若干定性PCR方法部颁标准在检测转基因混合样品中的应用分析

以若干定性PCR方法部颁标准对含有0.5％的4份不同转基因混合样品进行检测,先以通用元件标准中的CaMV35s启动子、NOS终止子对混合样品进行初步定性PCR筛选。结果表明,4份样品中都含有转基因成分。Bt基因特异性标准检测表明,3#和4#样品含有转基因抗虫水稻成分。构建特异性标准PCR检测表明,2#、3#和4#样品含有转基因GTS-40-3-2大豆成分。以MON810、Bt176、NK603转化体事件标准进行品系特异性PCR检测,结果证实：1#和4#样品中含有Bt176转基因玉米成分;3#样品中含有Mon810转基因玉米成分;4份样品中均不含NK603转基因玉米成分。说明农业部颁布的定性PCR方法标准能满足于对多种转基因混合样品的检测,且检测结果准确,可靠。     

种子DNA快速提取法在玉米转基因检测中的应用

利用一种快速玉米基因组DNA提取方法,分别对Bt11、MON810、NK603、MIR604和TC1507这5种转基因玉米种子的胚和胚乳进行DNA提取。根据不同转基因玉米重组结构点,分别设计特异性引物,参考农业部转基因检测标准,通过对PCR体系的优化整合后发现,该玉米DNA快速提起法获得的PCR检测结果准确、可靠,在此基础之上建立一套多重PCR,使转基因检测的效率得到很大提高。     

转基因玉米特异性检测阳性标准分子的构建与应用

试验用PCR方法从玉米中扩增内源基因zSSIIb片段,并将其克隆到pMD18-T载体上,获得中间载体pMD-zSSIIb;根据Bt11和MON810玉米转化体特异性序列,分别设计带酶切位点的引物,扩增出Bt11和MON810转化体特异性产物;用相应的酶对pMD-zSSIIb和两种PCR产物进行酶切,分别将Bt11和MON810产物克隆到pMD-zSSIIb上,获得阳性标准分子pMD-ZB和pMD-ZM,并对其进行特异性测试。结果表明,获得的阳性标准分子可以作为转基因产品检测时的阳性对照。     

PCR对转基因玉米MON810的鉴定检测

本研究成功建立了商业化种植的转基因玉米MON810的筛选检测和品种鉴定的PCR方法.该方法根据玉米自身IVR基因作为内源特异参照基因扩增226 bp片段,检查模板DNA提取的质量,避免了假阴性结果;同时扩增CaMV35S启动子基因195 bp片段,可以对MON810转基因玉米进行筛选检测;此外,根据MON810品种设计的特异性鉴定检测引物,可扩增Maize genome/CaMV35S基因170 bp片段和HSP/CryIA(b)基因194bp片段,具有很强的品种特异性,达到了对MON810转基因玉米的品种鉴定目的.PCR反应循环参数是94℃2 min;94℃40sec,55℃60sec,72℃60sec,35次循环;之后72℃延伸5 min。     

定性PCR方法检测转基因玉米MON810转化事件的研究

转基因玉米MON810(Yield Gard R)是孟山都公司通过DNA重组技术和微注射轰击研发的一种具有对欧洲玉米螟(ECB;Ostrinia nublialis)有特殊抗性的转基因玉米品系,目前在世界各地已经得到广泛种植,为加强对该品系玉米的安全管理,本研究旨在建立MON810品系玉米的转化事件特异性定性PCR检测方法。根据MON810的插入序列信息,在3′端的侧翼序列处设计定性PCR检测的引物,检测MON810在其他几种常见转基因作物混合样品的特异性。结果表明,该方法具有很好的特异性。同时检测该引物系统的扩增灵敏度,结果表明,检测引物的灵敏度可达0.1%。建立的MON810特异定性PCR检测方法经全国7家实验室的验证,进一步证实该方法能够特异地检测出样品中的MON810转化事件,检测方法的灵敏度可达0.1%,且检测结果具有良好的可重复性和可重现性。MON810转化事件定性PCR检测方法的建立可满足于抗虫转基因玉米MON810及其衍生品种生物安全管理的需要。     

抗虫玉米MON89034转化体特异性PCR检测技术研究

根据抗虫玉米MON89034外源插入片段5’端与植物基因组连接区序列设计特异性引物,并进行PCR扩增,预期产物大小为455 bp。以zSSIIb基因作为内标准基因,建立了转基因玉米MON89034转化体特异性定性PCR检测方法。对该方法进行重现性、特异性和灵敏度测试,结果表明:该方法能够特异性检测出MON89034转化体,以100 ngDNA为模板,该方法的检测灵敏度达到0.1%,约为40个起始模板拷贝。复合PCR检测结果还表明,在同一PCR反应管中可实现对zSSIIb基因和MON89034的同时检测。     

转基因玉米Bt蛋白降解规律及其与土壤动物互作研究

以转Cry11Ac基因玉米及其非转基因对照为试验材料,利用大、中、小不同孔径的凋落物分解袋,研究黑土区转基因玉米外源基因表达Bt蛋白的降解规律及其与土壤动物的互作关系。结果表明,第二年春天大孔径凋落物分解袋内转基因玉米残体Bt蛋白含量为初始含量的25.98%,至8月份Bt蛋白只剩下初始含量的0.35%。分解袋孔径对转基因玉米及其对照玉米凋落物分解率和Bt蛋白降解率均有显著影响,表现为大孔径中孔径小孔径,说明大型和中型土壤动物都可以促进凋落物分解和Bt蛋白的降解。与对照玉米相比,转基因玉米凋落物分解率和凋落物内土壤动物群落结构参数都没有显著差异(P0.05,t-test),说明导入Bt基因对玉米残体分解速率和土壤动物均无显著影响。     

多重PCR结合变性高效液相色谱技术转基因小麦检测方法的建立

为了满足对小麦转基因成分高通量快速检测的需要,建立了一种新的转基因小麦检测方法。通过对不同稀释倍数的转基因小麦品系B7361中的内源基因Wx012、外源基因ubiquitin、bar、nos、uidA进行单一PCR和多重PCR扩增,应用琼脂糖凝胶和变性高效液相色谱（DHPLC,denaturing high performance liquid chromatography）技术分离PCR 产物,进行灵敏度分析,并用非转基因小麦京花1号籽粒、大豆籽粒、麦片、转基因小麦B7361加工成的油炸制品为样本,对建立的检测体系进行检测。结果表明,样品经多重PCR扩增和DHPLC分离后,能够得到准确可靠的转基因图谱。与凝胶电泳方法比较,本研究所建立的多重PCRDHPLC具有更高的检测通量和灵敏度(能够同时检测5个基因,灵敏度达到1 ng·μL^-1),能够满足小麦转基因成分的高通量快速检测的要求,同时也为转基因产品检测提供了一种新的技术手段。     

转酸性蛋白酶pepB基因玉米的分子鉴定与农艺性状分析

通过分子生物学技术把酸性蛋白酶pepB基因转入受体玉米自交系基因组中,培育转酸性蛋白酶pepB基因玉米新品系。以耐盐基因badh作为筛选标记性基因,经过抗性筛选,对T1、T2、T3代转基因植株进行PCR检测,得到14个T1代转基因阳性植株,32个T2代转基因阳性植株和27个T3代转基因阳性植株。Southern杂交结果表明,外源酸性蛋白酶基因已经整合进玉米基因组中。RT-PCR结果表明,酸性蛋白酶基因在受体玉米中获得表达,获得外源酸性蛋白酶pepB基因遗传表达的T3代转基因玉米株系。通过对T2、T3代转基因玉米各品系农艺性状的分析结果表明,转基因玉米各品系在株高、茎粗、穗长等农艺性状上与受体亲本没有差异,但生育期均有不同程度的缩短。     

耐除草剂转基因玉米CM8401的获得及功能性状鉴定

耐草甘膦和耐草铵膦是转基因作物育种重要的目标性状。将耐草甘膦基因MC1-EPSPS构建到含有bar基因的植物表达载体pTF101.1中，通过农杆菌介导法转入玉米材料Hi-II中，从而获得兼具耐受草甘膦和草铵膦性状的转基因玉米材料 CM8401。目的基因PCR检测显示，MC1-EPSPS和bar基因稳定整合到玉米基因组中。目的蛋白试纸条检测结果显示，MC1-EPSPS蛋白和PAT蛋白在转基因玉米世代间中表达稳定。田间除草剂耐受性鉴定试验表明，转基因玉米CM8401对草甘膦和草铵膦都具有良好耐受性，可耐受4倍推荐中剂量的草甘膦和草铵膦。     

大豆和玉米加工产品中外源转基因成分检测技术的建立

采用PCR技术检测CaMV 35S启动子,并进一步通过PCR检测RoundUp Ready Soybean(RRS)和Btl76 Maximaizer的特异性DNA片段,判断大豆和玉米加工产品中是否含相应转基因成分.在1份豆粕和豆腐样品中检测到了RoundUp Ready大豆特异性的498 bp片段,而在玉米粒样品中检测到了Btl76特异性转基因成分.PCR检测的灵敏度达到0.1%,稳定性良好.结果表明,PCR技术检测外源基因是灵敏和准确的,可以广泛地应用到转基因作物及其加工产品的转基因成分检测中.     

应用单管巢式和半巢式PCR检测转基因玉米MON89034

根据MON89034玉米的5’端和3’端边界序列分别设计1组转化体特异性的巢式PCR引物,采用中途进退式PCR策略建立MON89034玉米的转化体特异性检测方法,扩增产物分别为491 bp和188 bp。以转基因玉米MON89034及8种其他转基因作物为材料,证明此方法对MON89034玉米具有高度特异性。灵敏度测试结果表明,此方法的相对检出限达到0.01%,绝对检出限为4个单倍体基因组拷贝数,比普通PCR提高了5倍。建立的单管巢式和半巢式PCR方法可准确、高效地检测转基因玉米MON89034及其产品。     

Development of real-time PCR systems based on SYBR® Green I,Amplifluor™ and TaqMan® technologies for specific quantitative detection of the transgenic maize event GA21

Maize GA21 line has integrated several tandemly repeated copies of the r-act 5-enol-pyruvylshikimate-3-phosphate synthase construct used for plant transformation. We were able to amplify a nucleotide sequence corresponding to the polylinker plasmid vector flanked by the r-act promoter and nopaline synthase 3′-terminator. A method for specific detection and quantification of Roundup Ready^® transgenic maize line GA21 DNA using conventional and real-time PCR and based on this transgenic sequence is described. GA21 specific primers and probe were designed targeting the vector–promoter junction region and amplifying a 72-bp DNA fragment. Quantification methods were optimized through three different real-time PCR chemistries, i.e. SYBR^® Green I, Amplifluor™ and TaqMan^®. All three methods proved to be specific, highly sensitive and reliable for both identification and quantification of GA21 DNA.Plasmid pGAivr containing single copies of the GA21 and invertase amplicons was constructed for use as external standard in calibration curves. Using pGAivr, a TaqMan^® based real-time PCR assay was optimized in duplex format targeting the maize species-specific ivr1 gene and the GA21 junction region. The detection limit of the method was 0.01% GA21, which is far below the established threshold for accidental presence of genetically modified organisms (GMO), this method therefore being suitable for use in routine GMO analysis.     

Genetic basis of Cry1F resistance in two Brazilian populations of fall armyworm,Spodoptera frugiperda

Large-scale adoption of transgenic crops expressing genes from Bacillus thuringiensis (Bt) imposes high selection pressure for evolution of field-relevant resistance that can reduce pest control efficacy, such as reported for Cry1F maize (Zea mays L.) in populations of fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), of Puerto Rico, Brazil, and the United States. As part of our effort to improve fall armyworm resistance management to Bt crops, here we determined the genetic basis of Cry1F resistance in two S. frugiperda strains originated from field collections in different regions of Brazil and further selected in the laboratory for high levels of resistance to Cry1F maize. Continuous exposure to the TC1507 event for 11 generations resulted in more than 183-fold resistance to Cry1F in the two strains studied, and such a high resistance level enabled the insects to complete larval development on the Bt maize plants. Genetic analyses using concentration-response bioassays with progenies from reciprocal crosses between resistant and susceptible insects indicated that the inheritance of the resistance is autosomal, recessive and without maternal effects. Backcross of the F₁ progeny with the parental resistant strains revealed that the resistance in the two selected strains is conferred by a single locus or set of tightly linked loci. These results support some of the assumptions of the strategy in use for fall armyworm resistance management to Bt Cry1F maize, but survival rates of heterozygotes on the Bt plants were higher than 5%, showing that the Cry1F maize does not produce a high dose of the insecticidal protein for S. frugiperda. Additionally, we detected a delay in larval development time that may favor assortative mating of individuals carrying resistance alleles. These findings are consistent with the rapid evolution of Cry1F resistance in certain field populations of fall armyworm. Implications for resistance management of S. frugiperda to Bt maize are discussed.     

转基因抗虫玉米对亚洲玉米螟的抗性评价

采用田间人工接虫鉴定方法,根据玉米抗螟性田间鉴定评价标准,研究转基因玉米Bt799和NC6304YGRR对亚洲玉米螟的抗性.心叶期接虫试验结果表明,以食叶级别作为评价参数,2种转基因玉米对亚洲玉米螟具有良好的抗性,食叶级别均为1级;2种非转基因玉米食叶级别均大于7级.吐丝期接虫试验结果表明,以单株虫孔数、单株活虫数、单株隧道个数及单株隧道长度作为评价参数,2种转基因玉米对亚洲玉米螟抗性均显著高于各自对应的非转基因玉米.转基因抗虫玉米Bt799和NC6304YGRR田间抗虫效果良好,均能保护玉米在整个生育期内不受亚洲玉米螟危害.     

Functional dominance of different aged larvae of Bt-resistant Spodoptera frugiperda (Lepidoptera: Noctuidae) on transgenic maize expressing Vip3Aa20 protein

Fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), is the main target pest of transgenic maize expressing insecticidal proteins from Bacillus thuringiensis Berliner (Bt) in Brazil. To optimize resistance management strategies, we evaluated the functional dominance of different aged larvae of Bt-resistant FAW on Vip3Aa20 maize. We measured the survival and development of Vip3Aa20-resistant, -heterozygote, and -susceptible strains on MIR162 (expressing Vip3Aa20) and Bt11 × MIR162 × GA21 (expressing Vip3Aa20 and Cry1Ab) maize. The resistant strain, from neonate to sixth instar, showed more than 72% survival on Vip3Aa20 maize. From surviving larvae, more than 64 and 54% developed to pupae and adults, respectively. In contrast, heterozygote and susceptible strains showed no larval survival up to fourth instar, and less than 25% larval survival in the fifth and sixth instar on Vip3Aa20 maize. These larvae produced less than 21% of pupae and adults. The development time of FAW strains from neonate-to-adult exposed to Vip3Aa20 maize was similar; however, the resistant strain showed an increase of ∼ 2 d when compared to those fed only non-Bt maize. In summary, the resistance of S. frugiperda to Vip3Aa20 maize is functionally recessive from neonate up to fourth instar larvae. However, high larval survival of resistant strain and some survival of heterozygote larvae in advanced instars on Vip3Aa20 maize were observed. These results will be important for designing insect resistance management to Bt maize plants expressing Vip3Aa20 protein in Brazil.     

Rove beetle (Coleoptera: Staphylinidae) communities in transgenic Bt (MON810) and near isogenic maize

Field experiments were conducted to investigate the mechanism of the underlying patterns (abundance, species richness, diversity and similarity) of rove beetles in transgenic Bt (MON810) and in near isogenic maize stands in Hungary. During the three-year (2001–2003) survey, 1538 individuals and 21 species were sampled with pitfall traps. The Cry1Ab protein expressed by the MON810 maize hybrid did not influence the overall community structure. After grouping staphylinids into guilds we found no significant differences for non-aphidophagous predators and parasitoids, whereas there were significantly and marginally significantly higher abundances for predators with aphids in their diet in isogenic maize stands in 2002 and 2003 respectively. The abundance of the prey Rhopalosiphum padi (L.) showed a high fluctuation between stands and years and was numerically higher only in isogenic stands in the second half of the maize-growing season. The abundance of predatory guilds including aphids in their diet did not correlate with the total annual number of R. padi in the same year, but there was a linear correlation in successive years.