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转基因玉米表达的Cry1Ab、PAT和EPSPS蛋白对日本通草蛉幼虫的安全风险评估
The risk assessment of Cry1Ab, EPSPS, and PAT proteins expressed by transgenic corn for Chinese green lacewing Chrysoperla nipponensis larvae
 [PDF]

高欣欣,全玉东,王振营,白树雄,张天涛,何康来,Gao Xinxin,Quan Yudong,Wang Zhenying,Bai Shuxiong,Zhang Tiantao,He Kanglai
- , 2018, DOI: 10.13802/j.cnki.zwbhxb.2018.2017003
Abstract: 为评价我国自主研发的转基因玉米表达的Cry1Ab、PAT和EPSPS蛋白对日本通草蛉Chrysoperla nipponensis幼虫生长发育的安全风险,通过将外源蛋白混入日本通草蛉人工饲料中的方法,以加入砷酸二氢钾(KH2AsO4)的饲料为阳性对照,研究了日本通草蛉幼虫取食后的生长发育状况。结果表明:日本通草蛉取食含Cry1Ab和EPSPS蛋白饲料的幼虫发育历期、茧期、结茧率、羽化率及成虫体重等生物学参数与取食正常饲料处理相比均没有显著差异;而取食含PAT蛋白饲料的幼虫发育历期和结茧率分别为10.7 d和96.6%,与取食正常饲料的对照11.1 d和89.9%差异显著,即饲料中添加PAT蛋白显著提高了幼虫的存活率和发育速率;取食含KH2AsO4饲料的日本通草蛉幼虫不能存活到茧期,说明KH2AsO4具有显著的杀虫活性。ELISA检测结果表明,取食了分别添加有3种外源蛋白饲料的日本通草蛉幼虫体内可以检测到相应的Cry1Ab、EPSPS和PAT蛋白,含量分别为2 758.8~5 210.7、35 018.0~54 426.6、16.8~149.8 ng/g。表明转基因玉米所表达的Cry1Ab、EPSPS和PAT蛋白对日本通草蛉幼虫没有显著的不利影响。
To understand the environmental safety of domestic biotech maize on natural enemies, the impact of Chinese biotech maize events C0030.3.5 expressing Cry1Ab and EPSPS proteins and C0010.1.1 expressing PAT and EPSPS proteins on the development and survival of Chrysoperla nipponensis larvae were assessed through diet incorporation bioassay, i. e. Tier-1 Approach. Diet mixed with potassium arsenate (KH2AsO4) was the positive control. The results showed that the biological parameters, including larval duration, pupation rate, pupation stage, eclosion rate, and adult fresh weight of C. nipponensis, had no significant difference between the tested C. nipponensis and the control when the artificial diet contained purified Cry1Ab, EPSPS. However, the larval duration and pupation rate were 10.7 d and 96.6%, respectively, when larvae were fed on diet containing PAT protein, which were significantly shorter and higher than those (11.1 d and 89.9%) of the control. In contrast, the larvae could not develop normally into pupae when they were fed on diet containing KH2AsO4, indicating that KH2AsO4 was toxic to the larvae. The ELISA results showed that the contents of Cry1Ab, EPSPS and PAT proteins were 2 758.8-5 210.7, 35 018.0-54 426.6, and 16.8-149.8 ng/g, respectively. The results indicated that the Cry1Ab, EPSPS and PAT proteins expressed by biotech maize events C0030.3.5 and C0010.1.1 had no adverse effects on the survival and development of C. nipponensis larvae.
Bt稻Cry1Ab蛋白的表达和降解及对采后季节土表灰橄榄长角跳虫发生的影响  [PDF]
闫瑞红,白耀宇,程家安,叶恭银
植物保护学报 , 2009,
Abstract: Bt水稻及其植株残体中的Bt杀虫蛋白对土壤跳虫具有潜在的生态风险。作者以Bt杂交稻东龙(DL)及非亲本对照汕优46(XY)和西农优1号(XN)为材料,采用ELISA法分析了DL各植株组织中Cry1Ab蛋白的田间表达和采后残留,并评价了采后季节DL植株秸秆还田对稻田土表跳虫发生量的影响。结果表明,在测定的各生长阶段,DLCry1Ab在叶、茎和根系中的表达量为2.49~16.13μg/gFW;在采后季节,DL各秸秆还田植株残体中的Cry1Ab可残留较长时间,在试验第132天地表茎叶、稻桩和根系中的残留量分别为0.072、0.074和0.033?μg/gDW;DL秸秆还田未对采后季节稻田土表发生量大的灰橄榄长角跳虫Entomobryagriseoolivata(Packard)种群数量产生显著影响,该跳虫种群密度动态在DL与对照XY和XN稻田基本相似,多数调查日期发生量无显著差异。
转cry1Ab基因水稻中毒蛋白的表达、分泌及其在土壤中的残留  [PDF]
吴立成,李啸风,叶庆富,汪海燕
环境科学 , 2004,
Abstract: 研究了转cry1Ab基因水稻(克螟稻)中Cry1Ab毒蛋白的表达、根系分泌及其在土壤中的残留规律.结果表明,分蘖始期至成熟期,克螟稻地上部和根部中的Cry1Ab毒蛋白的表达量(FW)分别为3.23~8.22μg/g和0.68~0.89μg/g.生育期间克螟稻根系分泌的Cry1Ab毒蛋白含量仅为1.66~48.02ng/(株·d).试验证实,克螟稻根际土中的Cry1Ab毒蛋白残留含量低于检测限(<0.5ng/g干燥土).生物测定还表明,克螟稻根际土及其提取液对棉铃虫初孵幼虫和3龄幼虫不产生致死性效应.相对于Cry1Ab毒蛋白的根系分泌转移,克螟稻秸秆还田对土壤环境的影响应引起重视.
Identification of Genetic Elements Associated with EPSPS Gene Amplification  [PDF]
Todd A. Gaines, Alice A. Wright, William T. Molin, Lothar Lorentz, Chance W. Riggins, Patrick J. Tranel, Roland Beffa, Philip Westra, Stephen B. Powles
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0065819
Abstract: Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world’s most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S) A. palmeri, and that only one of these was amplified in glyphosate-resistant (R) A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs) were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac) transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene.
转Bt基因水稻表达的毒蛋白Cry1Ab在害虫及其捕食者体内的积累动态  [PDF]
姜永厚,傅强,程家安,祝增荣,蒋明星,叶恭银,张志涛
昆虫学报 , 2004,
Abstract: 在室内对转基因水稻KMD1中的Cry1Ab毒蛋白经食物链在几种主要害虫及其捕食性天敌体内的积累进行了研究。结果表明:无论是水稻孕穗期还是成熟期,二化螟Chilosuppressalis连续取食KMD1或取食KMD1.36h后移至对照品种秀水11上取食不同时间后,幼虫体内的Cry1Ab含量均随取食时间延长逐渐下降。稻眼蝶Mycalesisgotama幼虫连续取食KMD1或在KMD1上取食两天后移至秀水11上继续取食不同时间,体内的Cry1Ab含量也都随取食时间延长而下降,但下降速度较二化螟更快。取食KMD1的二化螟和稻眼蝶幼虫的粪便中均检测到较高浓度的Cry1Ab,对照组中均无Cry1Ab。取食KMD1的二化螟幼虫血淋巴中检测到Cry1Ab,含量为3.5ng/g。取食KMD1的褐飞虱Nilaparvatalugens、稻蚜Sitobionavenae以及饲喂取食过KMD1的二化螟或稻眼蝶幼虫的拟水狼蛛Piratasubpiraticus体内都含有一定浓度的Cry1Ab,其中,拟水狼蛛体内的CrylAb含量以饲喂取食KMD1稻眼蝶幼虫的含量最高,约为饲喂取食KMD1二化螟幼虫的60倍。这些结果表明Cry1Ab可以沿水稻害虫天敌食物链传递。
bt水稻cry1ab杀虫蛋白表达的时间动态及其在水稻土中的降解  [PDF]
白耀宇?,蒋明星?,程家安?
生态学报 , 2005,
Abstract: 采用elisa技术检测了bt水稻克螟稻1号(kmd1)和克螟稻2号(kmd2)不同生育期茎和叶片中cry1ab基因的表达水平,以及植株组织(茎和叶片)室内埋于不同稻田土壤中其cry1ab杀虫蛋白的降解动态。结果表明,不论是kmd1还是kmd2,茎和叶片中cry1ab基因的表达量为3.7~9.1μg/g鲜重,均以拔节期和孕穗期相对较低,灌浆初期显著升高,黄熟期又明显下降(但仍然维持在较高的表达量)。kmd1茎和叶片中cry1ab蛋白在3种水稻土即青紫泥田、黄松田和黄筋泥田中的降解,均以前期(处理后12d内)较快,但中后期明显较慢;处理后42d,这两种组织中的cry1ab残留量均稳定在μg级水平,分别仅有0.20~0.85μg/g干重和0.35~1.81μg/g干重。kmd1茎和叶片中cry1ab蛋白降解动态与土壤类型密切相关,青紫泥田中降解最快,黄筋泥田中次之,黄松田中最慢。淹水可加快土壤中茎和叶的腐解,从而促进其中cry1ab蛋白的降解。kmd2粉碎叶中cry1ab蛋白在有菌水稻土中的降解快于无菌土中,表明土壤微生物存在可加快cry1ab蛋白的降解。在各种土壤淹水与非淹水、灭菌与非灭菌处理条件下的cry1ab蛋白降解过程均可用指数方程进行拟合,算得其削减半衰期为2.2~11.6d不等。讨论了土壤类型、淹水等影响crylab蛋白降解的可能机制,认为土壤微生物是其中起关键作用的一个因子。
转EPSPS基因大豆植株中蛋白的表达  [PDF]
邢珍娟,李飞武,刘娜,李葱葱,康岭生,宋新元,邵改革,张明
大豆科学 , 2009, DOI: 10.11861/j.issn.1000-9841.2009.06.981
Abstract: 采用ELISA定量测定法研究了转EPSPS基因大豆不同生长时期不同器官中CP4EPSPS蛋白含量的变化。结果表明:转EPSPS基因大豆不同器官在不同生育期CP4EPSPS蛋白含量表现出较大的差异,R8期籽粒中的CP4EPSPS蛋白含量在所有时期和所有组织中蛋白含量最高;上位叶和下位叶中CP4EPSPS蛋白除V3~V5期和R8期外的表达趋势一致,茎上部和茎下部中CP4EPSPS蛋白在不同时期表达动态趋势基本一致,根中CP4EPSPS蛋白含量在V3~V5期下降,然后逐渐升高,R1~R8期有一个大幅度的下降过程。从V1期至R8期,随着植株的不断生长,各组织中CP4EPSPS蛋白的含量有明显的变化。
转cry1Ab基因水稻对二化螟幼虫血细胞的影响  [PDF]
王世贵,叶恭银,胡萃
昆虫学报 , 2006,
Abstract: 研究了转cry1Ab基因水稻“克螟稻1号”对二化螟Chilosuppressalis幼虫细胞免疫系统的影响。结果表明,转cry1Ab基因水稻对二化螟幼虫的血细胞影响明显,取食转cry1Ab基因水稻后,二化螟幼虫各类血细胞都明显低于取食非转基因水稻“秀水11”的对照组(原血细胞和囊血细胞在取食初期例外),随取食时间延长,各类血细胞数量及血细胞总数均呈递减的趋势。从各类血细胞所占血细胞总数的百分比来看,原血细胞在取食36h后锐减,而浆血细胞和粒血细胞则比例增加,其余珠血细胞、囊血细胞的变化不明显。另外,血细胞还出现空泡化、肿胀等病态变化,致使血细胞快速破裂。由此推测转cry1Ab基因水稻自身表达的毒蛋白能严重干扰靶标昆虫二化螟幼虫的细胞免疫系统。
转cry1Ab基因水稻对非靶标昆虫白背飞虱种群增长的影响  [PDF]
周霞,程家安,娄永根
昆虫学报 , 2006,
Abstract: 通过室内饲养实验及水稻氨基酸和碳、氮含量测定,比较研究了转cry1Ab水稻克螟稻(KMD1和KMD2)与其亲本秀水11对非靶标害虫白背飞虱种群增长的影响。结果表明,以克螟稻为食可对白背飞虱的产卵期和每雌产卵量产生一定影响。以KMD2为食的白背飞虱的产卵期为7.6天,每雌产卵量为95.0粒,均显著低于以母本秀水11为食的白背飞虱的12.7天和167.5粒。但是,以KMD1为食的白背飞虱仅产卵期(8.6天)显著变短,其每雌产卵量与以其母本秀水11为食的白背飞虱无显著差异。稻苗氨基酸含量分析结果表明KMD2引起白背飞虱种群生殖力显著下降可能与其游离氨基酸的总量和丙氨酸含量的显著下降以及谷氨酸含量的显著上升有关。因此,转cry1Ab水稻可对白背飞虱种群增长产生影响,但其影响因转cry1Ab水稻品种而异。
Cry1Ab转基因水稻的杂种优势表现及抗虫性鉴定  [PDF]
戴正元,李爱宏,张洪熙,左示敏,舒庆尧
中国生态农业学报 , 2007,
Abstract: 以携带Cry1Ab基因的“克螟稻”为抗虫供体亲本,将目标基因转导到优良恢复系“R6547”、“R818”中,配制的杂交稻组合表现良好的杂种优势水平,克服了“克螟稻”在常规粳稻育种实践中常出现的农艺性状差、前期生长势弱的缺陷。可溶性蛋白含量检测结果显示目标基因在杂交稻中仍能高水平的表达,并在人工接虫和自然发生条件下对稻纵卷叶螟、二化螟等鳞翅目害虫表现优良抗性。
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