抗草甘膦转基因大豆生物与环境安全性

世界转基因作物发展速度迅猛,其中抗除草剂转基因大豆的种植面积和作物产量都占有较大比例,其安全性也受到人们极大关注。文章通过对抗除草剂转基因大豆多年的研究总结,并结合国内外抗除草剂转基因大豆的研究文献,阐述了抗草甘膦转基因大豆现状及其发展,并对其生物和环境的安全性问题进行评价。     

耐草甘膦大豆种质资源筛选的研究

通过田间试验结合室内生物测定的方法,对不同大豆品种(系)进行了耐草甘膦特性的筛选,以期获得耐草甘膦大豆育种的新种质资源。结果表明,在草甘膦常规剂量下,不同大豆品种(系)对草甘膦耐性程度有着较大差异。褐皮豆和绥无腥豆1号耐性程度较强;DS系列大豆对草甘膦的耐性明显高于其他品系。通过室内生物测定对耐性程度较高的品种(系)进行进一步研究发现,DS品系等4个品系的耐性程度较高,其耐性倍数分别为4.23、3.82、2.93、2.59和2.48。室内考种数据表明,草甘膦虽然对耐性品系的株高有不同程度的抑制;但耐性品系可以正常的成熟结实,在单株荚数、百粒重等重要指标上具有可利用价值。     

抗草甘膦转基因大豆粕对雄鼠运动机能的影响

在正常生理条件下,分别以抗草甘膦转基因大豆饲料和非转基因大豆饲料喂食雄性昆明小鼠,至30 d检测其运动能力、耐疲劳能力和耐缺氧能力,探讨抗草甘膦转基因大豆对雄鼠运动机能的影响。同时以环磷酰胺处理建立小鼠病理模型,探讨病理条件下抗草甘膦转基因大豆对雄鼠运动能力、耐疲劳能力和耐缺氧能力的作用。结果显示,在正常生理条件下,与非转基因对照组相比,喂食30 d抗草甘膦转基因大豆饲料组雄鼠的运动能力、耐疲劳能力和耐缺氧能力均无统计学差异（P>0.05）;同样,在环磷酰胺诱导的病理条件下,喂食抗草甘膦转基因大豆饲料30 d也不会对雄鼠运动能力、耐疲劳能力和耐缺氧能力造成不良影响。结果表明,喂食抗草甘膦转基因大豆饲料30 d对雄鼠运动机能无毒性效应。     

Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha~(–1) of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.     

抗草甘膦转基因大豆SHZD32-01田间节肢动物和杂草多样性研究

通过田间试验,采用直接观察法,比较抗草甘膦转基因大豆SHZD32-01和受体中豆32各处理田间节肢动物和杂草的数量及物种丰富度、多样性指数、优势集中性指数和均匀性指数等群落特征指数,评价抗除草剂转基因大豆田间节肢动物和杂草的多样性。节肢动物调查结果显示,SHZD32-01喷施草甘膦、SHZD32-01人工除草、中豆32人工除草3种处理田间烟粉虱、小绿叶蝉、苜蓿盲蝽和中华草蛉等主要节肢动物数量无统计学差异,节肢动物群落特征(物种丰富度、生物多样性指数、优势集中性指数和均匀性指数)差异不显著。杂草调查结果显示,人工除草管理下,SHZD32-01和中豆32田间杂草物种丰富度、生物多样性指数、优势集中性指数和均匀性指数差异不显著,反枝苋、藜、稗和鲤肠等杂草密度无统计学差异;与其他2个处理相比,喷施草甘膦45 d后,SHZD32-01田间杂草物种丰富度无显著变化,但多样性指数和均匀性指数显著下降,优势集中性指数显著上升,鲤肠等杂草密度显著减少(P0.05)。结果表明,SHZD32-01种植1年不影响田间节肢动物和杂草多样性,草甘膦喷施1次亦不影响大豆田间杂草的丰富度。     

Sexual compatibility of transgenic soybean and different wild soybean populations

The introduction of genetically modified(GM) soybean into farming systems raises great concern that transgenes from GM soybean may flow to endemic wild soybean via pollen. This may increase the weediness of transgenic soybean by increasing the fitness of hybrids under certain conditions and threaten the genetic diversity of wild soybean populations. Although pollen-mediated gene flow between GM crops and wild relatives is dependent on many factors, the sexual compatibility(SC)determined by their...     

Overexpression of a modiifed AM79 aroA gene in transgenic maize confers high tolerance to glyphosate

It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops (Cao et...     

Overexpression of a modified AM79 aroA gene in transgenic maize confers high tolerance to glyphosate

It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops(Cao et al. 2012). In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301 Ubi Sp AM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase(rbcS) small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize(Zea mays) immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.     

Developing transgenic maize (Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis(Bt) cry gene and epsps(5-enolpyruvylshikimate-3-phosphate synthase) gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.     

Developing transgenic maize(Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis(Bt) cry gene and epsps(5-enolpyruvylshikimate-3-phosphate synthase) gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.     

用作植物疫苗的转基因胡萝卜的分子生物学筛选与鉴定

以包含有不同外源病毒基因的转基因胡萝卜为试验材料,应用分子生物学手段,从DNA水平上和蛋白质水平上对这些材料加以鉴定,经PCR引物筛选后得到转基因胡萝卜的阳性率为38%左右;为保证实验的可靠性和可行性,防止检测过程中遗漏和错误的发生,用特异的PCR引物对检测结果进行复选,得到的植株阳性率为7%.在蛋白水平上应用ELISA检测法检测获得的阳性植株的结果与PCR检测结果相同,且同时发现外源基因在胡萝卜根中的表达量明显小于在叶中的表达量.     

大豆农杆菌介导转化系统的优化研究

对大豆子叶节再生系统及其农杆菌介导转化的适宜筛选剂浓度进行了优化 ;同时以发芽 5天的大豆无菌苗的下胚轴切段为外植体 ,以抗性愈伤为指标 ,对影响农杆菌介导的大豆转化频率的因子进行了研究。结果表明 :不同大豆品种对选择剂的敏感性存在一定差异。 4种选择剂比较 ,PPT抑制效果最好 ,品种之间比较稳定 ;潮霉素次之 ;卡那霉素的有效浓度最高。 4种选择剂的适宜浓度分别为 :卡那霉素 10 0～ 2 0 0 mg· L-1;G4 185 0～ 75 mg· L-1;潮霉素 2 0～ 30 mg·L-1;PPT5 mg· L-1。农杆菌感染时间和共培养时间均对大豆的转化有明显的影响。感染时间以 8分钟左右为宜 ;共培养时间以 2～ 4天为宜。在农杆菌感染和共培养阶段的乙酰丁香酮浓度和 p H值对大豆转化频率有明显影响。p H值以 5 .4 0～ 5 .6 0为宜 ;乙酰丁香酮的适宜浓度为 10 0 μmol· L-1左右。在感染农杆菌之前 ,让外植体在高渗培养基上预培养一段时间能提高大豆下胚轴切段的转化频率。最适预培养天数为 1d。     

转基因大豆、玉米和水稻外源基因检测通用标准分子的构建

将转基因大豆、玉米和水稻的主要外源Cry1A(B)基因、BAR基因、CP4-EPSPS基因、PAT 基因和内参RBCL基因目标片段分别克隆到克隆载体pMD18-T中,构建获得的质粒可作为定性检测3种转基因粮食作物的上述外源基因的通用标准分子质粒pMD18-T-PAT-CP4-EPSPS-Cry1A(B)-BAR-RBCL,长约4.7 kb.经过双酶切、测序及PCR扩增,获得与预期片断大小及序列一致的目的基因片段,证明所构建的标准分子质粒是正确的,可以用来作为不同品种转基因粮食作物定性检测CP4-EPSPS基因、Cry1A(B)基因、BAR基因和PAT基因的通用阳性标准分子.     

转双价抗真菌基因大豆主要农艺性状的调查与分析

对已获得的对疫霉根腐病抗性显著提高的双价转基因大豆株系G0431和G0433进行了产量性状、品质性状和形态性状的调查与分析。结果表明,在产量性状上,两个转基因大豆株系与对照相比无明显差异;在形态性状上,株系G0431结荚习性由亚有限结荚习性变成了无限结荚习性,株系G0433株高低于对照、生育期短于对照、结荚习性由亚有限结荚习性变成了无限结荚习性;在品质性状上,与对照相比,两个转基因大豆株系蛋白含量有所下降而油分含量有所上升。     

转基因抗旱大豆对土壤酶活性的影响

采用盆栽试验,在正常水分管理和干旱胁迫条件下研究了转DREB基因抗旱大豆对土壤脲酶、纤维素酶、磷酸酶和过氧化氢酶活性的影响。结果表明,转基因抗旱大豆和非转基因大豆在正常土壤水分管理下,根际土壤中脲酶、纤维素酶、磷酸酶和过氧化氢酶活性在大豆VE、R1、R4期无显著差异。在干旱胁迫下,转基因抗旱大豆和非转基因大豆在VE和R1期土壤脲酶活性显著降低,R1和R4期土壤纤维素酶活性显著降低,R1期土壤磷酸酶活性分别呈显著增高和显著降低趋势,VE和R4期磷酸酶活性分别表现显著降低和无影响的作用,对土壤过氧化氢酶活性无显著影响。     

以皖豆28为受体的抗草甘膦转基因大豆的获得及后代抗性鉴定与分析

研究以皖豆28(WD28)为受体,采用农杆菌介导法,将抗草甘膦g10-epsps基因转入到大豆中,先后获得6株T_0代植株,转化效率为1.23%。对获得的转化体的下代进行PCR检测,均获得目的片段。同时取其根、茎、叶、花、荚、种子进行Western blot检测,均有外源蛋白表达。T_1代通过喷施田间推荐浓度的草甘膦进行抗性鉴定,统计抗性单株数,进行X~2分析,除转化体WD28-3外,其余5个后代的抗感分离比均符合3∶1的比例。     

转TaDREB3a基因大豆抗旱筛选鉴定

研究利用转TaDREB3a基因株系T4代大豆植株与野生型植株开展PEG模拟干旱处理和PCR鉴定,获得5个阳性TaDREB3a过表达株系。于苗期和花期分别干旱处理,根据表型及相对含水量、相对电导率、叶绿素含量等生理指标,利用抗旱隶属函数值对5个转基因株系划分抗旱级别,同时分析株高、根长、荚数、粒数等农艺性状差异显著性。结果表明,大豆株系花期极其敏感,不适合在花期鉴定抗旱性,苗期适合抗旱材料鉴定;通过各生理指标与抗旱级别划分以及相关农艺性状显著性分析,最终确定株系HL16-16-6、HL16-17-1为高抗品系,为抗旱材料鉴定及抗旱品种培育奠定理论基础。     

转TaDREB3基因大豆的农艺性状研究

将抗逆基因TaDREB3转入东农50,得到稳定遗传株系,并将T4代株系种子播于盐碱地上。将其生育期、株高、主茎节数、分枝数、单株荚数、单株粒数、百粒重等性状和对照东农50进行比较,研究在较高盐碱条件下该基因对大豆农艺性状方面的影响。结果表明,在较高盐碱条件下,转基因株系和对照植株相比,生育期并没有改变,而主茎节数、单株荚数和分枝数等农艺性状呈显著或极显著提高,这表明转TaDREB3基因大豆对盐碱胁迫具有一定的抗性。     

缺磷胁迫下41％草甘膦异丙铵盐水剂对抗草甘膦大豆农艺性状和生理指标的影响

[目的]探明缺磷胁迫下草甘膦对抗草甘膦大豆农艺性状和生理指标的影响,为缺磷条件下抗草甘膦大豆种植时合理使用草甘膦提供理论依据.[方法]以转基因抗草甘膦大豆RR1为材料,采用溶液培养法,在RR1长出真叶时进行缺磷胁迫,第二复叶完全展开时进行4.98 mL/L的41％草甘膦异丙铵盐水剂处理,5d后测定各生理指标,10d后测量农艺性状.[结果]正常供磷条件下,草甘膦处理会降低RR1大豆的叶面积、总根长、主根长、地上鲜重、根鲜重、根冠比、根系活力、根系磷含量和叶片磷含量,而株高、叶片SOD活性和MDA含量增加,但与清水处理差异均不显著(P＞0.05).缺磷胁迫下草甘膦处理的RR1大豆的叶面积、总根长、地上鲜重、根系活力、叶片磷含量和根系磷含量降低,而株高、根鲜重、根冠比、叶片SOD活性和MDA含量增加,其中SOD活性变化达显著水平(P＜0.05).[结论]缺磷胁迫下喷施草甘膦,大豆的根鲜重、根冠比、叶片MAD含量和SOD活性增加,叶面积、地上鲜重、根系含磷量和叶片含磷量减少.