抗草甘膦转基因大豆AG5601对根际微生物群落功能多样性的影响

明确抗草甘膦转基因大豆AG5601对根际土壤微生物群落功能多样性的影响,为转基因大豆的安全性评价提供基础数据。采用BIOLOG的方法,对抗草甘膦转基因大豆AG5601、亲本大豆SNK500和普通栽培大豆中黄13成熟期和残茬期的根际土壤微生物的碳源利用特征进行比较分析。AG5601成熟期的根际土壤微生物群落碳源利用能力显著高于SNK500,但与中黄13无显著差异;在残茬期,AG5601、SNK500和中黄13的根际土壤微生物群落碳源利用能力均无显著差异。功能多样性分析发现,成熟期的AG5601与中黄13在根际土壤微生物McIntosh指数和Simpson指数均显著高于SNK500,而残茬期,3种大豆的根际土壤微生物多样性差异不显著。主成分分析表明,AG5601与SNK500成熟期和残茬期的根际土壤微生物碳源利用在两主成分轴上的差异较小,碳源利用模式基本相同。本研究表明,抗草甘膦转基因大豆AG5601对根际微生物群落功能多样性无长期、显著性影响。     

麦草畏室内生测方法建立及大豆对麦草畏耐受性分析

室内生物测定是植物对除草剂等化学物质耐受性鉴定的一种常用筛选方法,已广泛应用于大豆、稗草、棉花等植物对草甘膦、氯嘧磺隆等除草剂的耐受性研究,但麦草畏的室内生物测定方法和大豆对麦草畏耐受性相关研究尚未见报道。本研究以麦草畏对催芽大豆下胚轴伸长抑制率为评价指标,结合回归方程曲线分析和抑制中浓度分析,建立了大豆对麦草畏耐受性室内生物测定方法,确定以300μg/L麦草畏筛选浓度作为大豆室内生物测定临界筛选浓度。利用该方法对35份源自微核心种质的大豆品种进行鉴定,结果表明,随麦草畏浓度增加,不同品种对麦草畏的耐受性存在显著差异,大豆品种对麦草畏的耐受性降低,从中筛选出对麦草畏耐受性较高的大黄豆-1和什邡螺丝豆。本研究结果为培育抗麦草畏品种的亲本选配以及后代选择提供了理论依据、材料和技术支撑。     

转(Bt Cry1Ac+CP_4EPSPS)基因抗虫抗草甘膦棉花对草甘膦的耐受性研究

本研究比较了转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花与常规棉花在新疆棉区对草甘膦的耐受性差异。两年的研究结果表明,转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花对草甘膦有较好的耐受性,而对照棉花中棉所49对草甘膦耐受性较差。苗期喷施草甘膦后转基因抗棉铃虫抗草甘膦棉花生长发育没有受到影响,而对照棉花中棉所49喷施草甘膦后生长发育受到了明显的影响,个别植株甚至死亡。转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花株高、真叶数、蕾数、产量等指标与对照相比差异显著。转Bt Cry1Ac+CP_4EPSPS基因棉花对草甘膦的耐受程度显著高于非转基因棉花。草甘膦对转基因抗草甘膦棉花无负面影响。     

以草甘膦为筛选标记的大豆转基因体系的建立及抗除草剂转基因大豆的培育

抗草甘膦转基因大豆能显著提高大豆生产效率,具有重大的应用前景.本实验室前期研究建立了农杆菌介导、草胺膦为筛选剂的大豆转基因体系,转化效率在4%以上.在此基础上,利用G6-EPSPS和G10-EPSPS 2个具有自主知识产权的草甘膦抗性基因,通过优化转化体系,成功建立了以草甘膦为筛选剂的大豆遗传转化体系,转化效率达1%以上.浓度梯度实验发现,当草甘膦的筛选浓度为100 mg/L时,虽然丛生芽的再生率下降了50%～60%,但最终转化效率不受影响.进一步通过基因表达分析、Western blot、Southern blot和除草剂抗性鉴定等方法对转基因大豆进行了分子检测和验证,最后获得了分子特征明确、对草甘膦抗性稳定的抗草甘膦转基因大豆后代.结果对国内抗草甘膦转基因大豆转基因方法研究及抗除草剂新品种选育具有意义.     

抗草甘膦转基因大豆对非靶标节肢动物群落多样性的影响

在田间自然条件下,用直接观察法和吸虫器研究了抗草甘膦转基因大豆对豆田节肢动物群落的影响.结果表明,种植抗除草剂转基因大豆的豆田节肢动物群落和非转基因亲本大豆豆田节肢动物群落的物种数,优势集中性指数、多样性指数相似程度较高,它们之间差异不显著,说明抗除草剂转基因大豆对节肢动物群落多样性无明显影响.     

抗草甘膦转基因大豆对根际土壤细菌及根瘤菌的影响

转基因大豆是全球种植最广泛的转基因作物,抗除草剂是其最主要的转基因特性.微生物群落是土壤质量的监测指标之一,抗草甘膦转基因大豆及配施草甘膦是否影响大豆根际土壤细菌及根瘤菌群落尚不清楚.本研究基于大田试验,以非转基因亲本大豆‘中豆32’为对照(CK),分析转G10-epsps基因耐除草剂大豆SHZD32-01(GR)及配施草甘膦(GR+G)在大豆各生育时期对根际土壤细菌和根瘤菌的影响.结果表明: 与CK相比,GR、GR+G处理对苗期和成熟期根际土壤pH、总有机碳(TOC)、总氮(TN)、NH₄⁺-N含量等产生影响;GR处理显著增加结荚期根际土壤细菌群落丰度及多样性,GR+G处理显著增加结荚期根际土壤细菌群落多样性,但显著降低苗期和结荚期根际土壤细菌群落丰度;GR、GR+G处理改变了部分优势细菌类群的相对丰度,但不同生育期根际土壤优势细菌类群均为变形菌门、酸杆菌门、拟杆菌门、绿弯菌门、浮霉菌门和放线菌门;GR、GR+G处理改变了根瘤菌类群的相对丰度,但未影响慢生根瘤菌和中华根瘤菌两种主要大豆根瘤菌的相对丰度,且GR+G处理结荚期根际土壤根瘤菌相对丰度显著降低.环境因子分析显示,根际土壤放线菌和根瘤菌群落丰度主要受土壤pH影响.抗草甘膦转基因大豆或配施草甘膦显著影响结荚期根际土壤细菌和根瘤菌,但其影响随大豆的生长而消失.     

抗茎腐病转基因小麦新种质的筛选

为拓宽小麦茎腐病(又称茎基腐病)抗源种类,筛选抗茎腐病小麦新种质,对43份转TaPIMP1、AtNPR1和Gastrodianin基因小麦纯合株系,进行目的基因表达分析,以及茎腐病、纹枯病和赤霉病抗性鉴定。结果表明,转基因株系的目的基因均能正常表达;转基因株系间茎腐病抗性差异明显,24份转基因株系茎腐病抗性,比受体对照扬麦12显著提高;转基因株系茎腐病抗性与纹枯病抗性相关性显著,与赤霉病相关性不显著。结合农艺性状鉴定,筛选出5份抗茎腐病转基因株系,其中2份兼抗纹枯病和赤霉病,1份兼抗纹枯病,可作为长江中下游麦区茎腐病备用抗源。     

抗草甘膦转基因大豆对根际土壤氨氧化古菌群落多样性的影响

抗草甘膦转基因大豆是孟山都公司首次进行商业化生产的转基因作物。近年来其风险评价,包括土壤生物多样性风险评价受到越来越多的重视。氨氧化古菌是硝化过程的关键微生物,在氮素循环中起重要作用。本文以抗草甘膦转基因大豆(RRS)、非转基因亲本(RRS-S)、野生大豆(W-S)和栽培大豆(D-46)为材料,采用PCR-DGGE方法研究了根际土壤氨氧化古菌(AOA)群落多样性的变化,为转基因大豆的生态安全性评价提供理论依据。通过多样性指数与均匀度指数分析,RRS对根际土壤AOA群落多样性没有显著性影响;主成分分析结果表明,RRS与其他品种大豆(W-S和D-46)根际土壤AOA群落结构差异较大,但与亲本(RRS-S)差异不显著;amoA基因测序与系统发育树的构建表明,不同基因型大豆根际土壤中的AOA一部分属于文献中记录的土壤AOA类群(soil/sediment),一部分在现有文献中没有记录(soil),但被测序检测的AOA均不属于水生环境(water)或海洋底泥(sediment)中的AOA类群。另外,有众多已检测的AOA在文献中并无记录和归类,其中包括RRS的缺失条带1、20、25和特有条带3。综合分析,抗草甘膦转基因大豆对根际AOA多样性没有显著性影响,但改变AOA群落的组成。     

抗除草剂bar基因与EPSPS基因在转基因甘蔗中的应用研究

通过农杆菌介导法,分别将bar基因和EPSPS基因导入到甘蔗中,分别获得了抗草铵膦和草甘膦两种除草剂的转基因甘蔗。通过直接喷洒田间施用浓度的草铵膦和草甘膦除草剂,证明了转基因甘蔗T0、T1代、宿根1代和宿根2代外源基因遗传稳定,且均具有稳定的耐除草剂的功能。因此转耐除草剂基因的甘蔗,可以与转耐除草剂基因的玉米、大豆、油菜和棉花一样,有广阔的应用前景。     

转基因大豆叶片残体对白符跳的影响

转基因大豆种植后其外源基因表达产物会以叶片残体等形式暴露于土壤生态系统中,并对土壤动物存在潜在风险.本文利用我国自主研制的转hrpZm基因抗疫霉根腐病大豆B4J8049、转Cry1C基因抗食叶性害虫大豆A2A8001、转Cry1Iem基因抗食心虫大豆C802及非转基因对照亲本大豆Williams82,采用直接喂饲试验,通过60 d喂饲调查白符跳的存活率、繁殖率、体长变化等,研究3种转基因大豆材料对非靶标生物白符跳的影响.结果表明: 转基因大豆B4J8049、A2A8001和C802残体对环境指示生物白符跳的存活率、繁殖率及生长发育均无显著不良影响.初步确认转基因大豆B4J8049、A2A8001和C802在短时期内对环境无安全性风险,为其推广提供了生态安全基础数据.     

Assessment of gene flow from glyphosate-resistant transgenic soybean to conventional soybean in China

Glyphosate-resistant (GR) transgenic soybean has never been cultivated commercially in China. It is essential to develop the separation measures required to prevent out-crossing between GR and conventional soybean (Glycine max (L.) Merr.) by characterizing the transgene flow before GR soybean is released. In this study, the transgene flow from a GR transgenic soybean AG5601 to conventional soybeans was characterized. First, natural out-crossing rate was evaluated using 36 conventional soybean varieties interplanted with GR soybean AG5601 transformed with a cp4 EPSPS gene conferring the resistance to herbicide glyphosate in the field in 2007 and 2008 in China. Second, drift distance of cp4 EPSPS gene from GR soybean AG5601 to soybean cv. Zhonghuang13 was evaluated using the progenies harvested from eight directions at different distance. Third, the relationship of gene flow of GR soybean AG5601 with flowering synchronization days or insect pollinators of each variety was analyzed using regression analysis. Thirty-two of 36 tested conventional soybean varieties had surviving progenies after two times of sprays of glyphosate, and 49 of 41,679 progenies were verified to be glyphosate-tolerant heterozygous offspring. The out-crossing rates in positive varieties (having surviving offspring after two times of sprays of glyphosate) ranged from 0.039 to 0.934 %. The farthest distance (drift distance) between soybean AG5601 and cv. Zhonghuang13 at which out-pollinating was still able to be observed was 15 m, with an out-crossing rate of 0.012 %. Regression analysis showed that there was a positive relationship between cross-pollination frequency and flowering synchronization days or pollinator insects. Therefore, when GR soybean is released to the field, it should be critically separated with the conventional soybean in space and cultivation time with efficient insect control during flowering.     

Glyphosate-Tolerant Crops: Genes and Enzymes

This review focuses on the genes for the enzymes 5-enolpyruvyl-3-phosphoshikimlc acid synthase (EPSPS) and the glyphosate oxidoreductase (GOX). These genes have been used to genetically engineer plants that are resistant to the herbicide glyphosate. Overproduction of glyphosate-insensitive.EPSPS in transgenic crops has been used to overcome the deleterious effuts of this herbicide. The introduction into plants of GOX also confers glyphosate tolerance to plants and augments the tolerance of transgenic plants already expressing a glyphosate tolerant EPSPS. These genes also provide a method for selecting transformed plant tissue using the glyphosate tolerance as the selectable marker in the presence of inhibitory concentrations of glypllosate. Glyphosate tolerant transgenic plants of beet, corn, cotton, lettuce, poplar, potato, rapeseed. soybean, tobacco, tomato, and wheat have already been field tested and are entering agriculture.     

Efficient particle bombardment-mediated transformation of Cuban soybean (INCASoy-36) using glyphosate as a selective agent

Soybean is highly affected by weeds in tropical countries, causing significant losses in yields. Transgenic herbicide resistant soybeans have been produced in a limited number of varieties and parental lines. This study was conducted to obtain glyphosate herbicide resistant transgenic soybean plants through particle bombardment of embryonic axes in a Cuban variety. Shoot regeneration in 25 mg/L of glyphosate occurred within a short period and plantlets developed roots in a medium without selection pressure, which favored the in vitro growth of plants at a transformation frequency of 3.1–6.0?%. Expression and integration of the cp4epsps gene was confirmed in the progeny by an immune-detection assay, PCR and Southern blot. All greenhouse evaluated transgenic soybean lines (T₁) displayed tolerance to 1.25 Kg/ha of glyphosate. Growth and seed development of transformed plants was similar to untransformed plants. The regeneration procedure using embryonic axes combined with the efficient selection of shoots in glyphosate enabled the production of transgenic plants of this Cuban genotype, showing high tolerance to the herbicide, good efficiency and reproducibility.     

野生大豆资源对大豆疫病抗病性和耐病性鉴定

大豆疫病是大豆重要病害之一,在世界范围内导致严重经济损失。防治大豆疫病最有效方法是利用抗病或耐病品种。筛选抗性资源是发掘抗性基因和抗病育种的基础。本研究鉴定了野生大豆资源对大豆疫病的抗病性和耐病性,以期发掘优异抗源。苗期用子叶贴菌块方法鉴定104份野生大豆资源对两个不同毒力的大豆疫霉分离物PSJS2(毒力型:1a,1b,1c,1d,1k,2,3a,3b,3c,4,5,6,7,8)和PS41-1(毒力型:1a,1d,2,3b,3c,4,5,6,7,8)抗性,结果表明33份资源抗PS41-1,35份资源抗PSJS2,其中18份抗两个分离物。在抗病性鉴定基础性上,用菌层接种方法对选择的82份资源进行耐病性鉴定,发现7份高耐病性资源。这些结果表明,野生大豆中可能含有新的大豆疫病抗病和(或)耐病资源,这些抗病或耐病资源可以用于未来大豆抗病育种,以丰富大豆对大豆疫病的抗性遗传基础。     

Genetic Diversity of Soybean and the Establishment of a Core Collection Focused on Resistance to Soybean Cyst Nematode

Soybean cyst nematode （SCN; Heterodera glycines） Is one of the most Important pests affecting soybean production. The best method of control of SCN is through the development of resistant cultlvars. However, limited progress has been made in soybean breeding In China because most modern cultlvars have no resistance to SCN. The distribution and phenotype of 432 immune or highly resistant Chinese accessions were surveyed and a primary core collection was selected as a representative sample for further analyses. Using evenly distributed simple sequence repeat markers, five selection methods were applied to the primary core collection and the optimal method was chosen to establish a core collection, which consisted of 28 accessions. These encompassed 70.8% of the ailelic variation present in the overall resistant collection. The 28 accessions differed from the reference resistant accessions at the genomlc level, Indicating that Chinese resistant accessions are distinct from known resistant accessions. This applied core collection provides a rational framework for undertaking diversity surveys, using genetic variation for the investigation of complex traits and for the discovery of novel traits.     

Genetic diversity and association mapping in a collection of selected Chinese soybean accessions based on SSR marker analysis

For broadening the narrow genetic base of modern soybean cultivars, 159 accessions were selected from the Chinese soybean collection which contained at least one of seven important agronomic traits: resistance to soybean cyst nematode (SCN) or soybean mosaic virus (SMV), tolerance to salt, cold, or drought, high seed oil content or high protein content. Genetic diversity evaluation using 55 microsatellite loci distributed across the genome indicated that a large amount of genetic diversity (0.806) and allelic variation (781) were conserved in this selected set, which captured 65.6% of the alleles present in Chinese soybean collection (1,863 accessions). On average, 9.4 rare alleles (frequency <5%) per locus were present, which were highly informative. Using model-based Bayesian clustering in STRUCTURE we distinguished four main clusters and a set of accessions with admixed ancestry. The four clusters reflected different geographic regions of origin of the accessions. Since the clusters were also clearly different with respect to the seven agronomic traits, the inferred population structure was introduced when association analysis was conducted. A total of 21 SSR markers on 16 chromosomes were identified as significantly (P < 0.01) associated with high oil content (6), high protein content (1), drought tolerance (5), SCN resistance (6) and SMV resistance (3). Twelve of these markers were located in or near previously identified quantitative trait loci (QTL). The results for both genetic relationship and trait-related markers will be useful for effective conservation and utilization of soybean germplasm.     

A mini core subset for capturing diversity and promoting utilization of chickpea genetic resources in crop improvement

A core collection is a chosen subset of large germplasm collection that generally contains about 10% of the total accessions and represents the genetic variability of entire germplasm collection. The purpose of a core collection is to improve the use of genetic resources in crop improvement programs. In many crops the number of accessions contained in the genebank are several thousands, and a core subset consisting of 10% of total accessions would be an unwieldy proposition. In this article we have suggested a two-stage strategy to select a chickpea mini core subset consisting of only about 1% of the entire collection held in trust at ICRISAT’s genebank (16,991 accessions). This mini core subset still represents the diversity of the entire core collection. The first stage involves developing a representative core subset (about 10%) from the entire collection using all the available information on origin, geographical distribution, and characterization and evaluation data of accessions. The second stage involves evaluation of the core subset for various morphological, agronomic, and quality traits, and selecting a further subset of about 10% accessions from the core subset. At both stages standard clustering procedure was used to separate groups of similar accessions. A mini core subset consisting 211 accessions from 1,956 core subset accessions, using data on 22 morphological and agronomic traits, was selected. Newman- Keuls’ test for means, Levene’s test for variances, the chi-square test and Wilcoxon’s rank-sum non-parametric test for frequency distribution analysis for different traits indicated that the variation available in the core collection has been preserved in the mini core subset. The most important phenotypic correlations which may be under the control of coadapted gene complexes, were also preserved in the mini core. This mini core subset, due to its drastically reduced size, will prove to be a point of entry to proper exploitation of chickpea genetic resources. Received: 20 August 2000 / Accepted: 25 September 2000     

利用SSR分子标记检测黄淮夏大豆(Glycine max)初选核心样本的代表性

作物核心种质是用最小的样本代表其全部遗传资源的最大遗传多样性。为了检测大豆初选核心样本取样的代表性,本研究从黄淮夏大豆初选核心样本中,随机选取两个类群,其材料数分别为20份和14份;从保留种质的相应奥群中,分别随机选取6份和5份,共计45份材料,进行14个农艺性状和20对SSR引物的分析。对两组材料进行农艺性状聚类．保菌种质与初选核心种质聚在了一起;利用SSR分子标记数据聚类,也得到了相同的结果。初选核心样本两个类群材料的等位变异数分别为129个,136个;保留种质相应类群材料的等位变异分别为76个,71个;初选核心种质两个类群材料分别包合了整个资源86.00％和86．62％的遗传多样性。本研究为大豆核心种质构建及检测提供分子水平的依据。     

Effects of CO2 on stomatal conductance: do stomata open at very high CO2 concentrations?

Potato and wheat plants were grown for 50 d at 400, 1000 and 10000 micromoles mol-1 carbon dioxide (CO2). and sweetpotato and soybean were grown at 1000 micromoles mol-1 CO2 in controlled environment chambers to study stomatal conductance and plant water use. Lighting was provided with fluorescent lamps as a 12 h photoperiod with 300 micromoles m-2 s-1 PAR. Mid-day stomatal conductances for potato were greatest at 400 and 10000 micromoles mol-1 and least at 1000 micromoles mol-1 CO2. Mid-day conductances for wheat were greatest at 400 micromoles mol-1 and least at 1000 and 10000 micromoles mol-1 CO2. Mid-dark period conductances for potato were significantly greater at 10000 micromoles mol-1 than at 400 or 1000 micromoles mol-1, whereas dark conductance for wheat was similar in all CO2 treatments. Temporarily changing the CO2 concentration from the native 1000 micromoles mol-1 to 400 micromoles mol-1 increased mid-day conductance for all species, while temporarily changing from 1000 to 10000 micromoles mol-1 also increased conductance for potato and sweetpotato. Temporarily changing the dark period CO2 from 1000 to 10000 micromoles mol-1 increased conductance for potato, soybean and sweetpotato. In all cases, the stomatal responses were reversible, i.e. conductances returned to original rates following temporary changes in CO2 concentration. Canopy water use for potato was greatest at 10000, intermediate at 400, and least at 1000 micromoles mol-1 CO2, whereas canopy water use for wheat was greatest at 400 and similar at 1000 and 10000 micromoles mol-1 CO2. Elevated CO2 treatments (i.e. 1000 and 10000 micromoles mol-1) resulted in increased plant biomass for both wheat and potato relative to 400 micromoles mol-1, and no injurious effects were apparent from the 10000 micromoles mol-1 treatment. Results indicate that super-elevated CO2 (i.e. 10000 micromoles mol-1) can increase stomatal conductance in some species, particularly during the dark period, resulting in increased water use and decreased water use efficiency.