转Cry1Ab基因水稻分子特征及其特异性PCR检测方法

转Cry1Ab基因水稻Bt01为一种新型的转基因水稻,文章首先利用Southern blotting验证了外源基因Cry1Ab转入了Bt01中,且为单拷贝,再利用TAIL-PCR方法获得了其插入位点信息,根据获得的Bt01的5′端插入位点序列,设计了相应的定性与定量PCR检测体系的引物及探针,实验结果显示,定性PCR检测体系的最低检测极限(LOD)为10个拷贝,定量PCR检测体系的LOD为5拷贝,最低定量极限(LOQ)为10拷贝。同时为了验证建立的定量PCR体系的准确性,利用该体系检测已知转基因水稻Bt01含量分别为3%和0.5%的样品,定量结果分别为2.7%和0.47%。研究结果表明,该转化体特异性定性与定量检测方法具有高度的特异性和良好的灵敏性,为转基因水稻Bt01的身份识别和检测提供了有效的方法。     

转g10-epsps基因耐除草剂大豆ZUTS-33转化体特异性检测方法的建立

转g10-epsps基因耐除草剂大豆ZUTS-33是由浙江大学研发的耐除草剂大豆品系,目前已进入生产性试验阶段。到目前为止尚无文献报道对该转基因新品种的检测方法,因此亟需建立精准的定量检测方法为农业转基因生物安全管理提供技术支持。根据耐除草剂大豆ZUTS-33品系外源基因插入位点特异序列设计引物和TaqMan探针,利用优化的实时荧光定量PCR检测方法评价该引物对和探针的特异性、准确度、精确度和重复性,并确定此检测方法的检测极限（limit of detection,LOD）和定量极限（limit of quantity,LOQ）。实验结果显示,研究所建立的转基因大豆ZUTS-33转化体特异性实时荧光定量PCR检测方法具有高度的品系鉴定特异性,准确度、精确度均符合要求,重复性较好,且检测方法的LOD达到20拷贝,LOQ达到40拷贝。研究结果为转g10-epsps基因耐除草剂大豆ZUTS-33的身份识别和检测监测提供了有效的方法。     

转基因抗虫作物鉴定质粒标准分子的构建与应用

苏云金芽胞杆菌基因是转基因抗虫作物中通用的外源功能基因,在绝大多数抗虫转基因作物中均有存在,然而Bt基因检测标准样品的缺乏却限制了我国转Bt基因抗虫作物检测工作的发展。为了弥补传统基体标准样品的缺失,首先将Cry1Ab、Cry1Ac、Cry3A 3种常用Bt外源基因克隆到pUC57质粒上,通过测序、酶切和qPCR等技术对质粒的序列和扩增功能进行了验证,然后对扩增效率和实际应用情况加以测试,评价其转基因检测的适用性,构建了质粒标准分子。结果显示,制备的质粒标准分子测序结果与靶标序列完全符合,酶切结果、qPCR扩增结果和扩增效率等均符合预期,在Cry1Ab、Cry1Ac、Cry3A基因特异性检测中的应用符合阳性对照要求,表明制备的阳性质粒标准分子能够作为转Cry1Ab、Cry1Ac、Cry3A基因qPCR基因特异性检测的阳性标准样品。     

转Bt基因水稻对白符跳虫取食选择行为的影响

【目的】为探讨转基因Bt水稻种植对土壤动物的潜在生态风险性。【方法】本研究将3种转Bt基因水稻及其非转基因亲本水稻叶片残体饲养白符跳虫Folsomia candida,通过观察其粪便的数量与分布以分析白符跳虫对Bt水稻的取食选择行为。【结果】研究结果表明,Bt蛋白(Cry1Ab和Cry1Ac)不会影响白符跳虫的取食选择;而Bt基因插入后导致的水稻成分的变化可能影响了白符跳虫对水稻残体的偏好性。结果可为评估转Bt水稻对土壤生态系统影响提供参考价值,为转Bt水稻安全性评价提供科学的依据。     

适于转基因油菜RT73品系特异性检测的标准分子构建及其适用性鉴定

针对目前转基因产品检测标准物质缺乏的难题,构建适于转基因油菜RT73品系特异性检测的标准分子pEASY-RT73.其包含转基因油菜RT73品系3'端侧翼序列,油菜内标准基因HMG和PEP片段.对其在定性和定量检测中的适用性进行了实验室内部验证,结果表明,标准分子pEASY-RT173高度特异于转基因油菜RT73品系.以标准分子为标准品的普通PCR扩增中,3个目标片段的检测下限(LOD)均为10拷贝.实时荧光PCR检测的LOD均为25拷贝,定量下限(LOQ)均为50拷贝.以标准分子为标准品构建的标准曲线反应效率介于0.96-1.02间,相关系数均大于0.999.分别以PEP和HMG为内标准基因对5个盲样的测试结果显示,测量值与设定值间偏差(Bias)介于-14.13%-14.29%间,SD小于0.20,RSD小于18.0%.因此,标准分子pEASY-RT73可很好地替代植物来源阳性标准品用于转基因油菜Rt73及其来源产品品系特异性检测.     

利用花青素可视化跟踪表达系统快速筛选表达Cry1Ab/c基因的转基因玉米

以草甘膦抗性基因Epsps为标记基因, 在原核Kan^r基因两侧引入Cre(环化重组酶)基因识别的Lox-P位点, 同时以编码花青素合成转录因子的Bi和Cl基因为可视化选择报告基因, 构建了Bt杀虫蛋白基因Cry1Ab/c的可视化跟踪表达载体pBAC9017。用PDS1000/He基因枪转化玉米(Zea mays)自交系501的幼胚和胚性愈伤组织, 获得147个草甘膦抗性的玉米再生植株。其中106棵植株获得了结实种子, 16棵植株的结实种子有紫红色花青素基因的表达。经PCR检测表明, 外源Cry1Ab/c基因已经整合到玉米的基因组中。转基因植株种子蛋白粗提物用BT-Cry1Ab/1Ac金标免疫检测试纸条和ELISA检测, 结果表明, Cry1Ab/c在部分转基因植株后代中表达。     

转基因水稻“科丰6号”实时荧光PCR定性定量检测方法研究

旨在建立转基因水稻"科丰6号"外源基因和边界序列的实时荧光PCR检测方法,为科丰6号定性定量检测提供技术支持。根据外源基因和边界序列信息,设计实时荧光PCR探针引物,优化体系,对不同转基因产品和不同转基因含量的"科丰6号"水稻进行检测。结果显示,所设计的引物探针具有很好的特异性,与其他转基因水稻品系、转基因玉米、转基因棉花、转基因番茄和非转基因水稻均无非特异性反应,对转基因水稻"科丰6号"的检测灵敏度达到0.01%。建立的科丰6号实时荧光PCR检测方法重复性好、灵敏度高,能够达到目前国际上转基因产品定量检测的标准,为该水稻品系的定性定量检测提供技术支持。     

转基因水稻BPL9K-2事件特异性检测方法的建立

利用hiTAIL-PCR(high efficient thermal asymmetric interlaced PCR)法扩增获得了转基因水稻BPL9K-2的外源基因插入位点的左旁侧序列450bp,与水稻参考基因组数据比对发现其左边界插入在水稻基因组第10号染色体短臂的1 037 765位核苷酸残基之后。根据水稻参考基因组序列和外源基因右边界序列,设计引物扩增得到485bp的特异片段,通过数据库比对发现其右边界插入在水稻基因组第10号染色体短臂的1 037 825位核苷酸残基之前。因为外源基因插入和非正常重组,水稻基因组上缺失了59个核苷酸。基于左右旁侧序列,建立了转基因水稻BPL9K-2的事件特异性定性PCR检测方法,可以分别扩增到片段大小为449bp和485bp的特异条带。该方法特异性好,灵敏度高,能够在BPL9K-2基因组DNA相对含量为0. 1%的模板中检测出转基因成分。依据旁侧序列,建立了快速鉴定转基因后代植株外源基因型的三引物PCR检测方法。这些方法的建立,为转基因水稻BPL9K-2的应用和检测提供了技术支持。     

转基因水稻中转录水平crylAb基因的沉默及其阶段复活

以田间环境释放条件下农杆菌介导法转化而成的转crylAb基因水稻为研究对象,利用GUS组织化学染色法、Western杂交技术,在不抗虫转基因中8215株系后代中筛选到一个无Cry1Ab蛋白表达产物株系.分子杂交结果证实,转基因crylAb在中8215株系后代中发生了转录水平沉默,整合过程中基因重排使两个拷贝的ubiquitin启动子同时插入到水稻基因组中.甲基化分析证实ubiquitin启动子区域发生了甲基化,从而导致crylAb基因沉默.利用去甲基化试剂5-氮胞苷处理转基因沉默水稻种子,并在苗期、分蘖期、孕穗期、灌浆期及成熟期检测了其对沉默基因的复活效应,结果表明:5-氮胞苷处理使沉默的crylAb基因在灌浆期恢复表达活性,复活率约为8%～30%,且以低浓度(45 mg/L处理1,2 d)处理的复活率及恢复表达水平较高,复活基因表达的Cry1Ab蛋白最高可达可溶性总蛋白的0.147%.     

转基因玉米Bt176液相芯片检测方法的建立

为建立转基因玉米Bt176的液相芯片检测方法,根据已公布的转基因玉米Bt176外源插入基因CaMV35S启动子序列,外源基因3’端与玉米基因组DNA连接区序列,同时以玉米特异Zein内源基因序列为参照,利用Primer Premier5.0等软件设计特异性引物和探针。将探针与荧光编码微球偶联后,与PCR产物杂交反应,用液相芯片检测仪(Bio-plex 200)检测荧光信号。检测结果显示,该方法具有高特异性及灵敏度,各条探针之间无交叉反应,最低检测限可达0.01%。初步建立了检测转基因玉米Bt176的液相芯片技术,为其他转基因作物的快速高通量检测提供了借鉴和经验。     

Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins

Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258–1.407 μg/mL, the medium inhibition concentration (IC₅₀) were 0.727–0.892 μg/mL and detection limit (IC₁₀) were 0.029–0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21–5.94 × 10⁷ M⁻¹. The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%–100.8% with a CV at 2.5%–9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.     

Event-specific qualitative and quantitative PCR detection of roundup ready event GT73 based on the 3′-integration junction

With the development of genetically modified organisms, labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed state-of-art. This paper describes an event-specific PCR method for qualitative and quantitative of Roundup Ready canola event GT73. The 3′-integration junction was characterized by two methods: inverse-PCR and thermal asymmetric interlaced-PCR. In the conventional qualitative PCR assay, the event-specific primers designed were confirmed to be specific and the limit of detection (LOD) was 0.05% (approximates to ten haploid genome copies). In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were five and ten haploid genome copies, respectively. In addition, for further quantitative detection, a reference molecule which contained the canola endogenous gene and event-specific sequence was constructed and standard curves were set up. The goodness of the linearity and high efficiency of the PCR reaction indicated the usability of the plasmid and the established PCR system. Moreover, mixed samples with different GT73 content (6, 3, 1 and 0.5%) were quantified using the established real-time PCR system to evaluate the trueness and precision of the system. The trueness expressed as bias varied from 2.00 to 18.00%. The precision expressed as variation coefficient were different from 6.40 to 32.95%. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for GT73 in this study were acceptable and suitable for genetic modified canola detection. Rong Yang, Wentao Xu and Yunbo Luo contributed equally.     

Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.     

转Bt基因稻谷对印度谷螟生长发育的影响

为建立仓储阶段转Bt水稻安全性评价中靶标害虫抗性汰选研究体系,配制了含不同比例(70%,50%,30%,10%)转Bt基因(Cry1Ab/Cry1Abc)明辉63水稻谷粉(简称Bt谷粉)的人工饲料饲喂印度谷螟Plodia interpunctella(Hübner),测定其对1～3龄幼虫在72h内的急性毒力,及对印度谷螟种群生长发育的影响,并采用ELISA法检测转基因稻谷和末龄幼虫体内Bt蛋白含量。结果发现:4种比例人工饲料对幼虫的毒力作用均发生在取食48h后,72h后剂量效应明显。含Bt水稻较高比例的饲料对印度谷螟发育的负面效应明显:幼虫死亡率高,发育历期延长。Bt蛋白在幼虫体内含量与对应饲料中的含量基本成正比。综合考虑,将Bt杀虫蛋白含量2.35μg/g作为转Bt基因稻谷对印度谷螟的亚致死剂量最为合适。     

Fate of genetically modified maize and conventional rapeseed,and endozoochory in wild boar (Sus scrofa)

Feeding experiments were carried out to investigate the digestive fate of transgenic DNA and novel protein in wild boar applying polymerase chain reaction (PCR) and immunodiagnostic techniques. Furthermore, the dispersal of viable maize and rapeseed (endozoochory) was studied. A diet containing conventional rapeseed, and either genetically modified (GM) maize expressing Cry1Ab protein (Bt176) or non-GM isogenic maize was offered. By conventional and quantitative PCR both chloroplast-specific plant DNA (rubisco) and cry1Ab gene fragments were detected only in gastrointestinal content. Using an enzyme-linked immunosorbent assay (ELISA) positive signals of immunoactive Cry1Ab protein were detected in digesta samples. Analysis of endozoochory showed that excreted maize seeds retain their germination capacity only in extremely rare cases and no intact rapeseed was found in faeces. A possible dispersal of viable seeds by wild boars is highly unlikely.     

Geographic variation in susceptibility of Chilo suppressalis (Lepidoptera: Pyralidae) to Bacillus thuringiensis toxins in China

Geographic variation in the susceptibility of the striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), in China to Bacillus thuringiensis (Bt) insecticidal crystal proteins Cry1Ac and Cry1Ab was studied to establish baseline information for comparing the future response of populations with increased exposure to Bt products. Rice is the major host of C. suppressalis, and Bt rice ma) be released in China in the near future. Twelve populations of the pest were collected from the major rice-growing regions of China. LC50 estimates were determined for all populations for Cry1Ac and for eight populations for Cry1Ab. The bioassay results indicated that the range of LC50 in neonate larvae to Cry1Ac and Cry1Ab was from approximately 15 to approximately 157 mg (AI)/L and approximately 2 to approximately 34 mg (AI)/L, respectively. LC50 values were lower for Cry1Ab than for Cry1Ac, and there was a significant positive correlation between the two toxins tested.     

Dispersal behavior of neonate European corn borer (Lepidoptera: Crambidae) on Bt corn

European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), has historically been a significant economically important insect pest of corn (Zea mays L.) in the United States and Canada. The development in the 1990s of genetically modified corn expressing genes derived from Bacillus thuringiensis (Bt) that encodes insecticidal crystalline (Cry) proteins has proven to be effective in controlling this insect as well as other corn pests. The purpose of this study was to assess the movement and dispersal behavior of neonate European corn borer on Bt corn. We examined differences in neonate European corn borer dispersal behavior for the first 4 h after eclosion in the field among a stacked pyramid (Cry1F X Cry1Ab X Cry34/35Ab1) Bt corn, a Cry1F Bt corn, and a non-Bt sweet corn; and in the laboratory among a Bt corn hybrid containing Cry1F, a hybrid containing Cry1Ab, a pyramid combining these two hybrids (Cry1F X Cry1Ab), and a non-Bt near isoline corn. In field experiments, we found that dispersal was significantly higher on Bt corn compared with sweet corn. In laboratory experiments, dispersal was significantly higher on Cry1Ab Bt corn and Cry1F X Cry1Ab Bt corn than on non-Bt near isoline corn. Results indicated that neonate dispersal may be significantly greater in Bt cornfields compared with non-Bt cornfields. The findings on dispersal behavior in this study will be useful in evaluating the efficacy of a blended seed refuge system for managing European corn borer resistance in Bt corn.     

Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic rice borer Chilo suppressalis (Walker)

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.