菟丝子寄生加拿大一枝黄花后, 除了减少其资源获取外,还可能通过改变资源分配方式等途径进一步抑制其生长.     

长江三角洲主要超级稻CH4排放特征及其与植株生长特性的关系

采用盆栽试验研究了长江三角洲14个主要超级稻品种(6个粳型超级稻和8个籼型杂交超级稻)CH₄排放特征及其与植株生长特性之间的关系.结果表明: 粳型和籼型超级稻全生育期CH₄排放均呈双峰模式,排放峰值分别出现在分蘖盛期和孕穗期.粳型超级稻的平均CH₄排放总量比籼型超级稻高37.6%(P<0.01),品种间排放差异主要出现在生长后期.虽然两种类型超级稻的CH₄排放总量均与最大叶面积呈显著正相关,但CH₄排放与其他生长特性的关系因品种类型而异.在株高上,粳型超级稻CH₄排放总量与株高呈显著正相关,而籼型超级稻的相关不显著.在生产力上,籼型超级稻CH₄排放总量与其总生物量、籽粒产量和收获指数呈显著负相关,而粳型超级稻的相关不显著.籼型超级稻CH₄排放量低主要是由于其根系生物量显著高于粳型超级稻.     

不同遗传背景下杂交籼稻功能叶与产量构成因素的相关性

为明确水稻功能叶与产量构成因素间的相关性,以不同遗传背景下籼稻的10个不育系和16个恢复系为亲本,按照NCII设计配制两套双列杂交组合,对水稻12个功能叶性状与8个产量性状构成因素进行了相关分析,结果表明：3片功能叶叶长与叶面积、剑叶宽、倒2叶宽等性状之间均存在极显著正相关,功能叶夹角之间也存在极显著正相关,但不同遗传背景对夹角性状与9个形态性状之间的相关性则存在明显差异,在第1套组合中,其相关系数均为负值,且相关均不显著;而第2套组合则相反。8个产量构成因素中,单株穗数与平均穗长、着粒密度、穗实粒数以及穗着粒数之间存在极显著负相关,平均穗长与穗着粒数、结实率与单株产量呈显著或极显著正相关,遗传背景对产量组成上有较大影响,在第1套组合中单株产量主要由结实率、单株穗数以及穗实粒数等性状决定,而在第2套中则主要由穗实粒数和结实率等性状决定。在功能叶与产量构成因素的相关中,叶长、叶面积、剑叶宽、倒2叶宽与着粒密度、穗实粒数以及穗着粒教等3个性状之间存在显著或极显著正相关。12个水稻功能叶性状与8个产量构成因素之间的主成分分析表明,在不同的遗传背景下,产量构成因素均主要受叶面积和叶夹角影响,两种不同遗传背景中其累积贡献率分别为69．8％和84．0％。