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苏云金芽孢杆菌Ly30株cry1Ac基因的克隆及表达* 总被引：1，自引：0，他引：1
Bt菌Ly30株是中国自行分离的对多种害虫具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis,Bt),经CAPS(cleaved amplified polymorphic sequences)系统鉴定。它含有cry1Ac基因。以全长基因PCR产物的粘端定向克隆的方法,设计1对特异引物,分别引入SalⅠ和BamHⅠ酶切位点。以Ly30质粒DNA为模板扩增cry1Ac全长基因,与表达载体pET-21b相应的酶切产物连接,转化大肠杆菌(Eschrichia coli),获得含有cry1Ac基因重组质粒pEKLy1Ac。该基因的亚克隆和序列测定结果表明,其编码区为3534bp。编码蛋白分子量为133.5kD,含1177个氨基酸,等电点为4．8,与CrylAc3同源性最高,存在4个氨基酸的差异,与Cry1Ac10之间则有6个氨基酸的不同。该基因序列已在GenBank中登记注册为AF482767,并被国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry1Ac14该基因经诱导获得高效表达,SDS-PAGE电泳检测到明显的133．5kD蛋白带。室内生物测定结果表明,诱导表达的Cry1Ac蛋白对棉铃虫、甜菜夜蛾等鳞翅目害虫幼虫均有较高的杀虫活性,其LC5o值分别为19.236和3276μg／g饲料。 相似文献
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黑龙江凉水自然保护区是我国现有保存下来的较大片原始红松林基地之一,总面积6394hm^2,森林覆盖率95％以上。本研究从小兴安岭凉水自然保护区采集土样782份,采用醋酸钠培养基结合高温方法筛选土壤中的芽孢杆菌,通过光学显微镜观察鉴定产生伴胞晶体的苏云金芽孢杆菌（Bacillus thuringiensis．Bt）。总计分离得到芽孢杆菌和苏云金芽孢杆菌分别为1735株和33株,Bt菌株的分离率和出菌率分别为1．90％和4．22％。利用SDS-PAGE和PCR—RFLP方法对筛选获得的成分离株进行了杀虫晶体蛋白和基因型分析,结果表明14株产菱形伴胞的＆菌株,SDS-PAGE电泳分析芽孢后期产生分子量大小130kD蛋白带,PCR-RFLP分析初步鉴定为crylAc基因,其它产圆形或其它不定形晶体蛋白的＆分离菌中,芽孢后期主要蛋白大小为20～150kD不等,PCR—RFLP方法鉴定结合PCR片段测序分析这些菌株含有新型cry4、cry39和cry40基因等。本研究是“中国助资源收鉴与利用”项目组成部分之一,凉水自然保护区苏云金芽孢杆菌的收集与鉴定目的是要对整个东北地区成资源分布和多样性作一个初步评估,实验结果表明东北森林地区具有丰富多样的威菌株和杀虫基因资源。 相似文献
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苏云金芽孢杆菌cry1Aa14基因的分离、克隆及其表达 总被引：2，自引：1，他引：2
Bt25是中国自行分离的对小菜蛾(Plutella xylotella)具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis),经PCR-RFLP鉴定含有cry1Aa基因。以全长基因PCR产物的粘端定向克隆的方法,设计1对特异引物,以Bt25质粒DNA为模板扩增cry1Aa全长基因。序列测定结果表明,该基因编码区为3552bp,编码1183个氨基酸,分子量为133．7kD,pI4．755。该基因序列已在GenBank注册,登记号为AY197341,并获得正式命名cry1Aa14。在氨基酸序列918～1180间,和已知的11种cry1Aa存在22-23个氨基酸的差异(其中1094～1097的4个氨基酸无对应序列),而这段区域和Cry1Ab氨基酸序列的对应区域无差异。cry1Aa14全长基因插入Bt表达载体,获得了重组表达质粒pBYB1,转化Bt无晶体突变株HD73cry-,经过抗性筛选、DNA酶切分析和PCR检测,证实转化成功。SDS-PAGE分析表明,该基因在上述受体中能正常表达133kD蛋白。杀虫生物测定结果表明,cry1Aa14表达产物对小菜蛾幼虫具有显著的毒杀作用,与cry1Aa12进行比较,毒力无明显差异。这种单基因菌株的发现及其基因的获得,为害虫抗性研究和高效工程菌的构建提供了重要实验材料。 相似文献
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从广西大王岭和大明山两个自然保护区共采集到土样264份，共分离出597株芽孢杆菌，通过光学和电子显微镜检观察，16株分离株观察到伴胞晶体蛋白，初步确定为苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt），出菌率为6．06％。在16株肪分离株中，有4株在芽孢形成过程中能产菱形晶体蛋白，其余12株能产圆形和其他形状的晶体蛋白。利用PCR—RFLP方法和SDS．PAGE方法对16株助分离菌进行了蛋白和基因型的鉴定，结果表明，16株分离株中含有4株crylAc基因，表达约130kD的晶体蛋白，其中含有cry30基因和cry40基因的菌株分别1株和3株，表达大约75kD的晶体蛋白；另外8株戤菌株表达蛋白大小不一，其基因型尚不能确定，有待进一步分析。生物测定表明，产菱形晶体含有crylAc基因的4株励分离株对鳞翅目小菜夜蛾幼虫有很强的毒杀活性，而其它分离株对小菜夜蛾没有毒杀活性。 相似文献
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WB9是我国分离自武夷山的对多种重要农业害虫具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)菌株,经PCR-RFLP鉴定含有cry2Ac基因。根据cry2基因序列设计引物,以WB9质粒为模板扩增cry2Ac全长基因,与大肠杆菌(Escherichia coli)克隆载体pMD18-T连接获得含有cry2Ac全长基因的重组质粒pMD2Ac并测序。该基因在GenBank上登录号为DQ361267,被Bt国际命名委员会正式命名为cry2Ac4。通过亚克隆方法将cry2Ac4基因插入穿梭表达载体pHT315获得重组表达质粒pHT2Ac,将其转化大肠杆菌SCS110和Bt无晶体突变株HD73 Cry-,得到的工程菌能正常表达70 kD蛋白,形成方形晶体。生物测定结果表明,cry2Ac4基因表达产物对桔小实蝇(Bactrocera dorsalis Hendel)幼虫具有显著的毒杀作用,但对小菜蛾(Plutella xylostella)和致倦库蚊(Culex fatlgans)幼虫基本没有效果。 相似文献
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我们采用RT-PCR方法克隆了2个A州同源基因全长cDNA,分别命名为MAPl—1（GenBankac—cession No．FJ529206）和MAPl—2（GenBankaccession No．FJ529207）。MAPl—1编码247个氨基酸,开放阅读框长度为741bp,蛋白质分子量为28．54kD,等电点为8．31;MAPl—2编码248个氨基酸,开放阅读框长度为744bp,蛋白质分子量为28．78kD,等电点为8．70。同源性分析表明,它们的核苷酸序列与其它木本植物A纠同源基因的一致性为72％～81％。实验分析表明,MAPl—1和MAPl—2第1至第61个氨基酸含有一个MADS盒结构域,第88至第178个为K盒结构域;两个基因均定位于细胞核,且功能位点分布存在着不同,推测这两个基因在花器官发育过程中的功能存在差异。蛋白二级结构预测显示,MAPl—1蛋白有12个α-螺旋,4个8折叠区,14个β-转角;而MAP1—2蛋白有11个α-螺旋,5个B折叠区,15个β-转角;其大多数氨基酸具有亲水性。本研究有助于进一步了解芒果的开花分子机理及成花的生物学发育阶段。 相似文献
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苏云金芽胞杆菌cry1Ia基因的克隆、表达与活性研究 总被引：3，自引：0，他引：3
根据cry1Ia类基因的全长序列设计引物,以苏云金芽胞杆菌(Bacillus thuringiensis)菌株Btc008的总DNA为模板扩增出片段长为2.1kb的cry1Ia的全长基因,插入大肠杆菌(Escherichia coli)表达载体pET-21b,转化大肠杆菌BL21(DE3)菌株,诱导表达出81kD的蛋白。该蛋白由719个氨基酸组成,推导的分子量为81.2kD。该蛋白的氨基酸序列不同于已知的12种Cry1Ia蛋白,是一种新的Cry1Ia蛋白,该基因已被国际基因命名委员会正式命名为cry1Ia8。杀虫活性测定结果表明:Cry1Ia8对亚洲玉米螟(Ostrinia furnacalis)和小菜蛾(Plutella xylostella)有很强的杀虫活性,LC50分别为0.268μg/g和2.227μg/mL,其杀虫效果与Cry1Ab和Cry1Ac相当。对大豆食心虫(Leguminivora glycinivorella)也有较好的活性,但对鞘翅目叶甲科害虫榆兰叶甲(Pyrrhalta aenescens)没有活性。该基因的获得将为我国抗虫转基因作物和工程菌的研制提供新的基因来源,为筛选延缓昆虫抗性产生的基因组合提供了重要依据。 相似文献
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苏云金芽胞杆菌(Bacillus thuringiensis,Bt)是一种已被广泛用于农业害虫生物防治的昆虫病原细菌。本研究从永春天湖山矿区采集的159份样品中分离出1株Bt,光学显微镜检测发现菱形伴孢晶体。以cry1/7/9、cry2、cry3、cry4/10、cry5、cry6、cry8、cry11和cry1I的通用引物对该Bt分离菌进行Cry毒素基因分析,利用SDS-PAGE和双向电泳-质谱法(2-dimensional electrophoresis/mass spectrometry,2-DE/MS)对Bt分离菌进行蛋白鉴定。结果表明,分离菌含有cry1基因型,经克隆、测序后可知,菌株中的cry1亚基因型与cry1Ac有极高的同源性。分离株表达的晶体蛋白约为130 k D,通过蛋白质谱分析表明,杀虫蛋白为Cry1Ac,与PCR-RFLP所得出的结果一致;通过质谱分析还鉴定出分子伴侣Gro EL、F0F1型ATP-β亚基合成酶、延伸因子Tu和丙酮酸脱氢酶。生物信息学分析结果表明,这些蛋白主要参与帮助新生肽的正确折叠、能源生产和转换,以及提高细胞的应激能力以抵御外界不良刺激。本研究从煤矿中分离鉴定出Bt菌,为发现新资源、新型杀虫蛋白基因以及利用微生物治理重金属污染提供基础资料。 相似文献
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从土壤中分离得到营养期对鳞翅目害虫高毒的苏云金芽胞杆菌(Bacillus thuringiensis,Bt)LS1菌株,分子检测该菌株中存在营养期杀虫蛋白基因,进行了该基因的克隆和表达。设计全长基因引物扩增得到了约2.3kb的靶片段,序列分析证实为新vip3A基因,命名为vip3A-LS1,GenBank登录号为DQ016968,Bt基因命名委员会命名为Vip3Aa22。该基因推导蛋白与其它已知同源蛋白有8个氨基酸不同。构建vip3A-LS1基因的表达载体,SDS-PAGE检测约88kD目的蛋白大量表达;生物测定表明,胞内可溶性蛋白对棉铃虫(Helicoverpa armigera)和甜菜夜蛾(Spodoptera exigua)活性最高。纯化蛋白对初孵棉铃虫和甜菜夜蛾的LC50分别为73.6和32.2μg/g。 相似文献
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Indicated detection of two unapproved transgenic rice lines contaminating vermicelli products 总被引：3，自引：0，他引：3
Akiyama H Sasaki N Sakata K Ohmori K Toyota A Kikuchi Y Watanabe T Furui S Kitta K Maitani T 《Journal of agricultural and food chemistry》2007,55(15):5942-5947
We analyzed the DNA fragments extracted from four rice vermicelli products. The Bacillus thuringiensis (Bt) rice line, which has a construct similar to the GM Shanyou 63 line, was detected in some vermicelli products by identification of the junction region sequence between rice Act1 promoter and the Cry1Ac gene, and that between Cry1Ac and nos. In addition, we also detected a different Bt rice line by means of the junction region sequence between the maize ubiquitin promoter and cry1Ab gene and that between the cauliflower mosaic virus 35S promoter and the hygromycin phosphotransferase in some vermicelli products. Accordingly, we for the first time have detected the two transgenic Bt rice lines contaminating rice vermicelli samples. Furthermore, we developed a duplex real-time polymerase chain reaction (PCR) method for the simultaneous detection of both Bt rice lines. 相似文献
12.
国际转基因作物的安全性争论——几个事件的剖析 总被引：21，自引：4，他引：21
摘要：国际转基因作物安全性争论的实质并不纯粹是科学问题,而是经济和贸易问题。对安全性争论的几个典型事件从科学角度作了分析。 相似文献
13.
XU Jing-Liang WU Jun WANG Zhi-Chun WANG Kun LI Meng-Ying JIANG Jian-Dong HE Jian LI Shun-Peng 《土壤圈》2009,19(2):238-243
Methomyl, an extremely toxic pesticide, is widely used in agriculture. A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study. It could utilize methomyl as the sole carbon or nitrogen source. The optimal temperature and medium pH for its growth and methomyl biodegradation were 30-C and 7.0, respectively. It was identified as a Paracoccus sp. according to its morphological features, physiological and biochemical characteristics, and phylogenetic analysis based on the sequence of 16S rDNA. Gas chromatography-mass spectrometry (GC-MS) analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1. 相似文献
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The impact of transgenic plants containing Bacillus thuringiensis (Bt) toxin on soil processes has received recent attention. In these studies, we examined the influence of the lepidopterean Bt Cry1Ac toxin on mineralization and bioavailability of the herbicide glyphosate in two different soils. The addition of 0.25-1.0 microg g(-1) soil of purified Cry1Ac toxin did not significantly affect glyphosate mineralization and sorption in either a sandy loam or a sandy soil. In contrast, extractable glyphosate decreased over the 28 day incubation period in both soils. Our findings suggest that the reduction in the bioavailability of glyphosate was not influenced by the presence of Cry1Ac toxin but rather the results of aging or sorption processes. Results from this investigation suggest that the presence of moderate concentrations of Bt-derived Cry1Ac toxin would have no appreciable impact on processes controlling the fate of glyphosate in soils. 相似文献
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Hammond BG Campbell KW Pilcher CD Degooyer TA Robinson AE McMillen BL Spangler SM Riordan SG Rice LG Richard JL 《Journal of agricultural and food chemistry》2004,52(5):1390-1397
Fumonisins were monitored in corn grain collected from Bt hybrids grown in 107 locations across the United States in 2000-2002. Bt corn hybrids contain the Cry1Ab protein from Bacillus thuringiensis that controls European corn borers and other stalk-boring pests. Fumonisin levels were frequently lower in grain from Bt hybrids grown in field trials under conditions of natural (FACT trials) or manual insect infestation (university trials). Over three years of FACT trials, there were 126/210 comparisons when fumonisin levels in grain from control hybrids were >2 ppm, exceeding U.S. FDA guidance levels of 2 ppm for human food. Grain from Bt hybrids was at or below 2 ppm of fumonisins for 58 of the 126 comparisons. The use of Bt hybrids can increase the percentage of corn grain that would be suitable for use in food and feed. 相似文献
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一种检测转Bt基因抗虫棉新棉33B和GK-12的PCR方法 总被引：2，自引：1，他引：1
测定了转基因抗虫棉新棉33B和GK-12的Bt基因表达盒的序列,发现它们在Bt基因和Bt基因与终止子连接区的序列存在差异,而在启动子与Bt基因连接区的序列完全一致。基于这种结构上的差异,设计3条特异性引物MG-P1、MG-P2和MG-P3,建立了检测这两个转基因抗虫棉的双重PCR方法。采用建立的方法,检测了40个转基因棉花样品,其中,只含有新棉33B的Bt基因结构的样品数为32个;只含有GK-12的Bt基因结构的样品数为6个;同时含有两者结构的样品数为2个。 相似文献
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HUANG Tian-Pei XIAO Ying PAN Jie-Ru CHEN Zhi LI Li-Fen XU Lei ZHANG Ling-Ling GUAN Xiong 《土壤圈》2014,24(5):652-661
Chromium（Cr） may cause losses in the yield of field plant, which is one of the favorite habitats of Bacillus thuringiensis（Bt）. The purposes of our study were to assess the Cr（VI）-resistance and Cr（VI）-reducing abilities of an indigenous soil isolate of Bt and to determine the factors governing Cr（VI） reduction. Towards this end a novel dichromate-reducing Bt BRC-ZYR2, characterized with insecticidal crystal proteins（ICPs）, was isolated from a uranium deposit. Minimum inhibitory concentrations（MICs） of Cr（VI） were determined by broth dilution method and the concentrations of Cr（VI） and total Cr in the supernatant were quantified colorimetrically using 1,5-diphenylcarbazide（DPC） reagent and a mixture of sulfuric-nitric acids, respectively. The isolate contained five ICP genes（cry1Ba, cry1 Bb, cry1Be/cry1 Bf, cry9 Ca and cry9Da） and exhibited a high level of Cr（VI） resistance with MICs of 150 mg L-1at pH 7.0 and 30？C, and 500 mg L-1under optimal conditions（pH 9.0 and 40？C）. The total Cr concentration was similar to initial concentration of Cr（VI） under the optimal condition, suggesting that the essential removal of the Cr（VI） was dependent on Bt reduction. Under optimal conditions, the initial Cr（VI） concentrations from 25 to 75 mg L-1significantly decreased in 24 h after incubation. Addition of Mn2＋, Co2＋, Mo2＋and Cu2＋activated Bt-mediated Cr（VI） reduction, while Zn2＋, Ni2＋and glucose were found to inhibit the reduction. Our results indicated that this isolate could be a promising biopesticide with the potential for both insect biocontrol and Cr bioremediation in the field. 相似文献
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Growing Bt crops reduces the use of insecticides applied to them, but these crops could affect soil microorganisms and their activities. We evaluated the effects of Bt (Cry1Ab) corn (Zea mays L.) and deltamethrin ([S]-α-cyno-3-phenoxybenzyl [1R, 3R]-3-[2,2-dibromovinyl]-2,2-dimethylcyclopropane-1-carboxylate) insecticide application on soil microbial biomass C (MBC), β-glucosidase enzyme activity (final season only), bacterial functional diversity, and bacterial community-level physiological profiles (CLPPs) in corn monoculture in five seasons. We also determined if growing Bt corn in crop rotation would alter these effects. Statistical analysis of pooled data across seasons did not show any effects of Bt technology, insecticide application or crop rotation on soil microbial biomass or diversity even though differences between seasons and between the rhizosphere and bulk soil were observed. Annual analyses of results also showed that neither the Bt technology nor insecticide application affected soil MBC, enzyme activity, or functional diversity of bacteria in corn rhizosphere, but shifts in bacterial CLPPs due to Bt trait were observed in one year. Crop rotation effects on soil microbial properties were not observed in most cases. Where effects were observed, Bt corn grown in rotation resulted in greater MBC, enzyme activity and functional diversity than Bt corn grown in monoculture or conventional corn grown in rotation, and these effects were observed only in bulk soil. Therefore, the Bt technology is safe with respect to the non-target effects measured in this study. However, the effects of repeated use of Bt crops over many years on the soil environment should continue to be monitored. 相似文献