油菜抗咪唑啉酮类除草剂基因<italic>BnALS1R</italic>等位基因特异PCR标记的开发与应用

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胡茂龙, 龙卫华, 高建芹, 付三雄, 陈锋, 周晓婴, 彭琦, 张维, 浦惠明, 戚存扣, 张洁夫, 陈松. 油菜抗咪唑啉酮类除草剂基因BnALS1R等位基因特异PCR标记的开发与应用 . 作物学报, 2013, 39(10): 1711-1719
[HU Mao-Long, LONG Wei-Hua, GAO Jian-Qin, FU San-Xiong, CHEN Feng, ZHOU Xiao-Yin, PENG Qi, ZHANG Wei, PU Hui-Ming, QI Cun-Kou, ZHANG Jie-Fu, CHEN Song. Development and Application of Allele-Specific PCR Markers for Imidazolinone-Resistant GeneBnALS1R inBrassica napus . Acta Agronomica Sinica, 2013, 39(10): 1711-1719] 复制到剪切板

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油菜抗咪唑啉酮类除草剂基因BnALS1R等位基因特异PCR标记的开发与应用

胡茂龙, 龙卫华, 高建芹, 付三雄, 陈锋, 周晓婴, 彭琦^*, 张维^*, 浦惠明^*, 戚存扣, 张洁夫, 陈松

江苏省农业科学院经济作物研究所 / 国家油料作物改良中心南京分中心 / 农业部长江下游棉花与油菜重点实验室, 江苏南京 210014

^* 通讯作者(Corresponding author): 浦惠明, E-mail:puhuiming@126.com, Tel: 025-84390370

收稿日期:2013-05-25

基金:本研究由国家自然科学基金项目(31101174)项目, 国家高技术研究发展计划(863计划)项目(2011AA10A10403), 江苏省自然科学基金项目(BK2011679), 江苏省农业自主创新基金[cx(11)4012]和南京农业大学作物遗传与种质创新国家重点实验室开放基金(ZW2011006)资助。

摘要

油菜抗咪唑啉酮类除草剂基因BnALS1R是从抗性突变体M9中克隆获得, 抗性基因BnALS1R与野生型基因BnALS1存在1处SNP, 即乙酰乳酸合酶第638位丝氨酸残基被天冬酰胺酸替代。为获得油菜抗除草剂基因BnALS1R的分子标记, 根据该处点突变, 结合获得的BnALS3与BnALS1序列, 开发30条等位基因特异PCR (allele-specific PCR, AS-PCR)引物, 采用筛选出的3条AS-PCR引物在F₂、BC₁和BC₂群体中进行PCR扩增。结果表明, 该标记有效检测出群体中存在的3种基因型, 其分离比分别为1∶2∶1、1∶1、1∶1, 均遵循单基因遗传规律。应用该标记对获得的抗性恢复系进行PCR扩增, 结果发现所有抗性恢复系均能扩增出抗性基因BnALS1R目的条带, 表明3条标记引物可应用于抗性基因的检测。AS-PCR标记的获得将促进以抗性基因进行油菜抗除草剂分子标记辅助选择育种。

关键词: 油菜; 咪唑啉酮类除草剂; BnALS1R; 乙酰乳酸合成酶; 等位基因特异PCR

Development and Application of Allele-Specific PCR Markers for Imidazolinone-Resistant GeneBnALS1R inBrassica napus

HU Mao-Long, LONG Wei-Hua, GAO Jian-Qin, FU San-Xiong, CHEN Feng, ZHOU Xiao-Yin, PENG Qi, ZHANG Wei, PU Hui-Ming^*, QI Cun-Kou, ZHANG Jie-Fu, CHEN Song

Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences / Nanjing Sub-center, National Center of Oil Crops Improvement / Key Laboratory of Cotton and Rapeseed (Nanjing), Ministry of Agriculture, Nanjing 210014, China

Abstract

ABnALS1Rgene conferring resistance to the imidazolinone herbicides was previously isolated from an ALS (acetolactate synthase) inhabiting herbicide-resistant mutant line M9 inBrassica napus. A single nucleotide polymorphism leading to an amino acid substitution from serine to asparagine at site 638 of ALS was found between theBnALS1Rin the mutant andBnALS1in the wild type. We reported here the development of polymorphic molecular marker for allele-specific PCR (AS-PCR) assays to distinguish herbicide-susceptible and resistant ALS alleles in either homozygous or heterozygous genotypes. Thirty primers were designed according to the SNP (site 638, Ser was replaced by Asn) and the difference in DNA sequence between theBnALS1andBnALS3cloned from M9 and other susceptible cultivars. Three primers of them with polymorphism were obtained, which can differentiate the homozygous resistant M9 from susceptible rapeseed. Meanwhile, these PCR markers can effectively discriminate among three genotypes using polymorphic primers and were evaluated in F₂, BC₁, and BC₂populations. Observed segregation ratios fitted the expected 1∶2∶1, 1∶1, and 1∶1 ratios, respectively, which confirms the single-locus Mendel model. Furthermore, we used the markers for detecting the resistant gene and evaluating the size of DNA segments introgressed from the M9 donor in the resistant restoring lines containingBnALS1R. All the resistant restoring lines had the DNA banding pattern ofBnALS1R. Therefore, PCR-based assays using the markers could be used for the detection and characterization of the herbicide-resistant geneBnALS1Rin rapeseed. The validated AS-PCR markers will facilitate the breeding of herbicide-resistant rapeseed by usingBnALS1Rin marker-assisted selection.

Keyword: Rapeseed (Brassica napus L.); Imidazolinone herbicides; BnALS1R; Acetolactate synthase; Allele-Specific PCR

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引言

咪唑啉酮类除草剂是目前应用较广泛的一类除草剂, 具有杀草谱广、用量低、除草效率高等优点, 深受广大农户喜爱^[1]。研究表明, 乙酰羟基酸合酶(acetohydroxyacid synthase, AHAS), 也叫乙酰乳酸合酶(acetolactate synthase, ALS)是咪唑啉酮类除草剂的靶酶; 该除草剂通过与ALS形成复合物阻断底物进入酶活性位点通路, 抑制ALS活性, 导致支链氨基酸合成受阻, 使植物组织失绿、黄化, 最后逐渐死亡^[2]。ALS负责催化2个平行反应, 催化两分子丙酮酸缩合形成2-乙酰乳酸放出CO₂, 最终生成缬氨酸和亮氨酸, 催化一分子丙酮酸和一分子2-氧丁酸缩合形成乙酰羟基丁酸, 最终生成异亮氨酸^[3]。对拟南芥、烟草、水稻、小麦、向日葵、大麦等抗性植物的分子机制研究表明, 抗性植物是由于体内ALS基因核酸序列产生若干碱基位点的变异, 造成编码蛋白氨基酸残基位点改变, 从而引起除草剂与ALS结合方式的变化^[4,5,6,7]。

在油菜中, 加拿大Swanson等^[8]采用小孢子化学诱变获得2个抗咪唑啉酮类除草剂的油菜突变体PM1和PM2^[8]。PM1和PM2都由 ALS基因点突变而成, 其中PM1是基因 BnALS1的第653位Ser发生突变, PM2是基因 BnALS3的第574位Trp发生突变(以拟南芥的ALS氨基酸位置计算), 目前国外已商品化的抗咪唑啉酮油菜品种都是由这2个突变体转育而成^[9]。2004年, 高建芹等^[10,11]在油菜和大豆多年轮作的试验田中发现了1株抗咪唑啉酮油菜。通过连续多年自交及小孢子培养得到了抗性株系M9。抗性效应鉴定发现, M9抗除草剂的浓度是除草剂有效杀草浓度的2~3倍, 抗性稳定、具有极高的利用价值。胡茂龙等^[12]从M9中克隆到抗性基因 BnALS1R发现, 抗性基因 BnALS1R与野生型基因 BnALS1存在1处SNP, 导致ASL1的第638位丝氨酸残基被天冬酰胺酸替代(以油菜的ALS1氨基酸位置计算), 这与PM1碱基突变位点相同。但M9为大豆田中发现的天然突变株后代系选材料, 该材料抗性是由除草剂诱发的自然突变获得, 这与向日葵抗性突变体IMISUN-1、IMISUN-2、RW-B获得的途径相同, 而与PM1、PM2抗性获得的途径不同^[5,6,7]。国外抗性基因商业化利用需要交纳昂贵的专利费, 这将会提高油菜生产成本^[12]。而我们发现的抗性基因 BnALS1R具有自主知识产权, 不受国际知识产权保护的限制, 若能进一步找到与之紧密连锁的分子标记, 从而进行标记辅助选择, 区分出育种材料中抗性基因的不同基因型, 则可指导田间选育, 加快育种进程。

等位基因特异PCR (allele-specific PCR, AS- PCR)是基于等位基因的SNP设计特异引物, 通过PCR扩增来检测样本基因型的一种鉴定方法^[13]。该方法不受季节和时间限制, 具有快速、高效的优点, 已在水稻^[14]和大麦^[4]中成功运用。但迄今, 国内外关于检测甘蓝型油菜对咪唑啉酮类除草剂抗性基因的鲜见报道。前期研究发现, 突变体M9抗性基因 BnALS1R与正常敏感基因存在1处SNP差异, 用DNA测序技术可检测抗性基因, 但操作繁琐、费用相对昂贵。本研究依据SNP差异, 拟开发抗性基因位点的AS-PCR标记, 以期快速、准确检测抗性基因 BnALS1R,为利用抗性基因进行油菜抗除草剂分子标记辅助选择育种奠定基础。

1 材料与方法

1.1 试验材料及除草剂

油菜抗咪唑啉酮类除草剂突变体M9为浦惠明等^[15]在油菜和大豆多年轮作的试验田中发现的天然突变体及后代单株M9-2、M9-3、M9-11和M9-14; 敏感野生型对照品种宁油12、宁油14、宁油16、宁油18和宁油20为江苏省审定品种; BnALS1R基因型分离群体所用的敏感型亲本3075R为江苏省农业科学院经济作物研究所选育的MICMS细胞质雄性不育双低恢复系。F₂群体(3075R/M9-2)是以3075R为母本, M9-2为父本杂交、自交获得; BC₁群体[(3075R/M9-2)/3075R]和BC₂群体[(3075R/M9-2)/ M9-2]是由F₁(3075R/M9-2)为母本, 分别与3075R、M9-2回交获得。190个抗咪唑啉酮MICMS纯合恢复系分别是由(3018R/M9)和(3075R/M9)的F₁植株花粉经小孢子培养加倍获得的DH株系^[12]。咪唑啉酮类除草剂为山东先达化工有限公司生产的5.0%“豆施乐”水剂。试验在本院油菜隔离繁殖区进行, 严格控制花粉外漂, 不对外界环境产生污染。

1.2 菌株、试剂与克隆载体

大肠杆菌 E. coliDH5α由农业部长江下游棉花与油菜重点实验室保存, DNA分子量标准、DNA回收试剂盒、质粒提取试剂盒均购自天根生化科技北京有限公司, 普通 Taq DNA聚合酶、dNTPs、PCR产物平末端加A试剂盒和IPTG均购自TaKaRa宝生物工程有限公司(中国大连), 高保真性DNA聚合酶KOD-Plus及PCR试剂购自东洋纺(上海)生物科技有限公司, 克隆载体pEASY-T1购自北京全式金生物技术有限公司。引物由上海Invitrogen生命技术有限公司合成, DNA测序由南京金斯瑞生物科技有限公司完成。

1.3 除草剂处理与DNA提取

2007年油菜花期用M9株系的不同单株与MICMS双低恢复系3075R配制F₁组合, 秋播鉴定F₁和亲本的抗性表现。2008年春以F₁自交获得F₂世代, 同时用相应的抗性亲本和敏感型亲本分别对以敏感型亲本为母本的反交组合F₁回交, 获得BC₁、BC₂群体, 秋播鉴定分离世代F₂、BC₁、BC₂的抗性表现。在油菜三至五叶期用浓度为90 ga.i. hm^-2除草剂处理, 处理前取少许叶片保存于-80℃冰箱, 用于DNA提取。20 d后调查成活苗与死苗数, 统计分离比例, 进行卡方测验。采用CTAB法并稍作修改提取DNA^[16], 用1％( V/W)的琼脂糖电泳和分光光度法检测其纯度。

1.4 分子标记开发

此前研究发现, M9与常规油菜宁油16、宁油18编码ALS家族中 BnALS1的核苷酸序列存在1处SNP^[12], 根据该处SNP可开发AS-PCR标记检测抗性基因 BnALS1R。但由于油菜ALS基因家族核酸序列的高度同源性, 特别是 BnALS1和 BnALS3在核酸水平的同源性达98%^[17], 使得在编码区内很难开发出引物分别特异扩增 BnALS1和 BnALS3的基因片段。为此首先必须找到这2个基因在M9、宁油16、宁油18的序列差异, 通过比较 BnALS1和 BnALS3的序列差异, 设计特异引物扩增 BnALS1基因序列片段, 排除 BnALS3基因序列对检测抗性基因的干扰。由于此前申请的专利中已经获得M9、宁油16、宁油18的 BnALS1全长编码序列( BnALS1-NY16、 BnALS1-NY18)^[18], 因此为获得M9、宁油16、宁油18的 BnALS3全长编码序列, 根据NCBI中 BnALS3基因序列(GenBank登录号为Z11526), 利用Primer Premier 5.0软件在基因编码区段两侧且与 BnALS1的非同源区设计特异正向引物BnALS3-F: 5'-CGC GGTACCCTCTCTCTCTCTCATCTAACCAT-3'和反向引物BnALS3-R: 5'-CGCACTAGTCTCTCAGTAC TTAGTGCGACC-3', 通过PCR扩增全长的 BnALS3基因。PCR体系含DNA模板1 μL、10×酶反应缓冲液2 μL、25 mmol L^-1 MgSO₄ 1.2 μL、2 mmol L^-1 dNTPs 2 μL、10 μmol L^-1引物各2 μL、1 U L^-1KOD-Plus Taq酶0.8 μL, 加水至20 μL。反应程序为94℃预变性5 min, 94℃变性30 s, 55℃退火30 s, 72℃延伸2.5 min, 35个循环。按试剂盒使用说明书进行PCR产物平末端加A后, 经1.2% ( V/W)琼脂糖凝胶电泳分离, 用DNA凝胶回收试剂盒将目的片段回收纯化, 连接于克隆载体pEASY-T1上, 热激转化DH5α。通过蓝白斑筛选和菌落PCR鉴定, 将阳性克隆送公司测序。通过DNAMAN6.0、Sequencher、DNASTAR等软件分析野生型与突变体 BnALS1、 BnALS3基因序列的差异。根据序列差异, 设计30条分子标记引物(表1)。其中每条等位基因特异PCR引物的3末端分别与SNP位点对应的等位碱基完全匹配, 同时在特异引物3末端的倒数第3或第4位置引入1个错配碱基, 每个位置引入3种错配碱基,以期获得多个多态性标记。等位基因特异PCR引物AP3F~AP14F分别与3条公用引物AP1R、AP2R、A5R配对, 公用引物AP15F~AP17F分别与等位基因特异PCR引物AP18R~AP29R配对。以提取的油菜基因组DNA为模板, PCR筛选36个组合分子标记引物扩增产物多态性。理论上, 若样品中的等位位点与等位基因特异PCR引物的3末端相匹配, 便能进行有效扩增, 反之则不能进行有效扩增。

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	图1 不同来源的油菜 ALS1与 ALS3基因序列比对与多态性引物位置倒三角形表示突变位点, 箭头线表示引物位置和扩增方向。Fig. 1 Sequencing alignment of ALS1 and ALS3 genes from different species in rapeseed and sites of polymorphic lociThe single-site mutation is denoted by inverted triangle. The arrow lines represent sites and directions of primers.

表1 检测抗性基因的AS-PCR引物序列 Table 1 AS-PCR primer for assay of herbicide-resistant gene

PCR反应体系含: 10 ng μL^-1 DNA模板2.0 μL, 10 μmol L^-1引物的各2. 0 μL, 10×酶反应缓冲液2 μL, 25 mmol L^-1 MgC1₂ 1.2 μL, 2.5 mmol L^-1dNTPs 1.6 μL, 5 U L^-1 Taq酶0.1 μL, 加水至20 μL; PCR反应程序为94^oC预变性5 min, 94℃变性30 s, 60℃退火 30 s, 72℃延伸1 min, 共35个循环; 然后72℃延伸5 min, 12℃冷却10 min后, 将扩增产物加上样缓冲液终止反应。扩增产物在质量比浓度为1.2%的琼脂糖凝胶上电泳, 经溴化乙锭染色并于凝胶成像系统下观察、记载。用筛选的多态性标记引物在抗性油菜和敏感野生型品种中克隆, PCR产物测序。

1.5 多态性标记对分离群体中 BnALS1R基因型的检测

为探明多态性标记对甘蓝型油菜抗咪唑啉酮基因 BnALS1R基因型的检测效果, 提取油菜苗期3个 BnALS1R基因型分离群体植株叶片DNA, 利用多态性标记引物进行PCR扩增。DNA提取、PCR反应、产物检测方法同上。统计基因型分离比例, 进行卡平方测验。

1.6 应用多态性标记检测抗咪唑啉酮MICMS纯合恢复系

于苗期提取抗咪唑啉酮MICMS纯合恢复系的DNA, 利用多态性标记引物进行PCR扩增, 检测抗性恢复系中抗性基因 BnALS1R是否存在。DNA提取、PCR反应、产物检测方法同上。

2 结果与分析

2.1 检测抗咪唑啉酮基因 BnALS1R多态性分子标记的开发

为消除 BnALS3基因序列对特异性检测抗性基因 BnASL1R的干扰, 首先扩增M9及野生型宁油16、宁油18的 BnALS3基因, PCR产物与预期大小相同。测序发现, 3个序列与NCBI上公布的 BnALS3基因序列Z11526同源性达99%, 表明成功克隆到3个材料的 BnALS3基因, 分别命名为 BnALS3-M9、 BnALS3-NY16、 BnALS3-NY18。将3个基因序列与之前获得专利的 BnALS1的3个基因序列及Z11524、Z11526比对发现, 3个材料的 BnALS1和 BnALS3基因编码区的DNA序列高度同源, 仅存在1处9个碱基插入/缺失和一些单核甘酸变异(图1)。而9个碱基插入/缺失主要是重复序列, 不适合作为引物序列。因此, 根据此前专利中发现的突变体M9与宁油16、宁油18的 BnALS1核苷酸序列存在的单碱基突变(G/A), 将引物序列锚定在 BnALS1与 BnALS3单核甘酸变异上, 共设计30条AS-PCR引物(表1)。筛选36个组合AS-PCR标记多态性, 结果获得AP15F/AP18R、AP15F/AP19能分别特异扩增敏感基因 BnALS1、抗性基因 BnALS1R, 这3条引物在基因序列上位置如图1所示。引物对AP15F/AP18R特异扩增的是 BnALS1位点上第1933处核苷酸为G的等位位点, 即未突变的油菜野生型 BnALS1基因, 对除草剂敏感; 引物对AP15F/AP19R特异扩增的是 BnALS1位点上第1933处核苷酸为A的等位位点, 即抗性基因 BnALS1R, 对除草剂具有抗性。

用上述2对引物对含敏感基因 BnALS1的常规油菜宁油12、宁油14、宁油16、宁油18、宁油20与含有抗性基因 BnALS1R的抗咪唑啉酮油菜M9、M9-2、M9-3、M9-11、M9-14为模板进行PCR扩增。结果表明, 所有常规油菜由引物对AP15F/AP18R扩增的产物均有828 bp特征条带, 而AP15F/AP19无特征条带。反之, 所有抗性油菜AP15F/AP18R扩增产物无特征条带而AP15F/AP19R均有828 bp特征条带(图2)。特征条带经测序表明, PCR产物属于油菜 BnALS1的基因片段而非其他非特异性扩增, 且在抗性油菜与常规油菜中存在单碱基突变G/A (图3)。田间苗期喷施除草剂表明, 未发现抗性油菜死苗, 而5个常规油菜喷药后20 d全部死亡。以上结果表明, 用2对引物多态性分子标记可以准确检测油菜种质资源中是否含有抗咪唑啉酮基因 BnALS1R。

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图2 AS-PCR标记引物在抗性油菜和敏感油菜中PCR扩增结果M: DNA分子量标准(500、800、1200、2000、3000和4500 bp); 1~5: 敏感油菜宁油12、宁油14、宁油16、宁油18、宁油20; 6: H₂O; 7~11: 抗性油菜M9、M9-2、M9-3、M9-11、M9-14。Fig. 2 PCR products obtained using AS-PCR primers among resistant and susceptible lines in rapeseedM: DNA marker (500, 800, 1200, 2000, 3000, and 4500 bp); 1-5: Ningyou 12, Ningyou 14, Ningyou 16, Ningyou 18, and Ningyou 20; Lane 6: H₂O; 7-11: M9, M9-2, M9-3, M9-11, and M9-14.

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	图3 AS-PCR引物在抗性油菜和敏感油菜中PCR产物序列比对三角形表示突变位点。The single-site mutation is denoted by triangle.Fig. 3 Sequencing of PCR products obtained using AS-PCR primers among resistant and susceptible lines in rapeseed

2.2 多态性标记对分离群体中 BnALS1R基因型的检测

利用2对多态性标记在对3个分离群体F₂、BC₁和BC₂中的 BnALS1R基因型检测发现, F₂群体呈现3种特征条带(图4), 即不含抗性基因MICMS双低恢复系3075R特征条带(引物对AP15F/AP18R扩增产物有特征条带828 bp而AP15F/AP19无特征条带)为纯合敏感基因型单株, 如单株6、11、14、17、20; 含有抗性基因的M9-2特征条带(引物对AP15F/AP18R扩增产物无特征条带而AP15F/ AP19R有特征条带828 bp)为纯合抗性基因型单株, 如单株10、12、16、24; 同时具有双亲杂合带型(引物对AP15F/AP18R 和AP15F/AP19扩增产物都有特征条带828 bp)为杂合抗性基因型单株, 如单株4、5、7~9、13、15、18、19、21~23。在F₂群体中检测154个单株, 纯合敏感基因型单株35个, 纯合抗性基因型单株39个, 而具有杂合抗性基因型单株80个, 3种基因型数量符合1∶2∶1 (表2)。BC₁群体中检测的227个单株呈现2种条带(图5)。其中纯合敏感基因型单株共110个, 如单株5、9~10、12、14~15、19~20、23, 杂合抗性基因型单株117个, 如单株4、6~8、11、13、16~18、21~22、24, 2种基因型数量符合1∶1 (表2)。BC₂群体中检测的201个单株同样呈现2种条带(图6), 其中纯合抗性基因型单株113个(图6), 如单株4、6~7、9、12~13、16~17、21~22、24, 杂合抗性基因型单株98个, 如单株5、8、10~11、14~15、18~20、23, 2种基因型数量符合1∶1 (表2)。以上3个群体PCR检测结果与苗期喷施除草剂鉴定结果完全一致, 基因型分离比均遵循单基因遗传规律。因此, 2对引物的多态性标记可有效检测油菜抗咪唑啉酮基因 BnALS1R的3种基因型。

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	图4 AS-PCR引物在F₂群体中PCR扩增结果M: DNA分子量标准(同图2); 1: 3075R; 2: F₁(3075R×M9-2); 3: M9-2; 4~24: 部分F₂单株。Fig. 4 PCR products obtained using AS-PCR primers among F₂ population in rapeseedM: DNA marker (same as Fig. 2); 1: 3075R; 2: 3075R×M9-2 F₁; 3: M9-2; 4~24: plants randomly selected from F₂ population.

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	图5 AS-PCR引物在BC₁群体中PCR扩增结果M: DNA分子量标准(同图2); 1: 3075R; 2: M9-2; 3: F₁(3075R×M9-2); 4~24: 部分BC₁单株。Fig. 5 PCR products obtained using AS-PCR primers among BC₁ population in rapeseedM: DNA marker (same as Fig.2); 1: 3075R; 2: M9-2; 3: 3075R×M9-2 F₁; 4-24: plants randomly selected from BC₁ population.

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图6 AS-PCR引物在BC₂群体中PCR扩增结果M: DNA分子量标准(200、500、800、1200和2000 bp); 1: 3075R; 2: M9-2; 3: F₁(3075R×M9-2); 4~24: 部分BC₂单株。Fig. 6 PCR products obtained using AS-PCR primers among BC₂population in rapeseedM: DNA marker (200, 500, 800, 1200, and 2000 bp); 1: 3075R; 2: M9-2; 3: 3075R×M9-2 F₁; 4-24: plants randomly selected from BC₂population.

表2 SNP位点在F₂、BC₁、BC₂群体中的基因型分离 Table 2 Genotype frequencies at SNP site in F₂, BC₁, and BC₂ populations

2.3 应用多态性标记检测抗性恢复系中的抗性基因 BnALS1R

在此前的研究中, 我们已获得190个携带抗性基因 BnALS1R的MICMS纯合恢复系。为验证筛选的多态性标记AP15F/AP18R、AP15F/AP19R对抗性基因 BnALS1R的检测效果, 对获得的抗咪唑啉酮纯合恢复系进行2次PCR扩增, 电泳结果表明, 所有抗性恢复系中都含有抗性基因 BnALS1R特征条带(图7), 进一步证明2对引物多态性标记检测抗性基因的准确性。

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	图7 多态性标记对抗性恢复系中抗性基因 BnALS1R的检测M: DNA分子量标准(同图2); 1: M9; 2: 宁油18; 5: 水; 3~4、6~24: 抗性恢复系。Fig. 7 Assay results of resistant gene BnALS1R for resistant restoring lines using polymorphic markersM: DNA marker (same as Fig. 2); 1: M9; 2: Ningyou 18; 5: H₂O; 3-4, 6-24: some of resistant restoring lines.

3 讨论

基于基因序列SNP开发分子标记的方法很多, 如RFLP、CAPS或dCAPS等传统方法^[19,20,21]。本研究利用抗性油菜M9携带的抗性基因 BnALS1R与野生型基因的SNP差异, 在油菜 BnALS1与 BnALS3序列的非同源区设计30条标记引物, 从中筛选出3条引物的AS-PCR标记。与传统标记相比, 该方法不需要繁琐的分子杂交和限制性酶切实验, 仅需通过2次PCR反应就可鉴定出甘蓝型油菜抗除草剂基因 BnALS1R及其基因型。方法高效、快捷、价格低廉, 只要具有PCR仪及相关电泳设备的实验室就能完成。

与一般农艺性状连锁的分子标记不同, 本研究获得的标记是根据基因突变位点设计的功能性标记, 能直接反映植株的抗性, 不存在由于遗传交换而造成的错误鉴定。因此, 利用该标记方法能有效用于抗咪唑啉酮基因 BnALS1R引起的油菜对咪唑啉酮类除草剂抗性的标记辅助育种。常规育种方法是利用含 BnALS1R基因的M9与生产上推广的主栽品种进行一系列杂交, 然后在每个分离后代中喷施除草剂进行鉴定, 最终选择含抗咪唑啉酮基因 BnALS1R的抗性单株。在每个分离后代除草剂鉴定过程中, 如果除草剂剂量使用不当或喷施不均匀极易导致误选。另外, 多年连续喷施过量的咪唑啉酮类除草剂可能会导致土壤残留, 对下茬作物有一定影响。有时在抗除草剂基因研究过程中需要保存不抗的材料, 用作比较研究。因此, 利用 BnALS1R基因的功能性标记检测植株DNA, 可以在油菜任何时期鉴定出抗性基因的纯合基因型单株以及优良性状的不抗单株, 淘汰其他单株, 这样不仅节约育种成本、减少土壤残留, 而且可大大加速抗咪唑啉酮油菜品种的选择进程。

甘蓝型油菜为白菜型油菜和甘蓝天然杂交自然加倍而来, 包括A、C两套基因组, 其中A基因组起源于白菜型油菜, C基因组起源于甘蓝。基因组进化过程中高度重排导致甘蓝型油菜相关基因多以基因家族的形式存在^[22]。家族内基因核酸序列高度同源, 咪唑啉酮类除草剂的靶基因ALS也不例外。A、C基因组内3个具有功能的ALS基因核酸序列高度同源^[23], 特别是 BnALS1和 BnALS3在核酸水平的同源性达98%, 这给AS-PCR标记检测 BnALS1增加了难度, 因为在进行PCR扩增油菜 BnALS1过程中会受到高度同源序列 BnALS3的干扰。本研究中使用的30条引物组合成36对AS-PCR标记, 仅筛选出1个组合在抗性油菜和常规油菜间具有多态性, 而其他组合虽然都能有效扩增但无多态性, 可能是由于同源序列 BnALS3的干扰。另外一个原因可能来自AS-PCR引物的自身设计特点, 因为在设计AS-PCR引物时, 不管将公用引物设计成正向引物或者是反向引物, 其他2条等位基因特异PCR引物的3末端都必须锚定在SNP位点上, 即使在AS引物3末端倒数第3或第4位置人为引入1个特定的错配碱基, 降低AS-PCR引物与非匹配模板结合, 也很难保证在多倍体中每条引物均具有很好的PCR特异性^[24]。同时研究中发现, 开发甘蓝型油菜AS-PCR标记时公用引物序列的特异性非常重要。本研究筛选的多态性AS-PCR标记公用引物AP15F恰好锚定在 BnALS1和 BnALS3的4个SNP上(图1), 其分别与等位基因引物AP18R、19R配对才能准确鉴定抗性基因型。反之, 换成公用引物AP16F、AP17F, 则达不到鉴定效果。推测是由于AP16F、AP17F仅锚定在 BnALS1和 BnALS3的2个SNP上, 特异性较差。研究中还发现, 当使用共同公用引物AP15F时, AS引物3-末端引入错配碱基的替换方式和位置也尤为关键。因为仅在3末端倒数第3位引入错配碱基时, 3种碱基替换方式中的C/A类型替换才具有引物特异性。反之, 在倒数第4位引入错配碱基时, 任何1种碱基替换方式的AS引物都不具有引物特异性。Hayashi等^[25]在水稻中也发现, AS引物在3末端倒数第3位引入C/A类型的替换能显著提高引物的特异性, 他对49个SNP位点设计了特异引物, 其中67%的引物能可靠地区分2个等位基因。卫波等^[24]认为错配碱基的位置及替换方式对PCR结果的影响可能与3末端不同错配碱基产生的碱基堆积力、氢键及其立体结构的稳定性有关。还可能与错配碱基对和DNA聚合酶之间的相互作用造成的空间位阻有关。因此, AS引物3末端能否与其互补模板特异扩增而不与其非互补模板扩增的特性是人工引入错配碱基与等位基因3末端固有的SNP错配碱基共同作用的结果。

Kadaru等^[14]在水稻、Lee等^[4]在大麦中也都获得了检测抗咪唑啉酮基因的AS-PCR标记, 但与水稻染色体组(2 n=24)和大麦(2 n=14)不同, 甘蓝型油菜为异源四倍体(2 n=38), 基因组庞大, 重复序列较多。因此, 为明确研究获得的引物对AP15F/18R、AP15F/19R特异扩增的是油菜 BnALS1基因片段而非其他非特异扩增, 我们对2对引物分别在常规油菜和抗性油菜扩增的条带进行了测序, 结果表明特异扩增的是 BnALS1基因片段, 且在常规油菜和抗性油菜之间存在单碱基突变。同时, 用该引物组合对3个群体进行扩增, 发现基因型分离比遵循单基因遗传模式, 证明该AS-PCR分子标记可以有效、准确检测油菜抗咪唑啉酮基因 BnALS1R的3种不同基因型。另外, 陈吉宝等^[26]根据普通小麦( Triticum aestivum L.)品种旱选10和鲁麦14之间在 TaDREB1基因的SNP进行AS-PCR标记研究, 获得了1对AS-PCR标记可以区分2个品种, 并用该标记引物将 TaDREB1基因定位于3BS, 表明AS-PCR标记可以用在诸如油菜、小麦等多倍体作物中。当然, 利用本研究的标记定位油菜 BnALS1R的工作正在进行中。

抗咪唑啉酮类除草剂油菜已在加拿大、澳大利亚、欧洲国家广泛种植, 但我国由于缺乏自主知识产权的抗除草剂基因一直无法商业化应用^[27,28]。前期研究, 我们从油菜自然突变的抗性材料M9中克隆获得了抗性基因 BnALS1R, 虽然该基因的碱基突变位点与加拿大PM1突变位点相同, 但 BnALS1R是我国拥有自主知识产权的抗除草剂基因, 不受国际知识产权保护的限制, 也是国内首次报道的抗咪唑啉酮类除草剂基因^[12]。同时, 本研究根据该突变位点的SNP, 在国内外首次开发了检测该抗性基因的分子标记, 并在抗性恢复系中进行了检测, 发现该AS-PCR标记能特异扩增出抗性基因的目的条带, 表明基于此标记的PCR检测方法精确、可靠。显然, 以上这些研究为我国抗咪唑啉酮油菜的选育提供了基因来源和功能标记。

4 结论

从30条等位基因特异PCR引物中筛选、鉴定出3条AS-PCR引物, 通过2次PCR就可以快速、准确检测出油菜品种或品系中咪唑啉酮类除草剂抗性基因 BnALS1R的基因型。该AS-PCR标记的获得为利用抗性基因进行油菜抗除草剂分子标记辅助选择育种奠定了基础。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。

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[13]	Drenkard E, Richter B G, Rozen S, Stutius L M, Angell N A, Mindrinos M, Cho R J, Oefner P J, Davis R W, Ausubel F M. . A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis. Plant Physiol, 2000, 124: 1483-1492 [本文引用:1] [JCR: 6.535]
[14]	Kadaru S, Zhang W Q, Yadav A, James H O. . Development and application of allele-specific PCR assays for imazethapyr resistance in rice (Oryza sativa). Euphytica, 2008, 160: 431-438 [本文引用:2] [JCR: 1.554]
[15]	Pu H-M(浦惠明), Gao J-Q(高建芹), Long W-H(龙卫华), Hu M-L(胡茂龙), Zhang J-F(张洁夫), Chen S(陈松), Chen X-J(陈新军), Chen F(陈锋), Gu H(顾慧), Fu S-X(付三雄), Qi C-K(戚存扣). . Studies on inheritance of imidazolinones resistance in Brassica napus and its utilization. Chin J Oil Crop Sci(中国油料作物学报), 2011, 33(1): 15-19 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.1971]
[16]	Hu M-L(胡茂龙), Long W-H(龙卫华), Gao J-Q(高建芹), Chen X-J(陈新军), Zhang J-F(张洁夫), Chen S(陈松), Qi C-K(戚存扣), Pu H-M(浦惠明). . Cloning of glutathione peroxidase BnGPX1 gene from Brassica napus L. and its expression under abiotic stresses. Chin J Oil Crop Sci(中国油料作物学报), 2011, 33(4): 331-337 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.1971]
[17]	Rutledge R G, Quellet T, Hattori J, Miki B L. . Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family. Mol Gen Genet, 1991, 229: 31-40 [本文引用:1]
[18]	Hu M-L(胡茂龙), Pu H-M(浦惠明), Qi C-K(戚存扣), Zhang J-F(张洁夫), Gao J-Q(高建芹), Long W-H(龙卫华), Chen S(陈松), Chen X-J(陈新军), Chen F(陈锋), Gu H(顾慧). . Gene Cloning and Application of an ALS Inhabiting Herbicide-Resistant Mutant Line M9 in Brassica napus. ChinaPatent(CN201010232607. 4), 2012 [本文引用:1]
[19]	Konieczny A, Ausubel F. . A procedure for mapping Arabidopsis mutations using co-dominant ecotype specific PCR-based markers. Plant J, 1993, 4: 403-410 [本文引用:1] [JCR: 6.16]
[20]	Michaels S D, Amasino R M. . A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. Plant J, 1998, 14: 381-385 [本文引用:1] [JCR: 6.16]
[21]	Neff M M, Neff J D, Chory J, Pepper A E. . dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J, 1998, 14: 387-392 [本文引用:1] [JCR: 6.16]
[22]	Chen X, Martin T, Crystal L S, Aliaa E M, Saleh S, Weselake R J. . Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different expression patterns and functional divergence in Brassica napus. Plant Physiol, 2011, 155: 851-865 [本文引用:1] [JCR: 6.535]
[23]	Ouellet T, Rutledget R G, Miki B L. . Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression. Plant J, 1992, 2: 321-330 [本文引用:1] [JCR: 6.16]
[24]	Wei B(卫波), Jing R-L(景蕊莲), Wang C-S(王成社), Chang X-P(昌小平). . Assaying single nucleotide polymorphism in wheat (Triticum aestivum L. ) with allele-specific PCR. Sci Agric Sin(中国农业科学), 2006, 39(7): 1313-1320 (in Chinese with English abstract) [本文引用:2] [CJCR: 1.4522]
[25]	Hayashi K, Hashimoto N, Daigen M, Ashikawa I. . Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theor Appl Genet, 2004, 108: 1212-1220 [本文引用:1] [JCR: 3.297]
[26]	Chen J-B(陈吉宝), Jing R-L(景蕊莲), Yuan H-Y(员海燕), Wei B(卫波). . Development of allele-specific PCR. J Plant Genet Resour(植物遗传资源学报), 2005, 6(4): 469-47 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.1628]
[27]	Pu H-M(浦惠明), Qi C-K(戚存扣), Zhang J-F(张洁夫), Gao J-Q(高建芹), Fu S-Z(傅寿仲), Chen X-J(陈新军), Chen S(陈松), Zhao X-X(赵祥祥). . Studies on the gene flow from herbicide-tolerant GM rapeseed to its close relative crops. Acta Ecol Sin(生态学报), 2005, 25(3): 196-203 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.7477]
[28]	Pu H-M(浦惠明), Qi C-K(戚存扣), Zhang J-F(张洁夫), Gao J-Q(高建芹), Fu S-Z(傅寿仲), Chen X-J(陈新军), Chen S(陈松), Zhao X-X(赵祥祥). . The studies on gene flow from GM herbicide-tolerant rapeseed to cruciferous weeds. Acta Ecol Sin(生态学报), 2005, 25(4): 910-916 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.7477]

2005

2.251

0.0

Pest Manag. Sci.. 2005, 61(3):246-57

Imidazolinone-tolerant crops: history, current status and future.

Tan S, Evans RR, Dahmer ML, Singh BK, Shaner DL

BASF Corporation, 26 Davis Drive, Research Triangle Park, NC 27709-3528, USA. tans@basf-corp.com

Imidazolinone herbicides, which include imazapyr, imazapic, imazethapyr, imazamox, imazamethabenz and imazaquin, control weeds by inhibiting the enzyme acetohydroxyacid synthase (AHAS), also called acetolactate synthase (ALS). AHAS is a critical enzyme for the biosynthesis of branched-chain amino acids in plants. Several variant AHAS genes conferring imidazolinone tolerance were discovered in plants through mutagenesis and selection, and were used to create imidazolinone-tolerant maize (Zea mays L), wheat (Triticum aestivum L), rice (Oryza sativa L), oilseed rape (Brassica napus L) and sunflower (Helianthus annuus L). These crops were developed using conventional breeding methods and commercialized as Clearfield* crops from 1992 to the present. Imidazolinone herbicides control a broad spectrum of grass and broadleaf weeds in imidazolinone-tolerant crops, including weeds that are closely related to the crop itself and some key parasitic weeds. Imidazolinone-tolerant crops may also prevent rotational crop injury and injury caused by interaction between AHAS-inhibiting herbicides and insecticides. A single target-site mutation in the AHAS gene may confer tolerance to AHAS-inhibiting herbicides, so that it is technically possible to develop the imidazolinone-tolerance trait in many crops. Activities are currently directed toward the continued improvement of imidazolinone tolerance and development of new Clearfield* crops. Management of herbicide-resistant weeds and gene flow from crops to weeds are issues that must be considered with the development of any herbicide-resistant crop. Thus extensive stewardship programs have been developed to address these issues for Clearfield* crops.

PMID: 15627242

... 引言咪唑啉酮类除草剂是目前应用较广泛的一类除草剂, 具有杀草谱广、用量低、除草效率高等优点, 深受广大农户喜爱^[1] ...

2006

9.737

0.0

... 该除草剂通过与ALS形成复合物阻断底物进入酶活性位点通路, 抑制ALS活性, 导致支链氨基酸合成受阻, 使植物组织失绿、黄化, 最后逐渐死亡^[2] ...

2008

2.838

0.0

Plant Physiol Bioch. 2008, 46(3):309 - 324 DOI:10.1016/j.plaphy.2007.12.004

Structure and mechanism of inhibition of plant acetohydroxyacid synthase

Abstract

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure ofArabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.

... ALS负责催化2个平行反应, 催化两分子丙酮酸缩合形成2-乙酰乳酸放出CO₂, 最终生成缬氨酸和亮氨酸, 催化一分子丙酮酸和一分子2-氧丁酸缩合形成乙酰羟基丁酸, 最终生成异亮氨酸^[3] ...

2011

9.737

0.0

Proc. Natl. Acad. Sci. U.S.A.. 2011, 108(21):8909-13 DOI:10.1073/pnas.1105612108

Single nucleotide mutation in the barley acetohydroxy acid synthase (AHAS) gene confers resistance to imidazolinone herbicides.

Lee H, Rustgi S, Kumar N, Burke I, Yenish JP, Gill KS, von Wettstein D, Ullrich SE

Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA.

Induced mutagenesis can be an effective way to increase variability in self-pollinated crops for a wide variety of agronomically important traits. Crop resistance to a given herbicide can be of practical value to control weeds with efficient chemical use. In some crops (for example, wheat, maize, and canola), resistance to imidazolinone herbicides (IMIs) has been introduced through mutation breeding and is extensively used commercially. However, this production system imposes plant-back restrictions on rotational crops because of herbicide residuals in the soil. In the case of barley, a preferred rotational crop after wheat, a period of 9-18 mo is required. Thus, introduction of barley varieties showing resistance to IMIs will provide greater flexibility as a rotational crop. The objective of the research reported was to identify resistance in barley for IMIs through induced mutagenesis. To achieve this objective, a sodium azide-treated M(2)/M(3) population of barley cultivar Bob was screened for resistance against acetohydroxy acid synthase (AHAS)-inhibiting herbicides. The phenotypic screening allowed identification of a mutant line showing resistance against IMIs. Molecular analysis identified a single-point mutation leading to a serine 653 to asparagine amino acid substitution in the herbicide-binding site of the barley AHAS gene. The transcription pattern of the AHAS gene in the mutant (Ser653Asn) and WT has been analyzed, and greater than fourfold difference in transcript abundance was observed. Phenotypic characteristics of the mutant line are promising and provide the base for the release of IMI-resistant barley cultivar(s).

PMID: 21551103

... 对拟南芥、烟草、水稻、小麦、向日葵、大麦等抗性植物的分子机制研究表明, 抗性植物是由于体内ALS基因核酸序列产生若干碱基位点的变异, 造成编码蛋白氨基酸残基位点改变, 从而引起除草剂与ALS结合方式的变化^[4,5,6,7] ...

... 该方法不受季节和时间限制, 具有快速、高效的优点, 已在水稻^[14]和大麦^[4]中成功运用 ...

... Kadaru等^[14]在水稻、Lee等^[4]在大麦中也都获得了检测抗咪唑啉酮基因的AS-PCR标记, 但与水稻染色体组(2n=24)和大麦(2n=14)不同, 甘蓝型油菜为异源四倍体(2n=38), 基因组庞大, 重复序列较多 ...

2008

3.297

0.0

Theor Appl Genet. 2008, 118(1):105 - 112 DOI:10.1007/s00122-008-0880-6

Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower

Carlos A. Sala(1) Mariano Bulos(1) Mariel Echarte(1) Sherry R. Whitt(2) Robert Ascenzi(2)

1.NIDERA S.A. Biotechnology Department Ruta 8?km 376, Casilla de Correo 6 2600 Venado Tuerto Santa Fe Argentina
2.BASF Plant Science LLC 26 Davis Drive P.O. Box 13528 Research Triangle Park, Durham NC 27709 USA

A partially dominant nuclear gene conferring resistance to the imidazolinone herbicides was previously identified in the cultivated sunflower (Helianthus annuus L.) line CLHA-Plus developed by seed mutagenesis. The objective of this study was to characterize this resistant gene at the phenotypic, biochemical and molecular levels. CLHA-Plus showed a complete susceptibility to sulfonylureas (metsulfuron, tribenuron and chlorsulfuron) but, on the other hand, it showed a complete resistance to imidazolinones (imazamox, imazapyr and imazapic) at two rates of herbicide application. This pattern was in close association with the AHAS-inhibition kinetics of protein extracts of CLHA-Plus challenged with different doses of imazamox and chlorsulfuron. Nucleotide and deduced amino acid sequence comparisons between resistant and susceptible lines indicated that the imidazolinone-resistant AHAS of CLHA-Plus has a threonine codon (ACG) at position 122 (relative to theArabidopsis thalianaAHAS sequence), whereas the herbicide-susceptible enzyme from BTK47 has an alanine residue (GCG) at this position. Since the resistance genes to AHAS-inhibiting herbicides so far characterized in sunflower code for the catalytic (large) subunit of AHAS, we propose to redesignate the wild type allele asahasl1 and the incomplete dominant resistant alleles asAhasl1-1 (previouslyImr1 orAr_pur),Ahasl1-2 (previouslyAr_kan) andAhasl1-3 (for the allele present in CLHA-Plus). The higher tolerance level to imidazolinones and the lack of cross-resistance to other AHAS-inhibiting herbicides ofAhasl1-3 indicate that this induced mutation can be used to develop commercial hybrids with superior levels of tolerance and, at the same time, to assist weed management where control of weedy common sunflower is necessary.

... 但M9为大豆田中发现的天然突变株后代系选材料, 该材料抗性是由除草剂诱发的自然突变获得, 这与向日葵抗性突变体IMISUN-1、IMISUN-2、RW-B获得的途径相同, 而与PM1、PM2抗性获得的途径不同^[5,6,7] ...

2012

3.297

0.0

Theor. Appl. Genet.. 2012, 124(2):355-64 DOI:10.1007/s00122-011-1710-9

Inheritance and molecular characterization of broad range tolerance to herbicides targeting acetohydroxyacid synthase in sunflower.

Sala CA, Bulos M

Biotechnology Department, NIDERA S.A, Ruta 8 km 376, Casilla de Correo 6, 2600, Venado Tuerto, Santa Fe, Argentina. csala@nidera.com.ar

Ahasl1 is a multilallelic locus where all the induced and natural mutations for herbicide tolerance were described thus far in sunflower (Helianthus annuus L.). The allele Ahasl1-1 confers moderate tolerance to imidazolinone (IMI), Ahasl1-2, and Ahasl1-3 provides high levels of tolerance solely to sulfonylurea (SU) and IMI, respectively. An Argentinean wild sunflower population showing plants with high level of tolerance to either an IMI and a SU herbicide was discovered and used to develop an inbred line designated RW-B. The objectives of this work were to determine the relative level and pattern of cross-tolerance to different AHAS-inhibiting herbicides, the mode of inheritance, and the molecular basis of herbicide tolerance in this line. Slight or no symptoms observed after application of different herbicides indicated that RW-B possesses a completely new pattern of tolerance to AHAS-inhibiting herbicides in sunflower. Biomass response to increasing doses of metsulfuron or imazapyr demonstrated a higher level of tolerance in RW-B with respect to Ahasl1-1/Ahasl1-1 and Ahasl1-2/Ahasl1-2 lines. On the basis of genetic analyses and cosegregation test, it was concluded that tolerance to imazapyr in the original population is inherited as a single, partially dominant nuclear gene and that this gene is controlling the tolerance to four different AHAS-inhibiting herbicides. Pseudo-allelism test permitted us to conclude that the tolerant allele present in RW-B is an allelic variant of Ahasl1-1 and was designated as Ahasl1-4. Nucleotide and deduced amino acid sequence indicated that the Ahasl1-4 allele sequence of RW-B has a leucine codon (TTG) at position 574 (relative to the Arabidopsis thaliana AHAS sequence), whereas the enzyme from susceptible lines has a tryptophan residue (TGG) at this position. The utilization of this new allele in the framework of weed control and crop rotation is discussed.

PMID: 21959907

2012

3.297

0.0

Theor. Appl. Genet.. 2012, 124(2):385-96 DOI:10.1007/s00122-011-1713-6

Response to imazapyr and dominance relationships of two imidazolinone-tolerant alleles at the Ahasl1 locus of sunflower.

Sala CA, Bulos M, Altieri E, Weston B

Departamento de Biotecnología, Nidera S.A, Casilla de Correo 6, CP.: 2600, Venado Tuerto, Santa Fe, Argentina. csala@nidera.com.ar

Imisun and CLPlus are two imidazolinone (IMI) tolerance traits in sunflower (Helianthus annuus L.) determined by the expression of different alleles at the same locus, Ahasl1-1 and Ahasl1-3, respectively. This paper reports the level of tolerance expressed by plants containing both alleles in a homozygous, heterozygous and in a heterozygous stacked state to increasing doses of IMI at the enzyme and whole plant levels. Six genotypes of the Ahasl1 gene were compared with each other in three different genetic backgrounds. These materials were treated at the V2-V4 stage with increasing doses of imazapyr (from 0 to 480 g a.i. ha(-1)) followed by an assessment of the aboveground biomass and herbicide phytotoxicity. The estimated dose of imazapyr required to reduce biomass accumulation by 50% (GR(50)) differed statistically for the six genotypes of the Ahasl1 gene. Homozygous CLPlus (Ahasl1-3/Ahasl1-3) genotypes and materials containing a combination of both tolerant alleles (Imisun/CLPlus heterozygous stack, Ahasl1-1/Ahasl1-3) showed the highest values of GR(50), 300 times higher than the susceptible genotypes and more than 2.5 times higher than homozygous Imisun materials (Ahasl1-1/Ahasl1-1). In vitro AHAS enzyme activity assays using increasing doses of herbicide (from 0 to 100 μM) showed similar trends, where homozygous CLPlus materials and those containing heterozygous stacks of Imisun/CLPlus were statistically similar and showed the least level of inhibition of enzyme activity to increasing doses of herbicide. The degree of dominance for the accumulation of biomass after herbicide application calculated for the Ahasl1-1 allele indicated that it is co-dominant to recessive depending on the imazapyr dose used. By the contrary, the Ahasl1-3 allele showed dominance to semi dominance according to the applied dose. This last allele is dominant over Ahasl1-1 over the entire range of herbicide rates tested. At the level of enzymatic activity, however, both alleles showed recessivity to semi-recessivity with respect to the wild-type allele, even though the Ahasl1-3 allele is dominant over Ahasl1-1 at all the herbicides rates used.

PMID: 21964993

1995

0.0

Mol. Gen. Genet.. 1995, 246(4):419-25

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance.

Hattori J, Brown D, Mourad G, Labbé H, Ouellet T, Sunohara G, Rutledge R, King J, Miki B

Plant Research Centre, Agriculture Canada, C. E. F. Ottawa, Ontario.

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 Trp-->Leu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.

PMID: 7891655

... 在油菜中, 加拿大Swanson等^[8]采用小孢子化学诱变获得2个抗咪唑啉酮类除草剂的油菜突变体PM1和PM2^[8] ...

1989

3.297

0.0

... PM1和PM2都由ALS基因点突变而成, 其中PM1是基因BnALS1的第653位Ser发生突变, PM2是基因BnALS3的第574位Trp发生突变(以拟南芥的ALS氨基酸位置计算), 目前国外已商品化的抗咪唑啉酮油菜品种都是由这2个突变体转育而成^[9] ...

2010

0.0

1.1628

J Plant Genet Resour. 2010, (3):369 - 373

Identification of Imidazolidone-Resistant Oilseed Rape Mutant

抗咪唑啉酮油菜种质的发现与鉴定

GAOJian-qin;PUHui-ming;QICun-kou;ZHANGJie-fu;LONGWei-hua;HUMao-long;CHENSong;CHENXin-jun;CHENFeng;GUHui

高建芹;浦惠明;戚存扣;张洁夫;龙卫华;胡茂龙;陈松;陈新军;陈锋;顾慧

在喷施豆施乐除草剂的大豆试验田,发现抗咪唑啉酮类除草荆的自生油菜突变株,经连续3年自交纯合和抗性鉴定,该突变体抗咪唑啉酮类除草剂的浓度是除草剂有效杀草浓度的2倍以上,抗性性状稳定、抗性效应明显.该材料的发现对于开发具有自主知识产权的抗除草剂新基因,选育不受环境释放限制的油菜新品种,均具有重大的理论和现实意义.

Abstract：

A rapeseed mutant resistant to imidazolidone was found as a volunteer in the soybean field treated by spraying imidazolidone-herbicide.The resistance of this mutant was steadily inherited and effective,which had been testified by consecutive assessment of several generations in 3 years.The mutant showed resistant to a dose of imidazolidone which was two times of the effective concentration of this herbicide.It is of great theoretical and practical significance for the identification of new herbicide-resistant oilseed rape genes with independent intellectual property,also for the development of new varieties against environmental release restrictions.

... 2004年, 高建芹等^[10,11]在油菜和大豆多年轮作的试验田中发现了1株抗咪唑啉酮油菜 ...

2010

0.0

1.0171

Jiangsu Journal of Agricultural Sciences. 2010, (6):1186 - 1191

Resistance of Imidzolidone-resistant Oilseed Rape to Nonselective Herbicide Imazethapyr

抗咪唑啉酮油菜对非选择性除草剂的抗性效应

GAOJian-qin;PUHui-ming;LONGWei-hua;HUMao-long;QICun-kou;ZHANGJie-fu

高建芹;浦惠明;龙卫华;胡茂龙;戚存扣;张洁夫

探明抗咪唑啉酮油菜对非选择性除草剂咪唑乙烟酸的抗性效应及该突变体的应用价值,为选育具有自主知识产权且不受环境释放限制的抗除草剂油菜新品种提供科学依据.采用田间浓度梯度试验,在喷施咪唑乙烟酸后10 d、20 d、35 d、50 d和65 d取样,测定株高、叶片数、最大叶面积及十物质等,分析不同浓度药剂对油菜苗期生长的影响.油菜成熟时考察植株性状、主要经济性状及单株牛物学产量,测定菜籽的油分、蛋白质、硫苷含量和全脂肪酸组成,分析不同浓度处理对抗性油菜植株、经济性状和品质性状的影响.结果显示:高浓度(135 g/hm2,ai.和180g/hm2,ai.)处理对抗性油菜苗期叶片生长和主茎伸长有明显抑制作用,从而影响植株地上部干物质积累,对成熟油菜的植株性状和经济性状也有显著影响;低浓度(45 g/hm2,ai.和90 g/hm2,ai.)处理对油菜早期生长有一定的抑制作用,喷药后35 d叶面积已恢复到对照(不喷咪唑乙烟酸)的92.94%,喷药后65 d苗高已恢复正常,对成熟油菜的植株性状和经济性状虽有不同程度的影响,但差异均未达显著水平;不同浓度梯度的咪唑乙烟酸处理,对抗性菜籽品质均无不良影响;咪唑乙烟酸除草剂的最低有效剂量ED90为45 g/hm2,ai.,抗性油菜使用咪唑乙烟酸的最高安全剂量ED10为108.59～190.77 g/hm2,ai.,选择系数均在2以上,因此该抗性油菜的抗性较强,具有生产实用价值.

... 2004年, 高建芹等^[10,11]在油菜和大豆多年轮作的试验田中发现了1株抗咪唑啉酮油菜 ...

2012

0.0

1.4522

Scientia Agricultura Sinica. 2012, 45(20):4326 - 4334 DOI:10.3864/j.issn.0578-1752.2012.20.022

Inheritance and Gene Cloning of an ALS Inhabiting Herbicide- Resistant Mutant Line M9 in Brassica napus

油菜乙酰乳酸合成酶抑制剂类除草剂抗性突变体M9的遗传和基因克隆

HU Mao-Long, PU Hui-Ming, GAO Jian-Qin, LONG Wei-Hua, QI Cun-Kou, ZHANG Jie-Fu, CHEN Song

胡茂龙, 浦惠明, 高建芹, 龙卫华, 戚存扣, 张洁夫, 陈松

1.江苏省农业科学院经济作物研究所/国家油料作物改良中心南京分中心/农业部长江下游棉花与油菜重点实验室，南京 210014

【Objective】The objective of this study is to identify and characterize the resistant gene of a mutant line M9，which was previously found in the rapeseed (brassica napus L.) by spontaneous mutation, conferring resistance to acetolactate synthase or acetohydroxyacid synthase (ALS or AHAS) inhibiting herbicides, and understand the molecular basis for resistance to ALS-inhibiting herbicides.【Method】Reciprocal crosses were made in order to study the inheritance of resistance. Three genes BnALS1-BnALS3 encoding ALS were isolated from the mutant and wild type by using the homology-based candidate gene method. The resistant gene of M9 was transformed into Mutsu-Isuzu cytoplasmic male sterile (MICMS) restorer lines by hybridization, microspore culture, and PCR analysis. 【Result】The resistance of M9 was inherited as a single, dominant nuclear gene. Three genes BnALS1-BnALS3 encoding ALS were isolated. Molecular analysis identified a single-point mutation leading to an amino acid substitution from serine 638 (AGT) to asparagine (AAT) at the herbicide-binding site of the rapeseed BnALS1 gene. All the resistant restoring lines had the specific band of BnALS1R by sequencing. 【Conclusion】The resistance trait of M9 is controlled by a single dominant nuclear gene. The molecular basis for the resistance to ALS-inhibiting herbicides results from the point mutation (Ser638Asn).

【目的】分离和鉴定自然突变的油菜乙酰乳酸合成酶抑制剂类除草剂抗性突变体M9抗性基因，阐明抗性产生的分子基础。【方法】通过配置杂交组合研究其遗传规律，应用同源序列法克隆油菜ALS家族基因，采用杂交转育和小孢子培养技术将抗性基因转育到陆奥-五十铃细胞质雄性不育（MICMS）恢复系中，进行PCR鉴定。【结果】该突变体抗性由1个显性核基因控制。克隆获得BnALS1—BnALS3，核酸序列比对发现，M9抗性是由BnALS1的点突变使蛋白序列的第638位丝氨酸（AGT）残基被天冬酰胺酸（AAT）替代所致，将此抗性基因命名为BnALS1R。抗性基因BnALS1R转育到MICMS恢复系中的PCR检测表明，所有抗除草剂的恢复系都携带有BnALS1R。【结论】突变体M9抗性由1个显性核基因控制，BnALS1第638位丝氨酸突变成天冬酰胺酸是抗性产生的分子基础。

... 胡茂龙等^[12]从M9中克隆到抗性基因BnALS1R发现, 抗性基因BnALS1R与野生型基因BnALS1存在1处SNP, 导致ASL1的第638位丝氨酸残基被天冬酰胺酸替代(以油菜的ALS1氨基酸位置计算), 这与PM1碱基突变位点相同 ...

... 国外抗性基因商业化利用需要交纳昂贵的专利费, 这将会提高油菜生产成本^[12] ...

... 190个抗咪唑啉酮MICMS纯合恢复系分别是由(3018R/M9)和(3075R/M9)的F₁植株花粉经小孢子培养加倍获得的DH株系^[12] ...

... 4 分子标记开发此前研究发现, M9与常规油菜宁油16、宁油18编码ALS家族中BnALS1的核苷酸序列存在1处SNP^[12], 根据该处SNP可开发AS-PCR标记检测抗性基因BnALS1R ...

... 前期研究, 我们从油菜自然突变的抗性材料M9中克隆获得了抗性基因BnALS1R, 虽然该基因的碱基突变位点与加拿大PM1突变位点相同, 但BnALS1R是我国拥有自主知识产权的抗除草剂基因, 不受国际知识产权保护的限制, 也是国内首次报道的抗咪唑啉酮类除草剂基因^[12] ...

2000

6.535

0.0

Plant Physiol.. 2000, 124(4):1483-92

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis.

Drenkard E, Richter BG, Rozen S, Stutius LM, Angell NA, Mindrinos M, Cho RJ, Oefner PJ, Davis RW, Ausubel FM

Department of Genetics, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.

PMID: 11115864

... 等位基因特异PCR (allele-specific PCR, AS- PCR)是基于等位基因的SNP设计特异引物, 通过PCR扩增来检测样本基因型的一种鉴定方法^[13] ...

2008

1.554

0.0

Euphytica. 2008, 160(3):431 - 438 DOI:10.1007/s10681-008-9662-0

Development and application of allele-specific PCR assays for imazethapyr resistance in rice (Oryza sativa )

Suresh Kadaru(1) Weiqiang Zhang(1) Anirudh Yadav(2) James H. Oard(1)

1.Louisiana State University School of Plant, Environmental and Soil Sciences, LSU Agricultural Center Baton Rouge LA 70803 USA
2.Government, MVM Department of Botany Bhopal 16 MP India

Outcrossing or cross hybridization is a potential concern in herbicide-resistant crop management strategies such as in the Clearfield™ rice system. Recent studies have shown that the mutated acetolactate synthase (ALS) gene that confers resistance to imazethapyr (Newpath) herbicide can be transferred from Clearfield rice cultivars via cross pollination under field conditions to weedy red rice. Resistance of commercial Clearfield rice cultivars to imazethapyr is due to the presence of two point mutations in theALS gene that result in amino acid substitutions from serine to asparagine (S to D) and glycine to glutamic acid (G to E). We report here development of a DNA-based method that involves application of allele-specific PCR assays to distinguish herbicide-susceptible and resistantALS alleles in either homozygous or heterozygous genotypes produced from natural outcrosses between Clearfield varieties CL121, CL141, CL161 and weedy red rice. PCR assays that can distinguish between the homozygous and heterozygous imazethapyr-resistant S₆₅₃D and G₆₅₄E SNP alleles of the riceALS gene were developed and evaluated. A total of 483 individual red rice plants were successfully screened for the presence of S₆₅₃D SNP and another 145 F₂ individuals from natural red rice × CL121 hybridizations were screened for the presence of the G₆₅₄E SNP. The PCR-based assays produced during this study are simple, rapid, inexpensive, reproducible and require only standard PCR and electrophoretic instruments that can be applied toward outcrossing evaluation and effective weed management strategies for the Clearfield crop system.

... 该方法不受季节和时间限制, 具有快速、高效的优点, 已在水稻^[14]和大麦^[4]中成功运用 ...

2011

0.0

1.1971

... 1 试验材料及除草剂油菜抗咪唑啉酮类除草剂突变体M9为浦惠明等^[15]在油菜和大豆多年轮作的试验田中发现的天然突变体及后代单株M9-2、M9-3、M9-11和M9-14 ...

2011

0.0

1.1971

Chin J Oil Crop Sci. 2011, 33(4):331 - 337

甘蓝型油菜谷胱甘肽过氧化物酶基因克隆和非生物胁迫下的表达

胡茂龙，龙卫华，高建芹，陈新军，张洁夫，陈松，戚存扣，浦惠明*

利用RT-PCR（reverse transcription PCR）技术从甘蓝型油菜宁油16号（Brassica napus L.）中克隆了一个非生物胁迫下细胞抵御活性氧(reactive oxygen species，ROS)伤害的关键酶GPX（谷胱甘肽过氧化物酶)基因，命名为BnGPX1（GenBank登录号：HM130680）。BnGPX1的开放阅读框长度为711bp，推测编码蛋白含有236个氨基酸，分子量为26.10kD，等电点为9.37。BnGPX1酶具有GPX特有的3个结构域及半胱氨酸残基。通过PCR方法克隆得到BnGPX1基因组序列（GenBank登录号：HM130681）。该序列与拟南芥AtGPX1基因相似，由6个外显子和5个内含子组成，内含子的剪切位点符合真核生物GT-AG规则。半定量RT-PCR发现非生物胁迫下，BnGPX1在油菜茎、叶、角果和花中表达量高，根中表达量较低。在盐、干旱和高温胁迫下BnGPX1表达量升高，出现峰值的时间不同。该基因对低温胁迫没有响应。

... 采用CTAB法并稍作修改提取DNA^[16], 用1％(V/W)的琼脂糖电泳和分光光度法检测其纯度 ...

1991

0.0

... 但由于油菜ALS基因家族核酸序列的高度同源性, 特别是BnALS1和BnALS3在核酸水平的同源性达98%^[17], 使得在编码区内很难开发出引物分别特异扩增BnALS1和BnALS3的基因片段 ...

0.0

... 由于此前申请的专利中已经获得M9、宁油16、宁油18的BnALS1全长编码序列(BnALS1-NY16、BnALS1-NY18)^[18], 因此为获得M9、宁油16、宁油18的BnALS3全长编码序列, 根据NCBI中BnALS3基因序列(GenBank登录号为Z11526), 利用Primer Premier 5 ...

1993

6.16

0.0

... 3 讨论基于基因序列SNP开发分子标记的方法很多, 如RFLP、CAPS或dCAPS等传统方法^[19,20,21] ...

1998

6.16

0.0

... 3 讨论基于基因序列SNP开发分子标记的方法很多, 如RFLP、CAPS或dCAPS等传统方法^[19,20,21] ...

1998

6.16

0.0

... 3 讨论基于基因序列SNP开发分子标记的方法很多, 如RFLP、CAPS或dCAPS等传统方法^[19,20,21] ...

2011

6.535

0.0

Plant Physiol.. 2011, 155(2):851-65 DOI:10.1104/pp.110.169482

Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different expression patterns and functional divergence in Brassica napus.

Chen X, Truksa M, Snyder CL, El-Mezawy A, Shah S, Weselake RJ

Agricultural Lipid Biotechnology Program, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6H 2P5.

Brassica napus is an allotetraploid (AACC) formed from the fusion of two diploid progenitors, Brassica rapa (AA) and Brassica oleracea (CC). Polyploidy and genome-wide rearrangement during the evolution process have resulted in genes that are present as multiple homologs in the B. napus genome. In this study, three B. napus homologous genes encoding endoplasmic reticulum-bound sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) were identified and characterized. Although the three GPAT4 homologs share a high sequence similarity, they exhibit different expression patterns and altered epigenetic features. Heterologous expression in yeast further revealed that the three BnGPAT4 homologs encoded functional GPAT enzymes but with different levels of polypeptide accumulation. Complementation of the Arabidopsis (Arabidopsis thaliana) gpat4 gpat8 double mutant line with individual BnGPAT4 homologs suggested their physiological roles in cuticle formation. Analysis of gpat4 RNA interference lines of B. napus revealed that the BnGPAT4 deficiency resulted in reduced cutin content and altered stomatal structures in leaves. Our results revealed that the BnGPAT4 homologs have evolved into functionally divergent forms and play important roles in cutin synthesis and stomatal development.

PMID: 21173024

... 基因组进化过程中高度重排导致甘蓝型油菜相关基因多以基因家族的形式存在^[22] ...

1992

6.16

0.0

... A、C基因组内3个具有功能的ALS基因核酸序列高度同源^[23], 特别是BnALS1和BnALS3在核酸水平的同源性达98%, 这给AS-PCR标记检测BnALS1增加了难度, 因为在进行PCR扩增油菜BnALS1过程中会受到高度同源序列BnALS3的干扰 ...

2006

0.0

1.4522

Scientia Agricultura Sinica. 2006, 39(7):1313 - 1320

Assaying Single Nucleotide Polymorphism in Wheat (Triticum aestivum L.) with Allele-Specific PCR

用等位基因特异PCR检测普通小麦（Triticum aestivum L.）的单核苷酸多态性

,,,

卫波,景蕊莲,王成社,昌小平

中国农业科学院作科所

【Objective】Two hexaploid wheat cultivars (Triticum aestivum L.), Opata85 and W7984, their 111 recombinant inbred lines (RILs), and three diploid relative-species, T. urartu, Ae. Speltoides and Ae. Tauschii were used as plant materials to study the method of assaying single nucleotide polymorphism with allele-specific PCR in wheat. 【Method】 Two SNPs were discovered on B genome by aligning the TaDREB1 genes in two hexaploid wheat cultivars and three diploid relative-species. To type these SNPs, allele-specific primers and their complementary primers were designed using the SNPs as their 3′-end. In addition, we studied the effect of the mismatched bases at the 3′-end of the allele-specific primers on PCR and the optimum PCR system. 【Result】There were distinct effects of the mismatched bases at 3′-end different sites of the allele-specific primers on allele-specific PCR, so did different types of mismatched bases. Moreover, the concentrations of Mg2+, dNTP and Taq DNA polymerase in allele-specific PCR were higher than that in conventional PCR. 【Conclusion】It is feasible to assay SNPs by allele-specific PCR in hexaploid wheat, as long as proper mismatched bases were introduced at 3′-end proper sites of the allele-specific primers and the PCR system was optimized reasonably.

【目的】以2份六倍体小麦Opata85和W7984及其重组近交系（RIL）的111个株系和3份小麦二倍体野生近缘种为材料，研究用等位基因特异PCR检测普通小麦中单核苷酸多态性的方法。【方法】利用直接测序的方法检测2份六倍体小麦和3份小麦二倍体野生近缘种TaDREB1基因的DNA序列，在B基因组上发现了2个SNPs。以其为3?端，设计等位基因特异引物及其互补引物，对SNP进行分型，同时研究了特异引物3?端碱基错配对等位基因特异PCR的影响，优化了PCR反应体系。【结果】等位基因特异引物3?端不同位置的碱基错配及不同类型的碱基错配对PCR结果影响较大；在等位基因特异PCR中，Mg2+、dNTP及Taq DNA聚合酶的用量均大于普通PCR。【结论】只要在等位基因特异引物3?端加上合适的错配碱基，并且优化其PCR反应体系，用等位基因特异PCR方法检测六倍体小麦中的单核苷酸多态性是可行的。

... 另外一个原因可能来自AS-PCR引物的自身设计特点, 因为在设计AS-PCR引物时, 不管将公用引物设计成正向引物或者是反向引物, 其他2条等位基因特异PCR引物的3末端都必须锚定在SNP位点上, 即使在AS引物3末端倒数第3或第4位置人为引入1个特定的错配碱基, 降低AS-PCR引物与非匹配模板结合, 也很难保证在多倍体中每条引物均具有很好的PCR特异性^[24] ...

... 卫波等^[24]认为错配碱基的位置及替换方式对PCR结果的影响可能与3末端不同错配碱基产生的碱基堆积力、氢键及其立体结构的稳定性有关 ...

2004

3.297

0.0

Theor Appl Genet. 2004, 108(7):1212 - 1220 DOI:10.1007/s00122-003-1553-0

Development of PCR-based SNP markers for rice blast resistance genes at thePiz locus

K. Hayashi(1) N. Hashimoto(2) M. Daigen(2) I. Ashikawa(1)

1.National Agricultural Research Center 1-2-1 Inada, Joetsu 943-0193 Niigata Japan
2.Niigata Agricultural Research Institute 857 Nagakura, Nagaoka 940-0826 Niigata Japan

We assessed the utility of single-nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphisms (InDels) as DNA markers in genetic analysis and breeding of rice. Toward this end, we surveyed SNPs and InDels in the chromosomal region containing the Piz and Piz-t rice blast resistance genes and developed PCR-based markers for typing the SNPs. Analysis of sequences from a blast-susceptible Japanese cultivar and two cultivars each containing one of these genes revealed that SNPs are abundant in the Piz and Piz-t regions (on average, one SNP every 248 bp), but the number of InDels was much lower. The dense distribution of SNPs facilitated the generation of SNP markers in the vicinity of the genes. For typing these SNPs, we used a modified allele-specific PCR method. Of the 49 candidate allele-specific markers, 33 unambiguously and reproducibly discriminated between the two alleles. We used the markers for mapping the Piz and Piz-t genes and evaluating the size of DNA segments introgressed from the Piz donor cultivar in Japanese near-isogenic lines containing Piz. Our findings suggest that, because of its ability to generate numerous markers within a target region and its simplicity in assaying genotypes, SNP genotyping with allele-specific PCR is a valuable tool for gene mapping, map-based cloning, and marker-assisted selection in crops, especially rice.

... Hayashi等^[25]在水稻中也发现, AS引物在3末端倒数第3位引入C/A类型的替换能显著提高引物的特异性, 他对49个SNP位点设计了特异引物, 其中67%的引物能可靠地区分2个等位基因 ...

2005

0.0

1.1628

J Plant Genet Resour. 2005, (4):469 - 473

Development of Allele-specific PCR

等位基因特异PCR技术的研究与应用

CHENJi-bao^[1];JINGRui-lian^[2];YUNHai-yan^[3];WEIBo^[1]

陈吉宝^[1];景蕊莲^[2];员海燕^[3];卫波^[1]

生物的单核苷酸多态性(Single-nucleotide polymorphism,SNP)具有数量多、分布广、易于分型、稳定性强等优点,很适合于用做分子标记.等位基因特异PCR(Allele-specific PCR,AS-PCR)是根据SNP位点设计3'末端与SNP位点碱基互补或错配的特异PCR引物,通过凝胶电泳等方法检测PCR扩增产物的有或无,从而检测基因型中SNP的一种技术.经过不断地改进与完善,基于SNP的等位基因特异PCR标记已逐渐成为一种快速、简便、低成本、可靠、高通量的检测基因型SNP的方法.本文应用等位基因特异PCR技术,根据小麦TaDREB1基因在旱选10和鲁麦14的120(C→A)SNP成功地开发了一个SNP分子标记,证明了该方法的有效性和可行性.

... 另外, 陈吉宝等^[26]根据普通小麦(Triticum aestivum L ...

2005

0.0

1.7477

... 抗咪唑啉酮类除草剂油菜已在加拿大、澳大利亚、欧洲国家广泛种植, 但我国由于缺乏自主知识产权的抗除草剂基因一直无法商业化应用^[27,28] ...

2005

0.0

1.7477

Acta Ecol Sin. 2005, (4):910 - 916

The studies on gene flow from GM herbicide-tolerant rapeseed to cruciferous weeds

转基因抗除草剂油菜对十字花科杂草的基因漂移

PUHui-ming^[1];QICun-kou^[1];ZHANGJie-fu^[1];FUShou-zhong^[1];GAOJian-qin^[1];CHENXin-Jun^[1];CHENSong^[1];ZHAOXiang-Xiang^[2]

浦惠明^[1];戚存扣^[1];张洁夫^[1];傅寿仲^[1];高建芹^[1];陈新军^[1];陈松^[1];赵祥祥^[2]

以转基因抗除草剂油菜Q3为花粉供体材料,油菜远缘杂草为花粉受体材料,在自然传粉和人工辅助授粉条件下研究甘蓝型油菜与十字花科杂草间的基因漂移频率.结果表明,以转基因油菜为父本,十字花科杂草荠菜、碎米荠、播娘蒿、诸葛菜、风花菜、遏蓝菜和蔊菜为母本,杂交高度不亲和,基因漂移率为0%,无生态风险,但对野芥菜的基因漂移率高达0.885%.野芥菜是我国大部分地区的常见杂草,种类繁多,分布范围广,大面积种植转基因抗除草剂油菜对野生芥菜的基因污染应引起高度重视.

... 抗咪唑啉酮类除草剂油菜已在加拿大、澳大利亚、欧洲国家广泛种植, 但我国由于缺乏自主知识产权的抗除草剂基因一直无法商业化应用^[27,28] ...