大豆转基因体系的研究进展

从大豆的转基因方法和受体系统两个方面概述大豆转基因体系的最新研究进展,并讨论了大豆遗传转化的主要障碍及可能的解决途径。作者认为,以根癌农杆菌介导大豆子叶节和基因枪轰击大豆未成熟子叶是较有效的大豆遗传化系统。目前,在大豆遗传转化研究中存在着大豆组织培养体系有待于进一步完善、遗传转化率较低、重复性差、大豆受体基因型单一等问题,建立新的、高效和稳定的大豆组织培养体系,提高生产上栽培大豆品种的组织培养能力,改遗传转化现有的单一基因为多基因,可能是解决上述问题的有效途径。     

大豆转化技术

介绍近年来在大豆转基因研究中再生受体系统和转化方法等方面的研究进展。     

转g10-epsps基因耐除草剂大豆ZUTS-33对农田生物多样性的影响

转基因作物对农田生物多样性影响评价是其大田释放和获得环境安全证书之前的必要环节。本研究通过大田试验,比较喷施清水及除草剂对转g10-epsps基因耐除草剂大豆ZUTS-33及其受体大豆HC-3和主栽品种大豆ZH-13的大田节肢动物多样性、病害发生、根瘤菌数量及杂草多样性的影响。结果表明: 与对照非转基因大豆HC-3和ZH-13相比,转基因大豆ZUTS-33田间节肢动物多样性指数(百株虫口数、Shannon多样性指数、Simpson优势度指数和Pielou均匀性指数)无显著差异,大豆主要病害发病率和病情指数无显著差异,根瘤菌数差异不显著,大豆田杂草多样性无显著差异;转基因大豆ZUTS-33喷施除草剂与转基因大豆ZUTS-33、非转基因对照HC-3和ZH-13喷施清水相比,节肢动物多样性、病害发生以及根瘤菌数量等差异均不显著,但杂草数量显著降低。     

转HSSP基因大豆的分子检测和遗传稳定性分析

HSSP是用大豆密码子改造的10 kD玉米醇溶蛋白基因。在前期研究中,从获得的转基因大豆中筛选到1份单拷贝转基因材料GSDH5。该研究采用染色体步移法获取转基因大豆GSDH5的T-DNA插入位点的左边界旁侧序列,对获得的左边界旁侧序列进行分析,设计特异性引物,建立转基因大豆GSDH5特异性检测方法;采用Real-time PCR检测外源基因在转基因大豆不同组织部位(根、茎、叶、花和种子)中的表达量,采用RT-PCR和Western blot检测外源基因在转录和翻译水平上的遗传稳定性,并对转基因大豆GSDH5中的粗蛋白、含硫氨基酸含量及主要农艺性状进行测定分析,为培育高含硫氨基酸转基因大豆新品种奠定基础。结果表明:(1)分子鉴定显示,外源基因HSSP和筛选标记基因Bar成功整合到受体大豆‘东农50’基因组中,且以单拷贝的形式整合到大豆基因组中。(2)HSSP基因成功插入到大豆基因组1号染色体非编码区52 873 883 bp处。(3)HSSP基因在转基因大豆GSDH5的种子中特异性表达,且在T_2～T_4代转基因大豆中能够稳定遗传并表达。(4)‘东农50’粗蛋白含量在41.53%～43.32%之间,GSDH5粗蛋白含量在40.18%～43.03%之间,两者相比无显著差异;GSDH5种子中硫氨基酸占种子干样的比例为1.35%,占种子蛋白的比例为3.14%,与转基因受体品种‘东农50’相比,占比显著升高,分别增加了11%和16%。(5)转基因大豆GSDH5植株与受体品种‘东农50’在单株荚数、百粒重、株高、结荚习性、花色、叶形等方面均无显著差异,证明HSSP基因的插入对大豆植株的生长发育无不良影响。研究认为,转基因大豆GSDH5材料具备进一步培育成高含硫氨基酸大豆新品种的潜力。     

耐除草剂转基因大豆ZH10-6和亲本大豆中黄10的营养成分比较

目的：比较北京、石家庄、三亚三个产地的耐除草剂转基因大豆ZH10 6和亲本大豆中黄10的营养成分。方法：对三产地耐除草剂转基因大豆ZH10 6和亲本大豆中黄10的营养成分：水分、灰分、蛋白质、脂肪、膳食纤维、氨基酸、维生素、矿物质、脂肪酸等进行检测和分析。结果：三产地转基因大豆的钙、钾、叶酸含量高于亲本大豆,但均在ILSI推荐的参考范围内;个别产地、个别营养成分转基因大豆与亲本大豆营养成分存在差异,但属于自然变异;其余各营养成分转基因大豆和亲本大豆之间均无显著性差异。结论：耐除草剂转基因大豆ZH10 6和亲本大豆中黄10在营养成分上具有实质等同性。     

Chelex-100快速提取用于转基因检测DNA模板的研究

目的:建立从转基因作物中快速提取DNA的方法.方法 :采用Chelex-100法提取抗草甘膦大豆和非转基因大豆、转基因抗虫玉米Bt176和非转基因玉米中的DNA,使用PCR扩增大豆和玉米的内源基因(Lectin,zSSⅡb)及外源特异性序列(CaMV35S,Bt176)评价提取核酸的质量.结果 :Chelex-100法能够快速在1h之内从大豆和玉米中提取DNA,所提取的DNA可以直接用于PCR扩增反应,PCR扩增产物电泳条带清晰,转基因抗草甘膦大豆样品和转基因抗虫玉米Bt176检测均出现强阳性结果.结论 :Chelex-100法提取DNA可以作为转基因检测的模板,该方法具有经济、简便、快速的特点,适合于转基因检测工作.     

根癌农杆菌介导的大豆遗传转化

农杆菌介导法是大豆遗传转化的重要方法之一 ,许多实验室应用该方法得到了转基因大豆 ,但目前使用该方法进行转化的效率还比较低 ,尚需深入研究。农杆菌菌株、大豆基因型、组织培养条件、T-DNA的转移效率和转化后的筛选模式都会影响大豆转化的效率。概述了近年来根癌农杆菌介导的大豆遗传转化的一些重要成果 ,以及转化过程中大豆的易感性与农杆菌的转化能力、乙酰丁香酮促进vir基因活化、转化的受体系统和巯基混合物减轻受体材料的褐化、提高T DNA的转移效率等几个重要因素的研究进展 ,并介绍了转化中常用的几个筛选标记基因 (nptⅡ、hpt、bar基因和突变的ahas基因 )及通过共转化法去除标记基因的方法 ,同时对今后研究的重点进行了讨论.     

实时荧光定量PCR方法快速检测转基因大豆

针对转基因大豆中普遍含有的35S启动子进行引物设计,以双链DNA染料SYBR GreenⅠ为荧光标记物,利用实时荧光定量PCR方法对大豆样品进行检测。该法检测转基因大豆的检测低限为0.005 nmol/L的35S启动子,线性范围达3个数量级,可快速区分转基因大豆和非转基因大豆,具有快速、简便、灵敏、安全、高通量、低成本等优点,可推广用于转基因植物产品的快速定量检测。     

高等植物转基因研究

1　转基因的研究意义转基因是指通过基因工程技术将外源基因导入受体细胞内的过程。自 1984年转基因烟草问世以来 ,转基因技术已经在 2 0 0多种植物中获得成功。转基因物种涉及 5 0多个 ,特别是转基因棉花、水稻、大豆、玉米、烟草、蔬菜等已在生产上发挥重大作用。利用转基因技术可以冲破物种的界限 ,实现物种间的遗传育种 ,并能改良现有物种的生物性状 ,最大限度为人类服务。不仅如此 ,转基因技术还提供了科学的研究手段 ,使人类科技实现新的飞跃成为可能。将外源基因导入受体只是转基因的第一步 ,其在受体中的命运受到一系列生理生化过程…     

转基因大豆豆粕外源基因和蛋白在SD大鼠体内消化吸收分析

为了评估转基因抗草甘膦除草剂大豆的食用安全性,以20%的比例将转基因抗草甘膦除草剂大豆GTS40-3-2和其亲本非转基因大豆A5403豆粕分别添加到基础饲料中喂养两代Sprague-Dawley(SD)大鼠,采用定性、定量PCR和ELISA方法检测转基因大豆成分相关基因和蛋白在长期饲喂的大鼠体内代谢残留状况。结果表明,大鼠喂养转基因大豆豆粕后,除了大鼠肠粪和盲肠内容物检测到有转基因成分的残留,肠道菌群和实质脏器均未发现相关基因和蛋白。结果提示,长期饲喂转基因抗草甘膦除草剂大豆GTS40-3-2与亲本A5403大豆豆粕对SD大鼠具有同样的食用安全性。     

T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method

Designing transformation experiments for either functional genomics or crop improvement requires knowledge of the transgene locus structure, number, transmission and expression resulting from a specific transformation method. We recently reported an improvement to the soybean [Glycine max (L.) Merrill] cotyledonary-node transformation method that resulted in the efficient production of transgenic plants. To characterize the transgene loci resulting from this method, we analysed 270 independent T0 plants and 95 randomly selected T1 progenies for T-DNA locus complexity using Southern analysis. The lines were transformed with Agrobacterium tumefaciens strains LBA4404 or EHA105 carrying the binary plasmids pGPTV, pTOK233, pCAMBIA1303 or pCAMBIA1309, and regenerated in medium supplemented with or without silver nitrate (AgNO3). Analysis in the T0 generation showed that the number of hpt-hybridizing fragments per plant ranged from 1-15, with 31.5% of the lines having a single hpt-hybridizing fragment. Each primary soybean transformant had, on average, 2.0 unlinked transgene loci and that half of the segregating loci in the T1 progenies were single, simple T-DNA insertions. Of the loci containing multiple T-DNA fragments, a low frequency had tandem and inverted repeat T-DNA structures. Integration of binary plasmid backbone sequences occurred in 37% of primary transformants. A. tumefaciens strain, binary plasmid and thiol treatment had no significant effect on transgene locus structure, numbers or expression. Interestingly, exposure of soybean explants to AgNO3 throughout shoot induction and elongation increased T-DNA locus complexity in the primary transformants and decreased silencing of gusA expression in the T1 generation.     

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots

As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.     

Real-time PCR method using capturing oligo-immobilized PCR tubes to determine the specific gene for soybean and genetically modified soybean in food matrices

A new real-time PCR method using capturing oligo-immobilized PCR tubes is described. This method was used to detect specific genes for soybean and genetically modified (GM) soybean in food matrices. In a standard reaction using soybean genomic DNA and a capturing oligo for the lectin gene (Le1) immobilized on the tube, we examined the effects of such hybridization conditions as the location, length, and amount of the capturing oligo, and the incubation time and temperature. Under optimized conditions, the copy number of Le1 was determined in a concentration-dependent manner from soybean genomic DNA and soybean lysate (DNA 10-1000 ng, r=0.99; lysate 1-100%, r=0.99). The copy number of a Roundup Ready soybean (RRS) gene was also successfully detected in a concentration-dependent manner (1-100%, r=0.99) from GM soybean lysate, using PCR tubes with an immobilized capturing oligo for the transgene. Our data indicate that this is a rapid and simple method to determine specific genes for soybean and GM soybean in food matrices.     

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。     

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.     

抗除草剂转基因大豆后代遗传分析

分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3：1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒（SMV）并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。     

Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants

Production of stable transgenic plants is one factor that limits rapid evaluation of tissue specific transgene expression. To hasten the assessment of transgenes in planta, we evaluated the use of chimeric soybean seedlings expressing transgenic products in roots. Tap roots from four-day old seedlings (cultivars ‘Jack’ and KS4704) were excised and hairy roots were induced from hypocotyls via Agrobacterium rhizogenes-mediated transformation. Inoculated hypocotyls were screened on a MS-based medium containing either 200 mg/L kanamycin or 20 mg/L hygromycin. Beta-glucuronidase (GUS) activity assay indicated that highest GUS expression was observed in hypocotyls exposed to a 4-d pre-inoculation time, a neutral pH (7.0) for the co-cultivation medium. A 170-bp of the Fib-1 gene and 292-bp of the Y25C1A.5 gene fragments, both related to nematode reproduction and fitness, were cloned independently into pANDA35HK vector using a Gateway cloning strategy. The resulting RNAi constructs of the genes fragments were transformed into soybean using the chimeric hairy root system and evaluated for its effect on soybean cyst nematode (Heterodera glycines) fecundity. Confirmation of transformation was attained by polymerase chain reaction and Southern-blot analysis, and some potential for suppression of H. glycines reproduction was detected for the two constructs. This method takes on average four weeks to produce chimeric plants ready for transgene analysis.     

A transgenic locus in soybean exhibits a high level of recombination

Summary We have examined transgene inheritance in over 300 progeny of a line of soybean (Glycine max) transformed by particle bombardment with a construct containing bovine β-casein under the control of the soybean lectin 5′ and 3′ regulatory elements. Four copies of the casein transgene, located at a single locus, exhibit a high frequency of recombination that resulted in novel patterns in approximately 16% of the progeny in both the T₁ and T₂ generations. Characterization of the transgene locus using restriction enzymes that do not cut the transformation plasmid showed that all four transgene copies are at a single locus no larger than approximately 40 kb in size. Therefore, the recombination events resulting in the loss of transgene DNA are taking place within a limited physical distance on the host chromosome. This is the first report extensively documenting transgene instability at the DNA level in a plant transformed via particle bombardment. As this report indicates, a seemingly simple phenotype (presence of the foreign protein) may conceal inherent genetic instability at the DNA level.     

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.