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Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0-1.5mm in size, C58c1 of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production,shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptⅡ ELISA (nptⅡ enzyme assay), PCR, Southern blot and leaf bleach,29 positive plants were identified from two genotypes of Bobwhite and Yanglmai 10, with an average transformation efficiency of 0.82 %. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration. 相似文献
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采用两种方法筛选以npⅡt为选择标记基因的转基因小麦植株,结果表明,由未成熟种子获得的植株在无菌条件下进行卡那霉素筛选时(A方法),50 mg/L的卡那霉素能有效抑制非转化小麦幼苗的正常生长;由成熟种子自然发芽获得的植株在滤纸上进行卡那霉素滴注筛选时(常规方法,B方法),80~100 mg/L的卡那霉素能较好地抑制非转化小麦幼苗的正常生长。相对于B方法的常规筛选方法,A方法具有材料对卡那霉素敏感性高、筛选周期短而且筛选结果可靠性高等优点,是一种高效的以npⅡt为选择标记基因的转基因小麦的筛选方法。 相似文献
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小麦规模化转基因技术体系构建及其应用 总被引:3,自引:0,他引:3
在主要农作物中,小麦属于遗传转化比较困难的作物,转化效率较低,重复性较差,转化规模较小,优良转基因材料较少,基因工程育种进程明显落后于大豆、玉米、棉花、水稻等作物。目前,应用于小麦中的转基因技术主要包括基因枪介导法和农杆菌介导法,有些实验室也采用花粉管通道、离子束注入、激光微束穿刺、PEG、花粉介导和农杆菌浸花等方法。在外植体利用方面,多数研究主要利用小麦幼胚及其愈伤组织作为起始转化材料,以成熟胚、幼穗、花药愈伤组织为材料转化成功的报道还比较少,需要进一步探索。在转化效率方面,基因枪报道为0.1%-16.7%,农杆菌报道为0.7%-44.8%,变化幅度较大。在目标基因转化方面,除了nptⅡ、bar、hpt、GUS、GOX、pmi、ALS等筛选基因和报告基因外,转化的功能基因主要涉及小麦品质、抗病性、耐旱性、抗蚜虫和抗除草剂等性状改良。农杆菌介导和基因枪介导转化小麦幼胚的转化效率除与受体基因型有关外,还与受体材料的生理状态有关,供体植株生长期间的温度条件、光照条件、营养条件和水分条件对转化效率有至关重要的影响,开花到幼胚取样期间适宜的昼夜温度有利于转化后胚性愈伤组织诱导和候选转基因植株的获得。从整体水平看,中国小麦转基因技术研究虽然取得了较大进展,如建立了小麦成熟胚高频率再生体系并应用于小麦转化,改进了小麦幼胚再生体系和转化体系,将一批抗病、耐旱和品质改良相关基因转入小麦,初步建立了小麦规模化转基因技术体系,但与国际先进水平相比,尤其与一些跨国生物技术公司相比,在转化规模和转化效率方面仍然存在较大差距。认为转化效率较低、基因型依赖性强、人工气候条件不够先进、转化队伍不稳定是限制中国小麦规模化转基因技术发展的瓶颈;建立主栽品种转化体系、提高转化效率、开展多基因转化、开发安全型转化技术、避免载体骨架序列插入、减少基因沉默、实现定点整合等是小麦转基因技术研究的发展趋势;通过对组织培养技术、植株再生和转化相关基因的研究,以及优良受体基因型筛选、培养基改良和各个转化影响因素的优化、集成等,克服农杆菌转化小麦的瓶颈,提高小麦转化效率,扩大转化规模。文章重点综述了基因枪和农杆菌转化技术在小麦中的应用和发展,回顾了近5年中国小麦规模化转基因技术研究进展,对于促进转基因小麦新品种培育和小麦功能基因组学研究具有一定参考价值。 相似文献
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【目的】分析小麦不同受渍时期的土壤氧含量变化特征,构建基于土壤氧含量的小麦受渍指标,为准确及时鉴别小麦受渍程度提供理论基础。【方法】选取小麦耐渍品种扬麦11与不耐渍品种郑麦7698,采用盆栽试验,分别在拔节—开花、开花—成熟2个时期进行渍水试验,设持续渍水0(CK)、5、12和20 d共4个处理,通过测定土壤氧含量和小麦产量,分析不同品种、不同处理冬小麦低氧胁迫受渍持续时长与产量的关系,筛选出以土壤氧含量为特征值的小麦受渍指标。【结果】土壤氧含量随着时间及生育期的推移逐渐降低,其高低起伏与自然降雨密切相关;拔节—开花时期受渍土壤氧含量减少量大于开花—成熟时期。CK的土壤氧含量随着生育期变化,在播种至拔节期以前主要保持在20 mg/L以上;从拔节期至灌浆期降至10 mg/L左右;灌浆期以后在10 mg/L以下。渍水处理阶段,2个生育时期小麦土壤氧含量低于5 mg/L;确定在4月下旬以前,可用土壤氧含量作为小麦受渍指标,受渍土壤氧含量的受渍阈值为5 mg/L。扬麦11和郑麦7698的产量与受渍处理天数均呈极显著负相关(P<0.01),不管品种是否耐渍,渍害持续时间越长,对小麦产量影响越大。生育前期土壤相对水含量对土壤氧含量影响较大,渍水下的土壤相对水含量使土壤氧含量维持在5 mg/L以下,而在生育后期,相对水含量对土壤氧含量影响不明显。分析小麦产量与土壤氧含量低于5 mg/L持续时长的关系,得出基于土壤氧含量的小麦受渍害指标标准,即在拔节—开花时期,扬麦11土壤氧气含量低于5 mg/L分别持续7、17和25 d以上时,郑麦7698分别持续8、18和24 d以上,认定小麦受轻度、中度、重度渍害影响;扬麦11与郑麦7698在开花—成熟时期持续11、18和27 d以上,认定小麦受轻度、中度、重度渍害影响。【结论】以小麦受渍时土壤氧含量的变化特征作为渍害判别标准符合江汉平原小麦种植实际生产现状,确定在4月下旬以前,可用土壤氧含量作为小麦渍害受渍指标,受渍土壤氧含量的阈值为5 mg/L。 相似文献
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【目的】通过研究扬麦系列品种的品质特性并进行品质聚类,明确扬麦各品种品质类型,为小麦品种区域化种植和品质育种亲本选用提供依据;通过对不同类型品种品质性状和系谱分析提出简单实用的品质选择指标和亲本选配原则。【方法】以扬麦24个品种为材料,于2015—2017年连续2年在江苏里下河地区农业科学研究所试验基地进行种植,采用随机区组设计,2次重复,统一田间种植管理。成熟后收获晒干,测定籽粒硬度、蛋白质含量、面粉湿面筋含量、SDS沉淀值、溶剂保持力(solvent retention capacity,SRC)、粉质仪参数和快速黏度仪参数等品质指标。【结果】扬麦品种硬度变幅为13.48—62.12,可以明显地分成硬麦和软麦2种类型,硬麦大于54,软麦小于32;沉淀值为6.33—13.75 mL;蛋白质含量为12.60%—14.61%,湿面筋含量为29.74%—38.13%,面筋指数为38.86%—82.83%;水溶剂保持力为54.69%—78.56%,碳酸钠溶剂保持力为71.40%—107.73%,蔗糖溶剂保持力为95.66%—127.27%;粉质仪吸水率为54.77%—67.40%,形成时间1... 相似文献
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PANG Jun-lan XU Hui-jun DU Li-Pu YE Xing-guo LI Lian-cheng XIN Zhi-yong MA You-Zhi DIAO Ai-po Adams M J 《中国农业科学(英文版)》2003,2(4)
CWMV-CP1 target gene and bar selection gene were co-transferred into commercial wheat vari-ety of Yangmai158 by particle bombardment. In total, 145 resistant plants to 3 - 5 mg L-1 Bialaphos were ob-tained, 21 plants were identified to be positive in T0 generation by PCR-Southern test, and the transformationfrequency had 0. 99%. T1 plants were further tested by PCR and Southern hybridization. Results demonstra-ted that the alien resistance gene had been integrated into the wheat genome. The segregation ratio of CP1+ toCP1- in T1 generation was 1.0 to 1.3, and didn't agree with Mendelian rule. RT-PCR result from T2 plantsshowed that the alien gene CWMV-CP1 had stable expression in wheat genetic background. 相似文献
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XU Qiong-Fang LI Lian-cheng CHEN Xiao Tian fang MA You-Zhi YE Xing-guo ZHANG Zeng-Yan XU Hui-jun XIN Zhi-yong 《中国农业科学(英文版)》2002,1(1):25-29
The immature embryos of wheat plants, cv. Jing 411, 12 - 14 days after pollination, were cultured on SD2 medium for callus induction. After 10 days culture, 800 wheat calli were bombarded by biolistic particle coated with theDNA of plasmid pBI121-2 harboring both Galanthus nivalis agglutinin gene and bar gene. 67 green plants were finally regenerated from the bombardment calli on selection medium containing 4mg/L Basta. The results of bioassay by both inoculating wheat aphids onto the plants and applying Basta solution of 50 mg/L and 75 mg/L onto the wheat leaves in the field, and the molecular analysis, such as PCR and Southern blotting, indicated that 8 T2 plants contaning the target genes were obtained. 相似文献
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基因枪共转化将拟南芥DREB2A基因和bar基因导入小麦 总被引:1,自引:0,他引:1
为了提高小麦的抗旱耐盐性,采用PCR方法克降了拟南芥DREB2A(AtDREB2A)基因,构建了由水稻Actin启动子驱动的组成型表达载体,通过基因枪共转化途径,将AtDREB2A基因和抗除草剂筛选bar基因同时导入小麦品种Alondra和扬麦12,bar基因、AtDREB2A基因的转化频率以及两基因的共转化频率分别达到2.4%、0.4%和0.2%.对部分阳性植株进行RT-PCR分析表明,导入的AtDREB2A基因和bar基因均获得稳定表达.本研究获得的共转化植株为今后剔除筛选标记基因、培育安全的抗旱耐盐小麦新种质奠定了基础. 相似文献
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小麦成熟胚培养再生率比较低,尤其对优良小麦品种成熟胚再生性和农杆菌侵染敏感性还缺乏研究,一定程度阻碍了转基因小麦有效开展和产业化进程。以扬麦18、扬麦15、扬麦16、新冬18、小偃166和周麦22等12个优良小麦新品种为材料,研究了其成熟胚再生率及其对农杆菌侵染的敏感性。结果表明,12个小麦新品种成熟胚再生率0~35.3%,周麦27最高,扬麦19次之(27.9%),新冬18、石麦B07-4056分别为211%和20.0%,其余品种再生率均低于20.0%;12个小麦品种成熟胚愈伤组织农杆菌侵染后GUS基因瞬间表达率0~33.3%,周麦18最高,扬麦15次之(23.8%),扬麦18、周麦22、周麦27和石麦B07-4056虽有表达但频率较低(6.7%~9.1%),其余品种没有观察到GUS基因瞬间表达。认为周麦27、扬麦19等品种适合进行成熟胚培养,周麦18、扬麦15等品种成熟胚适合进行农杆菌转化。优良小麦品种成熟胚再生性能和农杆菌敏感性评价为进一步利用细胞工程和基因工程途径改良小麦奠定了基础。 相似文献
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【目的】比较糯小麦籽粒可溶性糖含量和淀粉含量与非糯小麦的差异,研究其花后籽粒中糖降解和淀粉积累特点,为糯小麦的育种和生产应用提供参考。【方法】2004~2005年度测定不同来源的17个糯小麦品系和4个非糯对照品种籽粒可溶性糖和淀粉含量,2005~2006年度选择其中3个糯小麦品系和2个非糯对照品种研究其花后籽粒中糖降解和淀粉积累动态。【结果】15个糯小麦品系籽粒可溶性糖含量显著高于所有对照品种,另2个糯小麦品系籽粒可溶性糖含量显著高于对照扬麦12和扬麦5号,而与扬麦158和扬麦9号差异不显著。4个对照品种之间淀粉含量差异不显著,扬麦9号最高,扬麦158最低;供试的17个糯小麦品系籽粒淀粉含量均低于对照扬麦9号,其中13个糯小麦品系低于扬麦158。籽粒中糖降解和淀粉积累动态研究表明花后10~17d糯小麦籽粒可溶性糖含量下降速率小,开花17d后3个糯小麦品系先后都经历一个淀粉含量增长的相对“停滞”阶段。【结论】糯小麦成熟期籽粒中可溶性糖含量高,淀粉含量低,可能与其灌浆前期籽粒中可溶性糖转化效率低,淀粉积累中后期经历一个相对“停滞”阶段有关,这主要是由于GBSS的缺失导致糯小麦不能合成直链淀粉改变了糯小麦糖转化和淀粉积累特性。在糯小麦育种中应加强对花后籽粒中可溶性糖转化效率和淀粉积累动态的研究,选择灌浆前期糖转化效率高,淀粉含量增长“停滞”阶段出现较迟的品种,以改良糯小麦籽粒饱满度。 相似文献
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基因枪法获得GNA转基因小麦植株的研究 总被引:27,自引:1,他引:27
以小麦品种京 411作为基因枪转化的靶材料 ,取其未成熟胚诱导愈伤组织 ,经过 10 d左右培养后 ,用含有雪花莲凝集素 (Galanthus nivalis agglutinin ,GNA)和 bar基因的质粒 p BI12 1- 2轰击 80 0个胚性愈伤组织 ,在含有4mg/ L Basta溶液的培养基上进行筛选 ,分化及生根培养 ,获得 6 7棵再生植株。田间涂抹 Basta溶液 (5 0、75 m g/ L)检测和接麦蚜实验 ,提取转基因植株基因组 DNA,用扩增 GNA基因的引物经 PCR扩增 Southern杂交 ,结果表明利用基因枪转化已从 T2 代获得了 8株含有编码 bar/ GNA基因的转基因植株。 相似文献
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【目的】全蚀病是小麦的毁灭性病害。创制转PvPGIP2和TaLTP5双价基因小麦,分析外源PvPGIP2和TaLTP5在转基因小麦中的遗传与表达情况,选育抗全蚀病的转基因小麦新种质。【方法】采用基因重组技术构建了PvPGIP2和TaLTP5双价表达转基因载体pA25-PvPGIP2-TaLTP5,利用基因枪介导法将该载体转入小麦品种扬麦18,采用PCR、RT-PCR与qRT-PCR方法分析转基因小麦T0—T4植株中目的基因及其表达量,并对T3和T4逐株进行全蚀病接种与抗性鉴定。【结果】创制并选育出稳定的抗全蚀病转PvPGIP2和TaLTP5小麦株系6个。PvPGIP2和TaLTP5能够在这6个转基因小麦株系中稳定遗传,并高水平表达。对转PvPGIP2和TaLTP5小麦的全蚀病抗性鉴定结果表明,与受体扬麦18相比,转PvPGIP2和TaLTP5小麦对全蚀病抗性明显提高。【结论】创制了转PvPGIP2和TaLTP5抗全蚀病的小麦新种质,其对全蚀病具有一定的抗性。 相似文献
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为了建立栓皮栎体胚遗传转化体系,以未成熟合子胚诱导的体胚为转化受体,采用带有pCAMBIA1301质粒的根癌农杆菌菌株EHA105,其中含有hptⅡ和GUS基因,研究不同因子对遗传转化的影响。结果表明:选择培养中适宜的抗生素浓度为300 mg/L头孢霉素和30 mg/L潮霉素。预培养时间为15 d时,转化率达91.7%;菌液浓度为OD600=0.5,乙酰丁香酮浓度为200 μmol/L时,转化率可达100%。在添加潮霉素的选择培养基中培养4周后,组织化学染色和PCR检测证实,GUS报告基因已经转入到体胚中并正常表达。 相似文献
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[目的]阐明小麦杂交育种中LOX活性的组合类型、杂交后代F2群体的遗传分布等。[方法]选用小麦杂交组合"鲁麦22/扬麦9号"F2代284个单株进行LOX活性分析,研究其F2植株籽粒中LOX活性的次数分布。[结果]"鲁麦22/扬麦9号"组合中,F2代LOX活性低于4.00 n(kat)/g的有14株,占4.93%,高于20.00 n(kat)/g的有12株,占4.12%,这些单株可用于筛选LOX活性较高和较低品系的育种材料。[结论]该研究可为高、低LOX活性的小麦品种资源筛选和早代选择提供参考依据。 相似文献
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【目的】研究小麦品种籽粒灌浆与脱水特性,为培育灌浆快、脱水快的少(免)晾晒小麦品种提供选择方法和理论依据。【方法】2015—2016年以长江中下游地区7个主推小麦品种为试验材料,采用Logistic方程拟合、多重比较及相关分析等方法,测定灌浆与脱水指标,生理成熟期及收获期籽粒含水率等。【结果】籽粒灌浆呈“S”型“慢-快-慢”的增长趋势,但不同品种最大灌浆速率、平均灌浆速率及灌浆持续时间差异显著,扬麦11、扬麦158、扬麦16最大灌浆速率及平均灌浆速率较大,花后30 d籽粒干重均达35 g以上,灌浆持续期较短;扬麦15灌浆速率仅次于上述3个品种,但灌浆持续期最长;宁麦13、扬麦20、扬麦22灌浆速率较小。最大灌浆速率、平均灌浆速率以及渐增期、快增期和缓增期的灌浆速率均与千粒重极显著正相关,3个灌浆时期灌浆速率R2>R1>R3,花后30 d灌浆基本完成。籽粒灌浆完成后进入脱水阶段,生理成熟期和收获期籽粒含水率、生理成熟后籽粒脱水速率品种间差异显著,扬麦11、扬麦158、扬麦16生理成熟后籽粒脱水速率较高,扬麦15最低。收获期籽粒含水率与生理成熟期籽粒含水率、籽粒平均脱水速率、生理成熟后2 d籽粒脱水速率显著或极显著相关。【结论】扬麦11、扬麦158、扬麦16灌浆速率大,灌浆完成早,籽粒脱水快。花后30 d粒重>35 g可作为育种材料灌浆快慢的选择指标,生理成熟期后籽粒平均脱水速率可作为衡量小麦品种脱水快慢的选择指标。 相似文献
20.
Characteristics of Grain Filling and Dehydration in Wheat 总被引:2,自引:0,他引:2
ZHU DongMei WANG Hui LIU DaTong GAO DeRong Lü GuoFeng WANG JunChan GAO ZhiFu LU ChengBin 《中国农业科学》2019,52(23):4251-4261
【Objective】The characteristic of grain filling and dehydration in wheat was studied, which provided a selection method and a theoretical basis for breeding wheat variety with fast filling and dehydration, and its grain with safety moisture content without drying process at harvesting date. 【Method】 In 2015 and 2016, 7 main wheat varieties in the middle and lower of the Yangtze River Valley were used as tested materials. The grain filling traits, dehydration rate and grain moisture content at physiological maturity and harvest date were measured to explore their profiles by Logistic Growth Equation (LGE) fitting analysis and multiple comparison and correlation coefficient method. 【Result】 The results indicated that grain-filling of 7 varieties fitted LGE best, with an S-like breakthrough curve of slow-fast-slow trend, but there were significant differences in the maximum grain-filling rate, average grain-filling rate and grain-filling duration in different varieties. The maximum grain-filling rates and average grain-filling rates of Yangmai 11, Yangmai 158 and Yangmai 16 were higher, whose dry grain weights at 30 d after anthesis were more than 35 g and the grain-filling durations were shorter; The grain filling rate of Yangmai 15 was the fourth fastest, but the grain-filling duration was the longest among the 7 wheat genotypes; Ningmai 13, Yangmai 20 and Yangmai 22 had the smaller filling rates, respectively. The maximum grain-filling rate, average grain-filling rate, R1, R2 and R3 were significantly positively correlated with 1000-grain weight. The rate of grain filling was R2>R1>R3 in the three filling stages. The filling grain of wheat was basically finished at 30 d after anthesis. After the grain filling stage, it started the dehydration and drying stage. There were significant differences among the 7 genotypes in the grain moisture content at physiological maturity and harvest date, as well as the grain dehydration rate. The grain dehydration rates of Yangmai 11, Yangmai 158 and Yangmai 16 were higher than the others, whereas Yangmai15 was the lowest. The grain moisture content at harvest date was significantly (P<0.01, 0.05) correlated with the grain moisture content at physiological maturity date, the average dehydration rate after physiological maturity, and the grain dehydration rate at 2 d after physiological maturity.【Conclusion】In Yangmai 11, Yangmai 158 and Yangmai 16, the grain-filling was faster and completed earlier, and the grain dehydrated more quickly. The grain weight >35 g at 30 d after anthesis could be used as the selection parameter of the grain-filling rate. The average grain dehydration rate after physiological maturity could be a selection parameter that evaluates the dehydration property of wheat. 相似文献