产志贺毒素大肠杆菌毒素基因的敲除

根据GenBank上致仔猪水肿病大肠杆菌志贺毒素(Stx2e)A单位基因序列(M21534)以及Red重组系统试剂盒提供的FRT-PGK-gb2-neo-FRT序列设计1对引物;引物的5′端与Stx2e基因同源,3′端与FRT-PGK-gb2-neo-FRT序列同源。使用合成的引物利用PCR扩增FRT-PGK-gb2-neo-FRT序列片段。将扩增的PCR片段转化带有pRedET重组质粒的产志贺毒素大肠杆菌(STEC)S451521菌株(F18+)的感受态细胞中,通过同源重组技术替换Stx2eA单位基因的805～1 152bp区域。通过卡那霉素抗性以及PCR检测获得同源重组的STEC后,将708-FLPe质粒进一步转化重组菌,利用Flp重组酶去除了重组菌的卡那霉素抗性。另外,研究还发现Stx2e基因在STEC基因组中并非单拷贝。     

The Influence of Transgenic crylAb/crylAc,crylC and cry2A Rice on Non- Target Planthoppers and Their Main Predators Under Field Conditions

Transgenic Bt rice has been shown to be an effective means of controlling Lepidoptera pests of rice. However, the potential roles of transgenic rice on planthoppers and their predators need to be investigated before its commercialization. Population density, species dominance and population dynamics are important parameters of arthropods populations in field. So the impacts of three transgenic Bt rice strains expressing cry1Ab/cry1Ac, cry1C and cry2A on population density, species dominance and population dynamics of three species of planthoppers (Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus) and their three main predators (Cyrtorhinus lividipennis, Pirata subpiraticus and Theridium octomaculatum) were evaluated at three sites in Hubei Province, China, in the current study. The results showed that among three species of planthoppers, both in transgenic and non-transgenic rice field, the predominant species of planthoppers within phytophagous guild was S. furcifera at any site either growing season (46–50%). Significantly higher population density of N. lugens was observed in T2A-1 field relative to Minghui 63 field at Wuxue in 2010. The species dominance of predator, P. subpiraticus, in TT51 field was significantly higher than that in T1C-19 and T2A-1 fields in 2009 at Xiaogan site. Sampling date significantly influenced six arthropods except for P. subpiraticus in 2010. The interaction between rice strain×sampling date had no significant adverse effects on the population dynamics of three species of planthoppers and their predators, except for several individual species in 2009. The interaction among rice strain×sampling date×sampling site also had no significant effect on six arthropods except for S. furcifera in 2009. The results indicated that transgenic Bt rice expressing cry1Ab/cry1Ac, cry2A and cry1C had no significant adverse effects on the population dynamics of three planthoppers and their predators in most investigated data and sampling site.     

茄病镰刀菌毒素的研究Ⅰ．毒素的分离、鉴定及生物测定

将从豌豆根腐病病株分离获得的茄病镰刀菌（Ｆｕ）Ｄ－０１６菌株接种在ＰＳＤ培养液中，且２５℃恒温箱内黑暗静止培养１４天后，可获得具有毒性的培养滤液。培养滤液及提取的粗毒素经生物测定都具有植物毒性效应。粗毒素经分离纯化后进行的初步鉴定表明，该菌可产生茄病镰刀菌烯醇和Ｔ＿２、二醋酸草镰刀菌烯醇、Ｈ－Ｔ＿２等毒素。     

转基因抗虫玉米Bt毒蛋白的时空表达分析

对2个转Bt基因玉米抗虫系株系进行Bt毒蛋白表达量的分析测定。结果显示,转基因株系中的毒蛋白可以在其后代稳定的遗传和表达,毒蛋白的表达为组成性表达,在玉米的各个发育时期和植株的各个部位均有表达,但其表达的量有显著差异,毒蛋白表达量随着植株的生长而减少,比较植株的各个器官,叶片毒蛋白含量最高,茎次之,根、种子和花丝中的毒蛋白含量较少。     

禾谷镰刀菌液体培养产毒条件研究初报

禾谷镰刀菌在PS液体培养基中可以产生对小麦具有毒性作用的毒素。不同菌株的产毒能力有明显的差异,经测试的11个菌株中以29号菌株产毒能力最强。禾谷镰刀菌产毒的最适温度为20℃,初始pH值为5～6,光照可抑制毒素的产生,一般培养21～28天达到产毒高峰。试验还表明,培养滤液pH值的变化与毒性的变化有明显的相关性。     

玉米小斑病菌O小种毒素的生物活性测定

中国玉米小斑病菌(Bipolaris maydis)小种培养滤液含有活性较强的生理活性物质,对供试15个自交系种子根生长具有明显的抑制作用;可诱导幼苗和离体叶片产生典型的小斑病症状,自交系对培养滤液致病作用的抵抗性与幼苗对病菌的致病性密切相关;O小种培养滤液对玉米根冠细胞具有强烈的致死作用,根冠细胞对培养滤液的敏感性与幼苗对病菌的感病性间存在明显的正相关。据此,初步认为O小种滤液中的生理活性物质为O毒素。     

Bt水稻对蜘蛛优势类群及其与猎物相关性的影响

以转cry1Ab/cry1Ac基因水稻汕优63（以下简称Bt水稻）为供试材料,亲本汕优63（非Bt水稻）为对照,在田间自然条件下研究了Bt水稻对稻田蜘蛛优势类群消长动态及其与猎物相关性的影响。结果表明,Bt和非Bt稻田中蜘蛛优势类群发生数量相对大小依次为：皿蛛〉狼蛛〉园蛛〉肖蛸。2种类型稻田中蜘蛛优势类群消长动态基本一致,早期狼蛛和肖蛸占优势,随后园蛛和皿蛛成为田间优势蜘蛛类群,但Bt水稻田各类优势蜘蛛数量都显著高于相应非Bt水稻田。2种类型稻田中蜘蛛优势类群与2类主要害虫稻飞虱和叶蝉消长动态相关性存在一定差异。其中,狼蛛与2类主要害虫消长动态相关性在非Bt水稻上达到显著或极显著水平;而在Bt水稻上相关性不显著;肖蛸与2类主要害虫消长动态相关性在2类型稻田中都不显著。     

农杆菌介导法转化大豆体细胞胚获得转基因植株

以六个品种大豆体细胞胚为受体,以Bt基因为目的基因,应用根癌农杆菌介导法对大豆进行了遗传转化,经卡那霉素选择、诱导体细胞胚萌发及壮苗培养,获得了完整的抗性再生苗,经PCR、PCR-Southern和点杂交分析鉴定,初步证明外源基因Bt已整合到大豆的基因组中。     

2种防治棉铃虫措施对转Bt基因棉田绿盲蝽种群动态的影响

对河北省南皮县2种防治棉铃虫措施的转Bt基因棉田及其周边生境绿盲蝽的发生规律进行了系统调查。结果表明:绿盲蝽成虫在转Bt棉田有4个高峰期,绿盲蝽若虫有3个高峰期,整个发生期各棉田绿盲蝽成、若虫平均百株虫量分别为GK田33.44头1、6.24头,综防田8.10头、3.38头,化防田5.68头、2.00头,GK田分别比综防田、化防田高312.8%3、80.5%4、88.7%、712.0%,差异极显著(P<0.01),但综防田与化防田发生期绿盲蝽成、若虫平均百株虫量差异不显著(P>0.05)。这说明绿盲蝽已成为当地转Bt基因棉田的重要害虫。