转基因大豆非同源对照模板QC-PCR检测方法的建立

建立了转基因大豆CP4EPSPS基因的非同源对照模板QC-PCR检测方法。非同源竞争对照构建方法如下:利用CP4EPSPS基因特异引物,以大肠杆菌基因组DNA为异源模板,在低严谨度PCR扩增,回收适宜DNA片段并克隆到pMD-8载体上。该片段与CP4EPSPS基因相应序列除两端引物序列完全相同外,其他部分没有同源性。     

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.     

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.     

Impairment of carbon metabolism induced by the herbicide glyphosate

The herbicide glyphosate reduces plant growth and causes plant death by inhibiting the biosynthesis of aromatic amino acids. The objective of this work was to determine whether glyphosate-treated plants show a carbon metabolism pattern comparable to that of plants treated with herbicides that inhibit branched-chain amino acid biosynthesis. Glyphosate-treated plants showed impaired carbon metabolism with an accumulation of carbohydrates in the leaves and roots. The growth inhibition detected after glyphosate treatment suggested impaired metabolism that impedes the utilization of available carbohydrates or energy at the expected rate. These effects were common to both types of amino acid biosynthesis inhibitors. Under aerobic conditions, ethanolic fermentative metabolism was enhanced in the roots of glyphosate-treated plants. This fermentative response was not related to changes in the respiratory rate or to a limitation of the energy charge. This response, which was similar for both types of herbicides, might be considered a general response to stress conditions.     

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20 + species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.     

aroA-In融合基因载体的构建、表达及对烟草的转化

PCR扩增突变的5’-烯醇丙酮酸莽草酸-3-磷酸合成酶(5’-enolpyruvylshikimate-3-phosphate synthetase,EPSPS)cDNA全长序列,插入pLitmus28得到pLEPSPS,进而通过反向PCR在EPSPS235／236aa之间将其打断为无功能的片段。选用人工构建的具有顺式和反式剪接功能的mini型蛋白内含子Ssp DnaB和Rma DnaB,插入被打断的aroA(抗除草剂基因),构建了质粒pLEBC、pLEBT、pLERC和pLERT。将4种重组质粒中的aroA-In(蛋白内含子Intein插入aroA)融合基因插入pET-32得到表达载体pETLEBC、pETLEBT、pETLERC和pETLERT,lPTG诱导后,SDS-PAGE分析显示其能在DE。中有效表达并发生相应的蛋白剪接。将aroA-cis Ssp DnaB和aroA-cis Rma DnaB融合基因分别插入pLYM中进一步构建成植物表达载体,农杆菌叶盘法转化烟草。基因组PCR分析表明融合基因整合入植物核基因组;RT-PCR分析显示其可在高等植物细胞中成功表达。结果说明蛋白内含子基因可以转化高等真核细胞,蛋白剪接技术可应用于高等植物细胞,从而为防止植物转基因扩散提供了一条新的途径。     

抗草甘膦EPSPS基因的专利保护分析

抗草甘膦转基因作物是目前全球播种面积最大的转基因作物,抗草甘膦基因（EPSPS）的克隆、表达及其功能验证等也因此成为现代分子生物育种研究的重点,利用专利等知识产权保护措施将这些功能基因和转化技术转变成自己的独占产权是发达国家和生物技术公司普遍采取的发展策略。通过检索搜集全球范围内EPSPS基因的专利和转化品系信息,分析研究了EPSPS基因在全球的专利保护、核心技术专利分布与产业化运用状况。结果表明,近几年对EPSPS的专利申请量迅速增加,专利申请人主要集中在美国、法国、中国等国家,但是核心技术和产业化应用的绝大部分专利由孟山都、拜耳、先锋、先正达等跨国公司拥有,相关产业的发展主动权也由此被其掌控。     

Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroA_A.sp. A phylogenetic analysis revealed that AroA_A.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The K_M for PEP and IC₅₀ [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroA_A.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC₅₀ [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroA_A.sp. The specific activity of the wild-type AroA_A.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.     

根癌农杆菌介导的转aroAM12基因棉花植株的草甘膦抗性

以中棉35无菌苗下胚轴为外植体,采用农杆菌介导法将含有通过基因优化技术获得的草甘膦抗性突变基因aroAM12导入棉花中.以aroAM12为选择标记,利用草甘膦直接筛选获得65棵再生植株.PCR和Southerablot分析表明,经过草甘膦筛选出的To代植株均整合有aroAM12基因.Western blot分析表明整合进的aroAM12基因得到了有效表达,且不同植株之间的表达不尽相同.大棚喷洒的实验结果表明To代转化植株具有很高的草甘膦抗性.对T1代棉花的草甘膦抗性遗传分析表明,aroAM12基因以孟德尔方式遗传.     

Co‐expression of GR79 EPSPS and GAT yields herbicide‐resistant cotton with low glyphosate residues

Glyphosate‐resistant (GR) crops have been adopted on a massive scale by North and South American farmers. Currently, about 80% of the 120 million hectares of the global genetically modified (GM) crops are GR crop varieties. However, the adoption of GR plants in China has not occurred at the same pace, owing to several factors including, among other things, labour markets and the residual effects of glyphosate in transgenic plants. Here, we report the co‐expression of codon‐optimized forms of GR79 EPSPS and N‐acetyltransferase (GAT) genes in cotton. We found five times more resistance to glyphosate with 10‐fold reduction in glyphosate residues in two pGR79 EPSPS‐pGAT co‐expression cotton lines, GGCO2 and GGCO5. The GGCO2 line was used in a hybridization programme to develop new GR cottons. Field trials at five locations during three growing seasons showed that pGR79‐pGAT transgenic cotton lines have the same agronomic performance as conventional varieties, but were USD 390‐495 cheaper to produce per hectare because of the high cost of conventional weed management practices. Our strategy to pyramid these genes clearly worked and thus offers attractive promise for the engineering and breeding of highly resistant low‐glyphosate‐residue cotton varieties.