Maize Lc transcription factor enhances biosynthesis of ant
大豆根茎过渡区弯曲突变体Mrstz_的鉴定与基因定位
作物学报ACTA AGRONOMICA SINICA 2024, 50(5): 1091 1103 / ISSN 0496-3490; CN 11-1809/S; CODEN TSHPA9E-mail:***************DOI: 10.3724/SP.J.1006.2024.34142大豆根茎过渡区弯曲突变体M rstz的鉴定与基因定位苗龙1,**舒阔1,**李娟1黄茹1王业杏1 Soltani MuhammadYOUSOF 1许竞好1吴传磊1李佳佳1王晓波1,*邱丽娟2,*1 安徽农业大学农学院, 安徽合肥 230036;2 中国农业科学院作物科学研究所 / 农业农村部作物基因资源与遗传改良重大科学工程 /农业农村部作物基因资源与种质创制重点实验室, 北京 100081摘要: 植物根茎过渡区(root-stem transition zone, RSTZ)将根和茎相互连接, 其发育形态决定了大豆的地上部株型和抗倒伏潜力。
本研究通过EMS诱变获得一个RSTZ弯曲或旋转的大豆突变体M rstz, 其形态特征能够稳定遗传, 是探究大豆茎秆发育规律的特异材料。
将该突变体和栽培大豆中黄13杂交构建重组自交系群体, 对群体中直立和弯曲型后代的RSTZ进行解剖结构比较, 发现弯曲型株系比直立型株系的维管形成层较宽、次生木质部细胞层数较多、细胞形状不规则, 表明维管组织分化可能是导致RSTZ形态发生差异的重要因素之一。
进一步对化学组分测定发现,木质素和粗纤维含量越高越不易弯曲。
选取RIL群体中弯曲型和直立型2种极端株系进行BSA-Seq, 采用SNP-index和InDel-index关联分析方法鉴定到调控RSTZ形态的关联区域Chr19: 43030943~45849854, 该区间共含有319个基因。
结合生物信息学分析、基因注释信息和表达丰度分析筛选到7个候选基因, 分别为Glyma.19G170200、Glyma.19G201500、Glyma.19G187800、Glyma.19G178200、Glyma.19G197000、Glyma.19G179100、Glyma.19G196900。
Nature Immunology：发现参与免疫细胞形成的关键因子MAZR
Nature Immunology：发现参与免疫细胞形成的关键因子
MAZR
佚名
【期刊名称】《中国动物保健》
【年(卷),期】2010(0)5
【摘要】T细胞是淋巴细胞的一种，在免疫反应中扮演着重要角色。
按照功能的不同，T细胞可以分成细胞毒T细胞和辅助T细胞等很多种类。
其中细胞毒T细胞能够消灭感染细胞，而辅助T细胞可通过增生扩散来激活其他可产生直接免疫反应的免疫细胞。
细胞毒T细胞和辅助T细胞都产生于共同先驱细胞，即双阳性胸腺细胞。
维也纳医科大学病理生理学专家维尔弗里德·艾梅尔领导的研究小组发现，一种名为MAZR的转录因子是T细胞形成的一种关键因子。
【总页数】1页(P81-81)
【关键词】免疫细胞;转录因子;细胞毒T细胞;免疫反应;病理生理学;淋巴细胞;感染细胞;胸腺细胞
【正文语种】中文
【中图分类】S852.4
【相关文献】
1.Nature Genetics:中国科学家发现肺干细胞参与肺再生 [J], ;
2.奥研究人员发现参与一种免疫细胞形成的关键因子 [J],
3.尹玉新教授课题组在Nature Immunology发表研究成果 [J],
4.发现参与免疫细胞形成的关键因子MAZR [J],
5.福建农林大学在Nature Review Immunology上发表重要综述 [J],
因版权原因，仅展示原文概要，查看原文内容请购买。
植物激素的作用机理研究
• Dwarf
• Floral abnormalities • Embryo lethal
A, E and H : wild type. B, C, F and G: ask1-1 mutant, strong ASK1-Line 3, ASK1-Line 14, ASK1-Line 3. D : WT and strong ASK1 RNAi line . I and Q (left) : WT J to M : ask1 N to P : ASK RNAi line Q (right) to T : ASK11
植物
燕麦 燕麦 大麦 水稻 黍 小麦
组 织
营养组织 颖 颖 颖 颖 颖 种 种 种 种 种 果 果 果 果 果 子 子 子 子 子 颖果(已磨)
游离IAA
16 440 40 1703 366 123
IAA酯
5 7620 329 2739 3198 511
肽合IAA
69 未测到 未测到 未测到 未测到 未测到
control
abp1/abp1 ABP1/ABP1 abp1/abp1 abp1/abp1
BY-2 cell Division stage
Anti-ABP1
abp1/abp1
abp1/abp1 ABP1/ABP1
abp1/abp1
control
BY-2 cell elongation stage
Anti-ABP1
细胞分裂素
油菜素内酯
穗粒数显著增多
株型结构，光合能力
• 蔬菜、瓜果的产量、抗性
油菜素内酯
• 株型、根 生长素
植物激素的定义
1. 产生于植物体内的特殊部位，是植物在 正常发育过程中或特殊环境条件下的代 谢产物； 2. 能从合成部位运输到作用部位；
过表达玉米Zmsh1基因提高转基因烟草的生物量
生物技术进展２０１７年㊀第７卷㊀第３期㊀２０３~２１０ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ㊀ＩＳＳＮ２０９５￣２３４１研究论文Ａｒｔｉｃｌｅｓ㊀收稿日期:２０１７￣０２￣２１ꎻ接受日期:２０１７￣０３￣２０㊀基金项目:吉林省科技发展计划项目(２０１６０５２００６０ＪＨ)资助ꎮ㊀作者简介:顾韩雪ꎬ硕士研究生ꎬ研究方向为植物生物化学与分子生物学研究ꎮＥ￣ｍａｉｌ:ｇｕｈａｎｘｕｅ０１＠１６３.ｃｏｍꎮ∗通信作者:郝东云ꎬ研究员ꎬ研究方向为农艺性状相关基因挖掘ꎮＥ￣ｍａｉｌ:ｄｙｈａｏ＠ｃｊａａｓ.ｃｏｍ过表达玉米Ｚｍｓｈ１基因提高转基因烟草的生物量顾韩雪１ꎬ２ꎬ㊀刘㊀玥１ꎬ㊀陈子奇３ꎬ㊀刘艳芝２ꎬ㊀刘相国２ꎬ㊀郝东云１ꎬ２∗１.吉林农业大学生命科学学院ꎬ长春１３０１１８ꎻ２.吉林省农业科学院农业生物技术研究所ꎬ吉林省农业生物技术重点实验室ꎬ长春１３００３３ꎻ３.哈尔滨师范大学生命科学学院ꎬ哈尔滨１５００８０摘㊀要:蔗糖合成酶(ＳｕＳｙ)是调控植物体内蔗糖代谢的一类关键酶ꎬ而ＳＨ１是玉米ＳｕＳｙ的一种亚型ꎮ研究表明ꎬ玉米ＳｕＳｙ催化活性主要由ＳＨ１基因(Ｚｍｓｈ１)决定ꎬ该基因在玉米分子育种中的价值评估是人们关注的热点ꎮ利用农杆菌介导法将玉米Ｚｍｓｈ１转入模式植物烟草中ꎬ发现转基因烟草中ＳＨ１酶活力比野生型烟草平均提高了３５％ꎬ根和茎的糖代谢关键产物蔗糖和果糖的含量平均增加了２３％和２８％ꎻ同时ꎬＺｍｓｈ１的转入显著提高了转基因烟草的总生物量ꎮ研究结果为进一步在玉米中过表达Ｚｍｓｈ１ꎬ评估转基因玉米的产业化应用价值提供了重要的理论参考ꎮ关键词:蔗糖合成酶ꎻＺｍｓｈ１ꎻ转基因烟草ꎻ生物量ꎻ基因应用价值评估ＤＯＩ:１０.１９５８６/ｊ.２０９５￣２３４１.２０１７.０００８ＲｅｓｅａｒｃｈｏｎＥｎｈａｎｃｉｎｇｔｈｅＢｉｏｍａｓｓｏｆＴｒａｎｓｇｅｎｉｃＴｏｂａｃｃｏｂｙＯｖｅｒｅｘｐｒｅｓｓｉｏｎｏｆＺｍｓｈ１ＧｅｎｅｆｒｏｍＭａｉｚｅＧＵＨａｎｘｕｅ１ꎬ２ꎬＬＩＵＹｕｅ１ꎬＣＨＥＮＺｉｑｉ３ꎬＬＩＵＹａｎｚｈｉ２ꎬＬＩＵＸｉａｎｇｇｕｏ２ꎬＨＡＯＤｏｎｇｙｕｎ１ꎬ２∗１.ＣｏｌｌｅｇｅｏｆＬｉｆｅＳｃｉｅｎｃｅｓꎬＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙꎬＣｈａｎｇｃｈｕｎ１３０１１８ꎬＣｈｉｎａꎻ２.ＪｉｌｉｎＰｒｏｖｉｎｃｉａｌＫｅｙＬａｂｏｒａｔｏｒｙｏｆＡｇｒｉｃｕｌｔｕｒａｌＢｉｏｔｅｃｈｎｏｌｏｇｙꎬｌｎｓｔｉｔｕｔｅｏｆＡｇｒｉｃｕｌｔｕｒａｌＢｉｏｔｅｃｈｎｏｌｏｇｙꎬＪｉｌｉｎＡｃａｄｅｍｙｏｆＡｇｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓꎬＣｈａｎｇｃｈｕｎ１３００３３ꎬＣｈｉｎａꎻ３.ＣｏｌｌｅｇｅｏｆＬｉｆｅＳｃｉｅｎｃｅꎬＨａｒｂｉｎＮｏｒｍａｌＵｎｉｖｅｒｓｉｔｙꎬＨａｒｂｉｎ１５００８０ꎬＣｈｉｎａＡｂｓｔｒａｃｔ:Ｓｕｃｒｏｓｅｓｙｎｔｈｅｔａｓｅ(ＳｕＳｙ)ｉｓａｃｒｕｃｉａｌｅｎｚｙｍｅｆａｍｉｌｙｒｅｇｕｌａｔｉｎｇｓｕｃｒｏｓｅｍｅｔａｂｏｌｉｓｍｉｎｐｌａｎｔｓ.ＳＨ１ｉｓｏｎｅｏｆｔｈｅｓｕｂｔｙｐｅｓｏｆＳｕＳｙｉｎｍａｉｚｅꎬａｎｄｉｔｓｅｎｃｏｄｉｎｇｇｅｎｅ(Ｚｍｓｈ１)ｐｌａｙｓａｍａｊｏｒｒｏｌｅｉｎｔｈｅｃａｔａｌｙｔｉｃａｃｔｉｖｉｔｙｏｆＳｕＳｙ.ＴｈｕｓꎬｔｈｅｅｖａｌｕａｔｉｏｎｏｆＺｍｓｈ１ｉｎｂｉｏｔｅｃｈｂｒｅｅｄｉｎｇｈａｓａｔｔｒａｃｔｅｄａｇｒｅａｔｄｅａｌｏｆａｔｔｅｎｔｉｏｎｉｎｒｅｃｅｎｔｙｅａｒｓ.ＩｎｔｈｉｓｓｔｕｄｙꎬｗｅｔｒａｎｓｆｏｒｍｅｄＺｍｓｈ１ｉｎｔｏｔｏｂａｃｃｏｂｙＡｇｒｏｂａｃｔｅｒｉｕｍ￣ｍｅｄｉａｔｅｄｍｅｔｈｏｄ.ＴｈｅｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔꎬｅｎｚｙｍｅａｃｔｉｖｉｔｙｏｆＳＨ１ｉｎｔｈｅｔｒａｎｓｇｅｎｉｃｅｖｅｎｔｓｅｘｈｉｂｉｔｅｄａｎａｖｅｒａｇｅｏｆ３５％ｈｉｇｈｅｒｔｈａｎｔｈｅｗｉｌｄｔｙｐｅꎬａｎｄｃｏｎｔｅｎｔｓｏｆｔｈｅｋｅｙｍｅｔａｂｏｌｉｔｅｓｓｕｃｈａｓｓｕｃｒｏｓｅａｎｄｆｒｕｃｔｏｓｅｉｎｃｒｅａｓｅｄａｂｏｕｔ２３％ａｎｄ２８％ｒｅｓｐｅｃｔｉｖｅｌｙｉｎｔｈｅｒｏｏｔａｎｄｓｔｅｍｏｆｔｈｅｔｒａｎｓｇｅｎｉｃｅｖｅｎｔｓ.ＡｔｔｈｅｍｅａｎｔｉｍｅꎬｔｈｅｔｒａｎｓｆｏｒｍａｔｉｏｎｏｆＺｍｓｈ１ｓｉｇｎｉｆｉｃａｎｔｌｙｅｎｈａｎｃｅｄｔｈｅｂｉｏｍａｓｓｏｆｔｒａｎｓｇｅｎｉｃｔｏｂａｃｃｏ.ＴｈｉｓｐｒｅｌｉｍｉｎａｒｙｓｔｕｄｙｐｒｏｖｉｄｅｄｉｍｐｏｒｔａｎｔｒｅｆｅｒｅｎｃｅｆｏｒｐｒｏｖｉｎｇｃｏｎｃｅｐｔｏｆＺｍｓｈ１ｉｎｍａｉｚｅｂｉｏｔｅｃｈｂｒｅｅｄｉｎｇ.Ｋｅｙｗｏｒｄｓ:ＳｕＳｙꎻＺｍｓｈ１ꎻｔｒａｎｓｇｅｎｉｃｔｏｂａｃｃｏꎻｂｉｏｍａｓｓꎻｐｒｏｖｅｃｏｎｃｅｐｔｏｆｇｅｎｅ㊀㊀玉米是我国东北地区第一大作物ꎬ也是我国主要的粮食作物ꎮ玉米合成并在籽粒中大量积累淀粉是其主要的经济价值所在ꎬ蔗糖合成酶在这过程中起着关键作用ꎮ植物蔗糖合成酶(ｓｕｃｒｏｓｅｓｙｎｔｈａｓｅꎬＳｕＳｙ)是蔗糖进入淀粉代谢途径的首个关键酶ꎬ是１９９５年Ｃａｒｄｉｎｉ[１]首次在小麦胚芽中发现的ꎮ它是由分子量约为８３~１００ｋＤａ的亚基构成的四聚体[２]ꎮＳｕＳｙ由３个大家族组成ꎬ依次为单子叶植物ＳＵＳ族㊁双子叶植物ＳＵＳＹ１族和双子叶ＳＵＳＹＡ族[３ꎬ４]ꎮ玉米ＳｕＳｙ(即ＳＵＳ家族)包. All Rights Reserved.含３种亚型:ＳＨ１㊁ＳＵＳ１和ＳＵＳ３ꎬ其中ＳＨ１是３种亚型中的典型代表[５]ꎬ其编码基因为Ｚｍｓｈ１ꎮＺｍｓｈ１是由Ｃｈｏｕｒｅｙ[６]在玉米皱缩型胚乳突变体ｓｈ１(ｓｈｒｕｎｋｅｎ１)中发现的ꎬ其ｃＤＮＡ序列长度为２７４６ｂｐꎬＣＤＳ区序列长度为２４０９ｂｐꎬ编码８０３个氨基酸ꎬ蛋白分子量约为９１ｋＤａꎮ大多情况下ꎬＳｕＳｙ分解蔗糖(最适ｐＨ６.０~７.０)生成果糖和腺苷二磷酸葡萄糖(ＡＤＰＧ)ꎬ分解的产物参与植物淀粉合成以储存能量[７]ꎮ对于大多数植物来说ꎬ尤其是在以淀粉为主要储藏物质的组织器官中ꎬＳｕＳｙ的功能是非常重要的ꎮＢａｒｏｊａ￣Ｆｅｒｎｎｄｅｚ等[８]发现在马铃薯中过表达内源ＳｕＳｙ基因导致马铃薯块茎膨大ꎬＳｕＳｙ酶活升高ꎻＴａｎｇ等[９]在胡萝卜中反义表达内源ＳｕＳｙ基因ꎬ结果表明储藏组织中的蔗糖利用率显著下降ꎬ蔗糖大量积累ꎬ淀粉㊁葡萄糖㊁果糖和纤维素也有较少量的积累ꎬ转化植株表型也受到影响ꎬ表现为植株矮小㊁叶面积减少[１０]ꎮＷｏｒｒｅｌｌ等[１１]曾在番茄中过表达Ｚｍｓｈ１ꎬ发现转基因番茄果实中ＳｕＳｙ的活性提高ꎬ改变了碳水化合物的分配ꎬ果实重量增加ꎬ表明过表达Ｚｍｓｈ１可以改善作物品质ꎬ提高作物产量ꎮＺｍｓｈ１主要存在于发育的胚乳中ꎬ表达活跃时期与淀粉积累时期重合ꎬ并且ＳｕＳｙ９０％的活性主要由Ｚｍｓｈ１调控[１２ꎬ１３]ꎬ说明该基因在改变作物品质和增加作物产量方面具有潜在的应用价值ꎮ在不同植物中有关过表达Ｚｍｓｈ１的研究至今很少报道ꎬ然而ꎬ该基因在作物特别是玉米生物技术育种中的价值评估(ｐｒｏｖｅｃｏｎｃｅｐｔ)是人们关注的热点ꎮ依据转基因生物技术育种流程ꎬ特定基因的应用价值评估是首要环节ꎬ通常以模式植物为先行实验材料ꎮ本课题组在模式植物烟草中过表达Ｚｍｓｈ１ꎬ通过对转基因烟草进行分子检测和生理指标测定ꎬ研究转基因烟草生物量的变化ꎬ探讨该基因对烟草生长发育㊁碳水化合物代谢以及生物量的影响ꎬ为实验室评估该基因在转基因玉米增产和品质改良等方面的育种价值ꎬ研究该基因参与玉米籽粒淀粉合成代谢机制提供了理论参考ꎮ１㊀材料与方法１.１㊀材料受体材料:大叶烟草(ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ.)ꎬ由吉林省农业科学院生物技术研究所保存ꎮ大肠杆菌(Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ)感受态细胞ＤＨ５α和克隆载体ｐＥａｓｙ￣Ｂｌｕｎｔ购自北京全式金生物技术有限公司ꎻ农杆菌ＥＨＡ１０５和植物表达载体ｐＣＡＭ￣ＢＩＡ１３０２￣３５Ｓ￣ｇｆｐ由吉林省农业科学院生物所保存ꎮ试验试剂:限制性内切酶ＳｐｅⅠ㊁Ｔ４连接酶㊁ＰｒｉｍｅＳＴＡＲＨＳＤＮＡ聚合酶㊁ＰｒｉｍｅＳｃｒｉｐｔＲＴｒｅａｇｅｎｔ(ｇＤＮＡＥｒａｓｅｒ)反转录试剂盒㊁ＲＮＡｉｓｏＰｌｕｓＲＮＡ提取试剂盒㊁ＤＮＡＭａｒｋｅｒ均购自ＴａＫａＲａ公司ꎮ主要仪器设备:ＴＣ￣５１２ＰＣＲ仪(英国ＴＥＣＨＮＥ公司)㊁ＤＹＹ￣１０Ｃ型电泳仪(六一仪器厂)㊁ＣＨＢ￣１００恒温金属浴(杭州博日科技有限公司)㊁２￣１６ＰＫ台式冷冻高速离心机(德国ＳＩＧＭＡ公司)ꎮ１.２㊀Ｚｍｓｈ１基因克隆和植物表达载体的构建１.２.１㊀基因克隆㊀根据ＧｅｎＢａｎｋ(ｗｗｗ.ｎｃｂｉ.ｎｌｍ.ｎｉｈ.ｇｏｖ/ｇｅｎｂａｎｋ)提供的Ｚｍｓｈ１的核酸序列(ＧｅｎｅＩＤ:５４２３６５)设计克隆ＰＣＲ引物ꎬ提取玉米Ｂ７３叶片总ＲＮＡꎬ具体方法参照ＲＮＡｉｓｏＰｌｕｓ试剂盒说明书ꎮ反转录得到ｃＤＮＡꎬ具体方法参照ＰｒｉｍｅＳｃｒｉｐｔＲＴｒｅａｇｅｎｔ(ｇＤＮＡＥｒａｓｅｒ)反转录试剂盒说明书ꎮ以反转录ｃＤＮＡ为模版扩增ꎬ设计以限制性内切酶ＳｐｅⅠ为酶切位点的引物Ｚｍｓｈ１￣Ｌ:５ᶄ￣ＧＧＡＣＴＡＧＴＡＴＧＧＣＴＧＣＣＡＡＧＣＴＧＡＣＴＣＧ￣３ᶄ和Ｚｍｓｈ１￣Ｒ:５ᶄ￣ＧＧＡＣＴＡＧＴＡＴＣＧＡＡＧＧＡＣＡＧＣＧＧ￣ＡＡＣＣＴＧ￣３ᶄꎬＰＣＲ反应体系:１μＬ模板ＤＮＡꎬ上㊁下游引物各０.５μＬꎬ酶１０μＬꎬｄｄＨ２Ｏ８μＬꎮ程序:９５ħ５ｍｉｎꎻ９５ħ３０ｓꎬ５８ħ３０ｓꎬ７２ħ４０ｓꎬ３０个循环ꎻ７２ħ１０ｍｉｎꎮＰＣＲ产物经过胶回收纯化后连入克隆载体ꎬ转化到大肠杆菌感受态细胞中ꎬ培养后得到的菌液送大连宝生物公司测序ꎬ以确定克隆序列的正确性ꎮ利用ＣｌｕｓｔａｌＸ在线程序(ｈｔｔｐ://ｗｗｗ.ｃｌｕｓｔａｌ.ｏｒｇ/)比对出烟草(ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ.)和玉米(ＺｅａｍａｙｓＬ.)ＳｕＳｙ氨基酸序列同源性为７４.７８％ꎮ１.２.２㊀植物表达载体构建和遗传转化㊀利用限制性内切酶ＳｐｅⅠ分别切割已经纯化的含有Ｚｍｓｈ１基因的ＰＣＲ产物ꎬ以及拟连接的植物表达载体质粒ｐＣＡＭＢＩＡ１３０２￣３５Ｓ￣ｇｆｐꎬ用Ｔ４连接酶３７ħ共孵育连接并转化至ＤＨ５α中ꎬ筛选获得重组质粒ꎬ命名为ｐＣＡＭＢＩＡ１３０２￣３５Ｓ￣Ｚｍｓｈ１￣ｇｆｐꎮ体系和反应条件参照Ｔ４ＤＮＡ连接酶说明书ꎮ采４０２生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ. All Rights Reserved.用农杆菌介导的遗传转化方法转入烟草植株ꎮ获得Ｔ０代转基因植株ꎬＴ０代自交得到Ｔ１代转基因植株ꎮ１.３㊀转基因烟草的分子检测采用ＣＴＡＢ法[１４]ꎬ提取烟草叶片中的基因组ＤＮＡꎮ根据Ｚｍｓｈ１序列ꎬ设计ＰＣＲ特异性引物Ｚｍｓｈ１￣Ｌ:５ᶄ￣ＡＴＧＣＣＴＣＣＴＴＴＣＣＴＣＧＴＣＣＴ￣３ᶄꎻＺｍｓｈ１￣Ｒ:５ᶄ￣ＣＴＣＡＣＧＴＡＣＴＴＣＣＡＧＡＡＣＣＣＧ￣３ᶄꎮＰＣＲ扩增体系和反应程序参考大连宝生物公司技术说明书ꎮ扩增产物进行１％的琼脂糖凝胶电泳(电压１３５Ｖ)ꎬ在紫外凝胶成像仪下观察ＰＣＲ电泳结果ꎮ１.４㊀ＲＴ￣ＰＣＲ分析设计ＲＴ￣ＰＣＲ引物ꎬ提取转基因烟草总ＲＮＡꎬ方法参照ＲＮＡｉｓｏＰｌｕｓ试剂盒说明书ꎬ将ＲＮＡ反转录为ｃＤＮＡꎬ方法参照ＰｒｉｍｅＳｃｒｉｐｔＲＴｒｅａｇｅｎｔ(ｇＤＮＡＥｒａｓｅｒ)试剂盒说明书ꎮ正反方向引物分别为:ＲＴ￣Ｚｍｓｈ１￣Ｌ:５ᶄ￣ＡＴＧＣＣＴＣＣＴＴＴＣ￣ＣＴＣＧＴＣＣＴ￣３ᶄ和ＲＴ￣Ｚｍｓｈ１￣Ｒ:５ᶄ￣ＴＣＧＴＣＧＴＧＣ￣ＣＣＴＴＧＴＡＧＴＴＡＴＧ￣３ᶄꎮ１.５㊀蔗糖合成酶酶活分析酶活测定方法采用蛋白计量方法ꎬ具体参照Ｚｈｕ等[１５]的方法ꎮ１.６㊀蔗糖含量测定蔗糖含量用比色法测定ꎬ具体步骤参照Ｒｏｅ等[１６]的方法ꎮ１.７㊀果糖含量测定果糖含量用比色法测定ꎬ具体步骤参照李合生等[１４]的方法ꎮ１.８㊀数据分析用ＳＰＳＳ１７.０软件对试验数据进行统计学分析[１７]ꎮ２㊀结果与分析２.１㊀转基因烟草的ＰＣＲ验证选取３株Ｔ１代转基因烟草(分别为转化事件９＃㊁转化事件４＃和转化事件７＃)作为实验对象ꎮ为验证经农杆菌转化获得的烟草植株是否为阳性转基因材料ꎬ提取Ｔ１代烟草叶片的基因组ＤＮＡꎬ以转化质粒为阳性对照㊁Ｚｍｓｈ１基因部分序列为目标产物㊁野生型烟草植株为阴性对照㊁水为空白对照ꎬ进行ＰＣＲ检测ꎬ产物经１％琼脂糖凝胶电泳检测(图１)ꎬ目标产物条带大小为４４６ｂｐꎬ与预期结果一致ꎬ初步表明获得转基因阳性烟草ꎮ图１㊀转基因烟草Ｚｍｓｈ１基因ＰＣＲ产物电泳结果Ｆｉｇ.１㊀ＥｌｅｃｔｒｏｐｈｏｒｅｓｉｓｒｅｓｕｌｔｓｏｆＺｍｓｈ１ＰＣＲｐｒｏｄｕｃｔｓｉｎｔｈｅｔｒａｎｓｇｅｎｉｃｔｏｂａｃｃｏ.Ｍ:Ｍａｒｋｅｒ(ＤＬ２０００)ꎻ１:转化质粒(阳性对照)ꎻ２:野生型烟草(阴性对照)ꎻ３~５:转化事件９＃ꎻ６~８:转化事件４＃ꎻ９ꎬ１０:转化事件７＃ꎻ１１:水(空白对照)ꎮ２.２㊀Ｚｍｓｈ１基因表达分析为分析Ｚｍｓｈ１基因在转基因烟草植株中是否转录表达ꎬ提取Ｔ１代转Ｚｍｓｈ１基因烟草植株叶片总ＲＮＡꎬ将ＲＮＡ反转录成ｃＤＮＡꎬ进行ＲＴ￣ＰＣＲ分析ꎮ以烟草保守基因Ｌ２５作为内参基因ꎬＺｍｓｈ１为目的基因ꎬ野生型烟草植株为阴性对照ꎬ水为空白对照ꎮ结果(图２)表明ꎬ转基因烟草和野生型烟草的内参基因均具有较好的扩增效率ꎬ图２㊀转基因烟草中Ｚｍｓｈ１(Ａ)和内参Ｌ２５(Ｂ)基因的ＲＴ￣ＰＣＲ产物Ｆｉｇ.２㊀ＲＴ￣ＰＣＲｐｒｏｄｕｃｔｓｏｆＺｍｓｈ１(Ａ)ａｎｄｉｎｔｅｒｎａｌｃｏｎｔｒｏｌｇｅｎｅＬ２５(Ｂ)ｉｎｔｈｅｔｒａｎｓｇｅｎｉｃｔｏｂａｃｃｏ.Ｍ:ＤＬ２０００Ｍａｒｋｅｒꎻ１:转化事件９＃ꎻ２:转化事件４＃ꎻ３:转化事件７＃ꎻ４:野生型烟草ꎻ５:水(空白对照)５０２顾韩雪ꎬ等:过表达玉米Ｚｍｓｈ１基因提高转基因烟草的生物量. All Rights Reserved.说明ＲＮＡ质量完好(图２Ｂ)ꎮ阴性对照和空白对照均无条带ꎬ１~３泳道为转基因烟草ꎬ能扩增出４４６ｂｐ的目标片段(图２Ａ)ꎬ与目的基因理论片段大小一致ꎬ表明玉米Ｚｍｓｈ１基因在烟草植株中能够正常转录表达ꎮ进一步证明获得转基因阳性烟草ꎮ２.３㊀转基因烟草中蔗糖合成酶活力测定为验证转基因烟草中的蔗糖合成酶具有生物学活性ꎬ本实验结合前人蔗糖诱导光敏色素相互作用因子的表达实验[１８]ꎬ选取光合作用的主要场所 叶片(采摘自苗期Ｔ１代烟草)为实验对象ꎬ采用紫外分光光度法测定ＯＤ值ꎬ对照标准曲线ꎬ由公式计算得到蔗糖合成酶活力ꎮ结果表明:转基因烟草中蔗糖合成酶活力均比野生型烟草高ꎬ转化事件９＃的蔗糖合成酶活力最高ꎬ约为野生型烟草的１.５倍(图３)ꎮ２.４㊀转基因烟草中蔗糖含量测定植物中蔗糖合成酶催化反应的底物是蔗糖ꎬ为探究转基因烟草中蔗糖合成酶活力升高对蔗糖含量的影响ꎬ以进一步验证Ｚｍｓｈ１在转基因烟草中的功能ꎬ本研究应用比色法测定现蕾期和开花期Ｔ１代转基因烟草不同部位的蔗糖含量ꎮ结果表明ꎬ转基因烟草与野生型烟草的叶和叶脉中蔗糖含量均高于根和茎ꎮ现蕾期的转基因烟草根中的蔗糖含量高于野生型烟草ꎬ在叶和叶脉中蔗糖含量低于野生型烟草ꎻ开花期的转基因烟草根㊁茎㊁叶㊁叶脉中蔗糖含量均高于野生型烟草(表１)ꎮ２.５㊀转基因烟草中果糖含量测定为验证蔗糖合成酶活力升高能否促进植物中蔗糖代谢ꎬ生成果糖ꎮ本研究应用比色法测定现蕾期和开花期Ｔ１代烟草根㊁茎㊁叶㊁叶脉中果糖的图３㊀蔗糖合成酶的活性测定Ｆｉｇ.３㊀Ａｃｔｉｖｉｔｙｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｓｕｃｒｏｓｅｓｙｎｔｈａｓｅ.ＷＴ:野生型烟草ꎻ９＃ꎬ４＃ꎬ７＃:转基因烟草ꎮ表１㊀转基因烟草蔗糖含量Ｔａｂｌｅ１㊀Ｓｕｃｒｏｓｅｃｏｎｔｅｎｔｉｎｔｈｅｔｒａｎｓｇｅｎｉｃｔｏｂａｃｃｏ.野生型烟草(ｍｇ/ｇ)转基因烟草９＃(ｍｇ/ｇ)４＃(ｍｇ/ｇ)７＃(ｍｇ/ｇ)现蕾期根５８.３ʃ３.２７６.７ʃ５.６∗５９.６ʃ２.４６３.１ʃ６.１茎６１.９ʃ１１.８９５.４ʃ１５.３∗５２.３ʃ９.７７２.３ʃ４.４∗叶３１９.５ʃ１１.９２７０.５ʃ１４.８∗３００.３ʃ１５.９２９８.３ʃ８.７∗叶脉１５６.６ʃ１.６１３４.３ʃ４.３１３６.８ʃ８.２１４１.２ʃ７.４开花期根５８.０ʃ２.６６８.７ʃ１０.５６０.１ʃ８.１６５.９ʃ７.７茎４２.３ʃ１０.４６５.７ʃ６.６∗４６.１ʃ４.２４８.９ʃ７.６叶２８９.８ʃ６.６３７２.６ʃ３.１∗３１０.６ʃ８.５３２９.９ʃ４.６叶脉１９３.７ʃ１１.５２０５.０ʃ４.３２００.６ʃ６.１２０１.５ʃ５.２㊀注:数据为平均值ʃ标准误ꎬ∗表示与野生型相比在Ｐ<０.０５水平上差异显著ꎮ６０２生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ. All Rights Reserved.含量ꎮ结果表明(表２)ꎬ现蕾期的野生型烟草和转基因烟草植株中ꎬ茎>叶脉>根>叶ꎬ转基因烟草相比野生型烟草ꎬ各部位果糖含量均较高于野生型烟草ꎬ茎中相差较大ꎻ开花期时ꎬ野生型烟草植株中果糖含量:叶>茎>根>叶脉ꎻ在转基因烟草中:茎>叶>叶脉>根ꎮ２.６㊀转基因烟草的生理指标观测为检验Ｚｍｓｈ１对烟草生长和发育的影响ꎬ本研究对Ｔ１代转基因烟草的生物量积累和与光合作用有关的生理指标进行观察ꎮ分别以转化事件４＃㊁７＃㊁９＃为观察对象ꎬ连续测量烟草整个生长期的株高变化ꎬ结果表明:转基因烟草生长表现为苗期生长缓慢ꎬ株高比野生型烟草矮ꎬ营养期和生殖期生长迅速ꎬ到生殖生长结束时株高比野生型烟草高(图４)ꎮ由于农杆菌介导法将外源基因转入烟草基因组时插入位点的随机性ꎬ导致３个转化事件９＃㊁４＃㊁７＃烟草的株高等表型出现差异ꎮ比较转基因烟草和野生型烟草生物量ꎬ结果表明ꎬ转基因烟草较野生型烟草生物量显著增加(表３和图６ꎬ彩图见图版二)ꎮ连续观测其生长期内１２０ｄ的叶绿素含量ꎬ结果表明ꎬ转基因烟草的叶绿素含量较野生型在生长初期含量较低ꎬ两个月后叶绿素含量持续升高并最终高于野生型(图５)ꎮ３㊀讨论本研究在烟草中过表达了玉米蔗糖合成酶基因Ｚｍｓｈ１ꎬ该基因的转入增加了转基因烟草中蔗表２㊀转基因烟草果糖含量Ｔａｂｌｅ２㊀Ｆｒｕｃｔｏｓｅｃｏｎｔｅｎｔｉｎｔｈｅｔｒａｎｓｇｅｎｉｃｅｖｅｎｔｓ.野生型烟草(ｍｇ/ｇ)转基因烟草９＃(ｍｇ/ｇ)４＃(ｍｇ/ｇ)７＃(ｍｇ/ｇ)现蕾期根５５.４ʃ６.６７９.３ʃ８.７∗５６.２ʃ３.７６８.７ʃ４.２茎１６０.７ʃ１０.７２４９.３ʃ１４.２∗１７１.３ʃ１３.５１８０.５ʃ９.１∗叶５０.６ʃ１.０８５.０ʃ５.１∗６０.１ʃ６.５７２.１ʃ５.１∗叶脉８２.７ʃ７.５１１１.１ʃ８.４∗８３.７ʃ６.２８９.６ʃ８.９开花期根３０.１ʃ１.０５９.４ʃ１０.５∗３２.６ʃ４.２４０.２ʃ６.５∗茎８９.４ʃ６.４１５９.５ʃ９.６∗９６.３ʃ１０.１９９.５ʃ７.９叶９３.９ʃ０.４８０.３ʃ１.０∗９０.１ʃ８.７８７.６ʃ８.１叶脉４１.１ʃ６.５６５.１ʃ０.６∗４６.８ʃ４.７４９.２ʃ６.４㊀注:数据为平均值ʃ标准误ꎬ∗表示与野生型相比在Ｐ<０.０５水平上差异显著ꎮ图４㊀转基因烟草株高Ｆｉｇ.４㊀Ｐｌａｎｔｈｅｉｇｈｔｍｅａｓｕｒｅｍｅｎｔｉｎｔｈｅｔｒａｎｓｇｅｎｉｃｅｖｅｎｔｓ.ＷＴ:野生型烟草ꎻ９＃ꎬ４＃ꎬ７＃:转基因烟草ꎮ７０２顾韩雪ꎬ等:过表达玉米Ｚｍｓｈ１基因提高转基因烟草的生物量. All Rights Reserved.图５㊀转基因烟草叶绿素含量Ｆｉｇ.５㊀Ｃｈｌｏｒｏｐｈｙｌｌｃｏｎｔｅｎｔｉｎｔｈｅｔｒａｎｓｇｅｎｉｃｅｖｅｎｔｓ.ＷＴ:野生型烟草ꎻ９＃ꎬ４＃ꎬ７＃:转基因烟草ꎮ图６㊀转基因烟草与野生型烟草植株的对比Ｆｉｇ.６㊀Ｍｏｒｐｈｏｌｏｇｉｃａｌｃｏｍｐａｒｉｓｏｎｂｅｔｗｅｅｎｔｈｅｔｒａｎｓｇｅｎｉｃｅｖｅｎｔｓａｎｄｉｔｓｗｉｌｄｔｙｐｅ.ＷＴ:野生型烟草ꎻ９＃ꎬ４＃ꎬ７＃:转基因烟草ꎮＡ:转基因烟草和野生型烟草植株比较ꎻＢ:９＃转基因烟草根部以上长５ｃｍ茎段ꎻＣ:野生型烟草根部以上长５ｃｍ茎段ꎮ(彩图见图版二)表３㊀转基因与非转基因烟草生物量相关生理指标对比Ｔａｂｌｅ３㊀Ｃｏｍｐａｒｉｓｏｎｏｆｂｉｏｍａｓｓ￣ｒｅｌａｔｉｎｇｃｈａｒａｃｔｅｒｉｓｔｉｃｓｂｅｔｗｅｅｎｔｈｅｔｒａｎｓｇｅｎｉｃｅｖｅｎｔｓａｎｄｉｔｓｗｉｌｄｔｙｐｅ.千粒重(ｇ)茎秆粗(ｃｍ)鲜重(ｇ/株)根茎叶干重(ｇ/株)根茎叶株高(ｃｍ)叶绿素(ＰＡＤ)９＃１.２ʃ０.２∗１.２ʃ０.１∗６.８ʃ０.２∗３２.３ʃ２.９∗１０５.３ʃ７.９∗１.１ʃ０.１∗４.４ʃ０.６∗８.１ʃ０.８∗４４.４ʃ８.８∗１８０２.４ʃ９.５４＃０.９ʃ０.１０.８ʃ０.１５.０ʃ０.４２４.３ʃ３.８７４.２ʃ９.３∗０.７ʃ０.１２.７ʃ０.３５.０ʃ１.３３７.２ʃ４.３１７４２.３ʃ７.９７＃０.９ʃ０.２０.９ʃ０.１５.５ʃ０.５２５.１ʃ３.４∗８５.１ʃ８.４∗０.８ʃ０.３２.９ʃ０.２５.３ʃ０.６３６.９ʃ６.６１７９０.１ʃ１２ＷＴ０.８ʃ０.１０.７ʃ０.１４.１ʃ０.１２０.４ʃ１.６６８.７ʃ４.５０.７ʃ０.１２.２ʃ０.１４.７ʃ０.３３０.３ʃ４.３１７０６.３ʃ９.３㊀注:９＃㊁４＃㊁７＃:转基因烟草ꎻＷＴ:野生型烟草ꎮ数据为平均值ʃ标准误ꎬ∗表示与野生型相比在Ｐ<０.０５水平上差异显著ꎮ８０２生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ. All Rights Reserved.糖合成酶的活性ꎬ在不同转化事件中该酶的活性表现程度有所不同ꎮ这可能是由于外源基因插入烟草基因组的位置不同所致ꎬ也可能由于玉米Ｚｍｓｈ１基因的表达通过某种机制影响了烟草内源ＳｕＳｙ等酶的活性ꎮ然而ꎬ外源基因是否影响植物內源基因的表达ꎬ还需要进一步实验证明ꎮ蔗糖作为主要的能量来源和调控植物生长发育的信号分子ꎬ在植物生长发育中起着举足轻重的作用[４ꎬ１９]ꎮ蔗糖作为光合作用的终产物ꎬ被蔗糖酶水解为单糖而参加器官建成[２０]ꎮＱｕｙｎｈ等[２１]研究表明:光合作用增强和光合性蔗糖的合成增加驱动了蔗糖含量增加ꎬ可能导致光合作用固定碳的增加ꎮＢａｒｏｊａ￣Ｆｅｒｎｎｄｅｚ等[８]研究发现:在马铃薯中过表达ＳＵＳ４ꎬ导致块茎中果糖增加１２％ꎮ本实验研究结果与前人研究结果相似ꎮ本研究数据表明:与野生型烟草相比ꎬ开花期的转基因烟草根㊁茎㊁叶的蔗糖含量均有显著升高ꎬ整株蔗糖含量平均升高１７％ꎮ转基因烟草果糖含量测定结果表明:转基因烟草不同部位中的果糖水平均显著高于野生型烟草ꎬ整株果糖含量平均升高２５％ꎮ在胡萝卜[９]㊁马铃薯[１８]㊁番茄[２２]中ꎬ抑制ＳｕＳｙ酶活性可能会导致叶片和根变小㊁块茎干重降低ꎬ植株矮小等不利表型ꎮ本研究过表达Ｚｍｓｈ１基因后ꎬ转基因烟草生长发育也会受到影响ꎮ转基因烟草苗期生长缓慢ꎬ较之野生型烟草矮ꎻ营养期和生殖期生长迅速ꎬ在生殖生长结束后ꎬ株高均高于野生型烟草ꎮ除此之外ꎬ本研究还发现过表达Ｚｍｓｈ１基因后ꎬ转基因烟草除株高外其他生物量也显著增多ꎬ如:转基因烟草的千粒重增加ꎻ茎秆更粗壮ꎻ根㊁茎㊁叶的干重增多ꎮ这可能是由于细胞中的蔗糖被ＳＨ１分解为淀粉合成底物ＡＤＰＧꎬ促进了各器官中淀粉的生成[２３]ꎮ在马铃薯中过表达ＳＵＳ４导致ＳｕＳｙ酶活增加ꎬ马铃薯干重增多[８]ꎮＷａｎｇ等[２４]在番茄中过表达ＳｕＳｙ基因增加了番茄果实鲜重ꎬ提高了产量ꎮ本研究发现ꎬ与野生型烟草相比ꎬ转基因烟草根㊁茎㊁叶的干重分别平均增加了２３％㊁５５％和４０％ꎬ千粒重增加了３３％ꎮ实验结果与前人研究相似ꎬ并且转Ｚｍｓｈ１烟草的生物量积累更多ꎮ上述研究为进一步应用Ｚｍｓｈ１基因改良玉米㊁大豆㊁甘蔗等经济作物ꎬ提高产量ꎬ优化果实糖分㊁淀粉等生物性状提供了重要的理论数据参考ꎮ参㊀考㊀文㊀献[１]㊀ＣａｒｄｉｎｉＣＥꎬＬｅｌｏｉｒＬＦꎬＣｈｉｒｉｂｏｇａＪ.Ｔｈｅｂｉｏｓｙｎｔｈｅｓｉｓｏｆｓｕ￣ｃｒｏｓｅ[Ｊ].Ｂｉｏｌ.Ｃｈｅｍ.ꎬ１９５５ꎬ２１４(１):１４９－１５５. [２]㊀ＭｏｒｉｇｕｃｈｉＴꎬＹａｍａｋｉＳ.Ｐｕｒｉｆｉｃａｔｉｏｎａｎｄｃｈａｒａｃｔｅｒｉｚａｔｉｏｎｏｆｓｕ￣ｃｒｏｓｅｓｙｎｔｈａｓｅｆｒｏｍｐｅａｃｈ(Ｐｒｕｎｕｓｐｉｒｓｉｃａ)ｆｒｕｉｔ[Ｊ].ＰｌａｎｔＣｅｌｌＰｈｙｓｉｏｌ.ꎬ１９８８ꎬ２９(８):１３６１－１３６６.[３]㊀ＬｉｎｇｌｅＳＥꎬＤｙｅｒＪＭ.Ｃｌｏｎｉｎｇａｎｄｅｘｐｒｅｓｓｉｏｎｏｆｓｕｃｒｏｓｅｓｙｎ￣ｔｈａｓｅｃＤＮＡｆｒｏｍｓｕｇａｒｃａｎｅ[Ｊ].ＰｌａｎｔＰｈｙｓｉｏｌ.ꎬ２００１ꎬ１５８(１):１２９－１３１.[４]㊀柴静ꎬ张会ꎬ姚丽丽ꎬ等.蔗糖合酶在植物生长发育中的作用研究[Ｊ].生命科学ꎬ２０１２ꎬ２４(１):８１－８８. [５]㊀ＤｕｎｃａｎＫＡꎬＨａｒｄｉｎＳＣꎬＨｕｂｅｒＳＣ.Ｔｈｅｔｈｒｅｅｍａｉｚｅｓｕｃｒｏｓｅｓｙｎｔｈａｓｅｉｓｏｆｏｒｍｓｄｉｆｆｅｒｉｎｄｉｓｔｒｉｂｕｔｉｏｎꎬｌｏｃａｌｉｚａｔｉｏｎꎬａｎｄｐｈｏｓ￣ｐｈｏｒｙｌａｔｉｏｎ[Ｊ].ＰｌａｎｔＣｅｌｌＰｈｙｓｉｏｌ.ꎬ２００６ꎬ４７(７):９５９－９７１. [６]㊀ＣｈｏｕｒｅｙＰＳꎬＮｅｌｓｏｎＯＥ.Ｔｈｅｅｎｚｙｍａｔｉｃｄｅｆｉｃｉｅｎｃｙｃｏｎｄｉｔｉｏｎｅｄｂｙｔｈｅｓｈｒｕｎｋｅｎ￣１ｍｕｔａｔｉｏｎｓｉｎｍａｉｚｅ[Ｊ].Ｂｉｏｃｈｅｍ.Ｇｅｎｅｔ.ꎬ１９７６ꎬ１４(１１):１０４１－１０５５. [７]㊀ＫｏｍａｔｓｕＡꎬＭｏｒｉｇｕｃｈｉＴꎬＫｏｙａｍａＫꎬｅｔａｌ..Ａｎａｌｙｓｉｓｏｆｓｕｃｒｏｓｅｓｙｎｔｈａｓｅｇｅｎｅｓｉｎｃｉｔｒｕｓｓｕｇｇｅｓｔｓｄｉｆｆｅｒｅｎｔｒｏｌｅｓａｎｄｐｈｙｌｏｇｅｎｅｔｉｃｒｅｌａｔｉｏｎｓｈｉｐｓ[Ｊ].Ｅｘｐ.Ｂｏｔ.ꎬ２００２ꎬ５３(３６６):６１－７１.[８]㊀Ｂａｒｏｊａ￣ＦｅｒｎｎｄｅｚＥꎬＭｕñｏｚｆＪꎬＭｏｎｔｅｒｏＭꎬｅｔａｌ..Ｅｎｈａｎｃｉｎｇｓｕｃｒｏｓｅｓｙｎｔｈａｓｅａｃｔｉｖｉｔｙｉｎｔｒａｎｓｇｅｎｉｃｐｏｔａｔｏ(Ｓｏｌａｎｕｍｔｕｂｅｒｏｓ￣ｕｍＬ.)ｔｕｂｅｒｓｒｅｓｕｌｔｓｉｎｉｎｃｒｅａｓｅｄｌｅｖｅｌｓｏｆｓｔａｒｃｈꎬＡＤＰｇｌｕｃｏｓｅａｎｄＵＤＰｇｌｕｃｏｓｅａｎｄｔｏｔａｌｙｉｅｌｄ[Ｊ].ＰｌａｎｔＣｅｌｌＰｈｙｓｉｏｌ.ꎬ２００９ꎬ５０(９):１６５１－１６６２.[９]㊀ＴａｎｇＧＱꎬＳｔｕｒｍＡ.Ａｎｔｉｓｅｎｓｅｒｅｐｒｅｓｓｉｏｎｏｆｓｕｃｒｏｓｅｓｙｎｔｈａｓｅｉｎｃａｒｒｏｔ(ＤａｕｃｕｓｃａｒｏｔａＬ.)ａｆｆｅｃｔｇｒｏｗｔｈｒａｔｈｅｒｔｈａｎｓｕｃｒｏｓｅｐａｒｔｉｔｉｏｎｉｎｇ[Ｊ].ＰｌａｎｔＭｏｌ.Ｂｉｏｌ.ꎬ１９９９ꎬ４１(４):４６５－４７９. [１０]㊀ＫａｔｏＴ.Ｃｈａｎｇｅｏｆｓｕｃｒｏｓｅｓｙｎｔｈａｓｅａｃｔｉｖｉｔｙｉｎｄｅｖｅｌｏｐｉｎｇｅｎｄｏ￣ｓｐｅｒｍｏｆｒｉｃｅｃｕｌｔｉｖａｒｓ[Ｊ].ＣｒｏｐＳｃｉ.ꎬ１９９５ꎬ３５(３):８２７－８３１. [１１]㊀ＷｏｒｒｅｌｌＡＣꎬＢｒｕｎｅａｕＪＭꎬＳｕｍｍｅｒｆｅｌｔＫꎬｅｔａｌ..Ｅｘｐｒｅｓｓｉｏｎｏｆａｍａｉｚｅｓｕｃｒｏｓｅｐｈｏｓｐｈａｔｅｓｙｎｔｈａｓｅｉｎｔｏｍａｔｏａｌｔｅｒｓｌｅａｆｃａｒ￣ｂｏｈｙｄｒａｔｅｐａｒｔｉｔｉｏｎｉｎｇ[Ｊ].ＰｌａｎｔＣｅｌｌꎬ１９９１ꎬ３(１０):１１２１－１１３０. [１２]㊀ＣｈｅｎＹＣꎬＣｈｏｕｒｅｙＰＳ.Ｓｐａｔｉａｌａｎｄｔｅｍｐｏｒａｌｅｘｐｒｅｓｓｉｏｎｏｆｔｈｅｔｗｏｓｕｃｒｏｓｅｓｙｎｔｈａｓｅｇｅｎｅｓｉｎｍａｉｚｅ:Ｉｍｍｕｎｏｈｉｓｔｏｌｏｇｉｃａｌｅｖｉ￣ｄｅｎｃｅ[Ｊ].Ｔｈｅｏｒ.Ａｐｐｌ.Ｇｅｎｅｔ.ꎬ１９８９ꎬ７８(４):５５３－５５９. [１３]㊀ＣｈｅｎｇＷＨꎬＴａｌｉｅｒｃｉｏＥＷꎬＣｈｏｕｒｅｙＰＳ.Ｔｈｅｍｉｎｉａｔｕｒｅｓｅｅｄｌｏｃｕｓｏｆｍａｉｚｅｅｎｃｏｄｅｓａｃｅｌｌｗａｌｌｉｎｖｅｒｔａｓｅｒｅｑｕｉｒｅｄｆｏｒｎｏｒｍａｌｄｅｖｅｌｏｐｍｅｎｔｏｆｅｎｄｏｓｐｅｒｍａｎｄｍａｔｅｒｎａｌｃｅｌｌｓｉｎｔｈｅｐｅｄｉｃｅｌ[Ｊ].ＰｌａｎｔＣｅｌｌꎬ１９９６ꎬ８(６):９７１－９８３.[１４]㊀李合生.植物生理生化实验原理和技术[Ｍ].北京:高等教育出版社ꎬ２０００.[１５]㊀ＺｈｕＹＪꎬＫｏｍｏｒＥꎬＭｏｏｒｅＰＨ.Ｓｕｃｒｏｓｅａｃｃｕｍｕｌａｔｉｏｎｉｎｔｈｅｓｕｇａｒｃａｎｅｓｔｅｍｉｓｒｅｇｕｌａｔｅｄｂｙｔｈｅｄｉｆｆｅｒｅｎｃｅｂｅｔｗｅｅｎｔｈｅａｃｔｉｖ￣ｉｔｉｅｓｏｆｓｏｌｕｂｌｅａｃｉｄｉｎｖｅｒｔａｓｅａｎｄｓｕｃｒｏｓｅｐｈｏｓｐｈａｔｅｓｙｎｔｈａｓｅ[Ｊ].ＰｌａｎｔＰｈｙｓｉｏｌ.ꎬ１９９７ꎬ１１５(２):６０９－６１６.[１６]㊀ＲｏｅＪＨ.Ａｃｏｌｏｒｉｍｅｔｒｉｃｍｅｔｈｏｄｆｏｒｔｈｅｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｆｒｕｃｔｏｓｅｉｎｂｌｏｏｄａｎｄｕｒｉｎｅ[Ｊ].Ｂｉｏｌ.Ｃｈｅｍ.ꎬ１９３４ꎬ１０７(１):１５－２２.９０２顾韩雪ꎬ等:过表达玉米Ｚｍｓｈ１基因提高转基因烟草的生物量. All Rights Reserved.[１７]㊀丁雪梅ꎬ徐向红ꎬ邢沈阳ꎬ等.ＳＰＳＳ数据分析及Ｅｘｃｅｌ作图在毕业论文中的应用[Ｊ].实验室研究与探索ꎬ２０１２ꎬ３３(３):１２２－１２８.[１８]㊀ＺｒｅｎｎｅｒＲꎬＳａｌａｎｏｕｂａｔＭꎬＷｉｌｌｍｉｔｚｅｒＬꎬｅｔａｌ..Ｅｖｉｄｅｎｃｅｏｆｔｈｅｃｒｕｃｉａｌｒｏｌｅｏｆｓｕｃｒｏｓｅｓｙｎｔｈａｓｅｆｏｒｓｉｎｋｓｔｒｅｎｇｔｈｕｓｉｎｇｔｒａｎｓ￣ｇｅｎｉｃｐｏｔａｔｏｐｌａｎｔｓ(ＳｏｌａｎｕｍｔｕｂｅｒｏｓｕｍＬ.)[Ｊ].ＰｌａｎｔＪ.ꎬ１９９５ꎬ７(１):９７－１０７.[１９]㊀ＲｕａｎＹＬ.Ｓｕｃｒｏｓｅｍｅｔａｂｏｌｉｓｍ:Ｇａｔｅｗａｙｔｏｄｉｖｅｒｓｅｃａｒｂｏｎｕｓｅａｎｄｓｕｇａｒｓｉｇｎａｌｉｎｇ[Ｊ].Ａｎｎｕ.Ｒｅｖ.ＰｌａｎｔＢｉｏｌ.ꎬ２０１４ꎬ６５:３３－６７.[２０]㊀ＳａｄｉｋＳꎬＯｚｂｕｎＪＬ.Ｈｉｓｔｏｃｈｅｍｉｃａｌｃｈａｎｇｅｓｉｎｔｈｅｓｈｏｏｔｔｉｐｏｆｃａｕｌｉｆｌｏｗｅｒｄｕｒｉｎｇｆｌｏｒａｌｉｎｄｕｃｔｉｏｎ[Ｊ].Ｃａｎ.Ｊ.Ｂｏｔ.ꎬ１９６７ꎬ４５(７):９５５－９５６.[２１]㊀ＮｇｕｙｅｎＱＡꎬＬｕａｎＳꎬＷｉＳＧꎬｅｔａｌ..Ｐｒｏｎｏｕｎｃｅｄｐｈｅｎｏｔｙｐｉｃｃｈａｎｇｅｓｉｎｔｒａｎｓｇｅｎｉｃｔｏｂａｃｃｏｐｌａｎｔｓｏｖｅｒｅｘｐｒｅｓｓｉｎｇｓｕｃｒｏｓｅｓｙｎｔｈａｓｅｍａｙｒｅｖｅａｌａｎｏｖｅｌｓｕｇａｒｓｉｇｎａｌｉｎｇｐａｔｈｗａｙ[Ｊ].Ｆｒｏｎ.ＰｌａｎｔＳｃｉ.ꎬ２０１６ꎬ６:１１１－１２６.[２２]㊀ＳｕｎＪꎬＬｏｂｏｄａＴꎬＳｕｎｇＪＳꎬｅｔａｌ..Ｓｕｃｒｏｓｅｓｙｎｔｈａｓｅｉｎｗｉｌｄｔｏ￣ｍａｔｏＬｙｃｏｐｅｒｓｉｃｏｎｃｈｍｉｅｌｅｗｓｋｉｉꎬａｎｄｔｏｍａｔｏｆｒｕｉｔｓｉｎｋｓｔｒｅｎｇｔｈ[Ｊ].ＰｌａｎｔＰｈｙｓｉｏｌ.ꎬ１９９２ꎬ９８(３):１１６３－１１６９.[２３]㊀ＰｒｅｉｓｓＪ.Ｂｉｏｌｏｇｙａｎｄｍｏｌｅｃｕｌａｒｂｉｏｌｏｇｙｏｆｓｔａｒｃｈｓｙｎｔｈｅｓｉｓａｎｄｉｔｓｒｅｇｕｌａｔｉｏｎ[Ｊ].ＯｘｆｏｒｄＳｕｒｖ.ＰｌａｎｔＭｏｌ.ＣｅｌｌＢｉｏｌ.ꎬ１９９１ꎬ７:５９－１１４.[２４]㊀ＷａｎｇＦꎬＳａｎｚＡꎬＢｒｅｎｎｅｒＭＬꎬｅｔａｌ..Ｓｕｃｒｏｓｅｓｙｎｔｈａｓｅꎬｓｔａｒｃｈａｃｃｕｍｕｌａｔｉｏｎꎬａｎｄｔｏｍａｔｏｆｒｕｉｔｓｉｎｋｓｔｒｅｎｇｔｈ[Ｊ].ＰｌａｎｔＰｈｙｓｉｏｌ.ꎬ１９９３ꎬ１０１(１):３２１－３２７.０１２生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ. All Rights Reserved.。
青稞ＨｖｎＷＡＫ_基因的克隆及其在条纹病胁迫下的表达
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水稻谷氨酰胺氨基转移酶
水稻谷氨酰胺氨基转移酶英文回答：Glutamine amidotransferase (GAT) is a key enzyme involved in nitrogen metabolism in rice. It catalyzes the transfer of the amide group from glutamine to 2-oxoglutarate, forming glutamate and 2-oxoglutarate. This reaction is essential for the synthesis of glutamate, which is a precursor for the biosynthesis of other amino acids, nucleotides, and chlorophyll.GAT is present in both the cytoplasm and chloroplastsof rice cells. The cytoplasmic form of GAT is responsiblefor the synthesis of glutamate for use in various metabolic pathways, while the chloroplastic form is involved in the assimilation of ammonia into glutamate for the synthesis of amino acids and chlorophyll.The activity of GAT is regulated by a number of factors, including the availability of substrates, the redox stateof the cell, and the presence of inhibitors. Theavailability of substrates is the most important factor regulating GAT activity. When the concentration of glutamine and 2-oxoglutarate is high, the activity of GATis increased. The redox state of the cell also affects GAT activity. When the cell is in a reduced state, the activity of GAT is increased. This is because the reduced state of the cell favors the formation of glutamate. Finally, the presence of inhibitors can also affect GAT activity. There are a number of inhibitors of GAT, including 2-aminoethyl cysteine, serine hydroxamate, and methionine sulfoximine. These inhibitors bind to GAT and prevent it from catalyzing the transfer of the amide group from glutamine to 2-oxoglutarate.The expression of the GAT gene is regulated by a number of factors, including the nitrogen status of the plant, the light intensity, and the temperature. The expression of the GAT gene is increased when the nitrogen status of the plant is low. This is because the plant needs to increase the synthesis of glutamate in order to meet the demand for amino acids. The expression of the GAT gene is alsoincreased when the light intensity is high. This is because the plant needs to increase the synthesis of glutamate in order to meet the demand for chlorophyll. The expression of the GAT gene is also increased when the temperature is low. This is because the plant needs to increase the synthesisof glutamate in order to protect itself from the cold.GAT is a key enzyme involved in nitrogen metabolism in rice. The activity and expression of the GAT gene are regulated by a number of factors, including theavailability of substrates, the redox state of the cell,the presence of inhibitors, the nitrogen status of the plant, the light intensity, and the temperature.中文回答：水稻谷氨酰胺氨基转移酶（GAT）是水稻氮代谢中的一种关键酶。
The Transcription Factor FUSCA3
Developmental Cell,Vol.7,373–385,September,2004,Copyright 2004by Cell PressThe Transcription Factor FUSCA3Controls Developmental Timing in Arabidopsisthrough the Hormones Gibberellin and Abscisic Acidanalysis using Arabidopsis thaliana has also identified genes that regulate the timing of juvenile and adult leaf transitions and those involved in the conversion from a vegetative to a reproductive mode of development (Poethig,2003;Bastow and Dean,2003).To date,how-Sonia Gazzarrini,1Yuichiro Tsuchiya,1Shelley Lumba,1Masanori Okamoto,2,3and Peter McCourt 1,*1Department of Botany University of Toronto ever,none of the genes identified through Arabidopsis 25Willcocks Street genetic screens appear to be conserved in nematodes,Toronto M5S 3B2which is perhaps not surprising since plants and animals Canada are thought to have evolved multicellularity indepen-2Lab for Reproductive Growth Regulation dently (Meyerowitz,2002).Plant Science Center,RIKEN In contrast to the lack of genetic conservation of de-Tsurumi-ku,Yokohama velopmental timing,there appears to be a linkage be-Kanagawa 230-0045tween terpenoid hormones and heterochrony.In Arabi-Japan dopsis ,the terpenoid-based hormone,gibberellin (GA),3Department of Biological Sciences can influence the time to flowering by stimulating the Tokyo Metropolitan University transcription of the LEAFY gene,and in many insects,Hachioji-shi the terpenoid,juvenile hormone (JH)can affect develop-Tokyo 192-0397mental transitions (Blazquez et al.,1998;Thummel,Japan2001;Wheeler and Nijhout,2003).The commonality of a relationship between terpenoid-based hormones and developmental timing might suggest that genetic dis-Summarysection of newly defined heterochronic pathways will eventually uncover a hormonal component.Although plants continually produce different organs In Arabidopsis ,the ability to easily distinguish cotyle-throughout their life cycle,little is known about the dons (embryonic leaves)from vegetative (postembry-factors that regulate the timing of a given develop-onic)leaves has allowed the identification of mutations mental program.Here we report that the restricted that result in either the replacement of cotyledons with expression of FUS3to the epidermis is sufficient to organs more similar to vegetative leaves or vice versa control foliar organ identity in Arabidopsis by regulat-(Baumlein et al.,1994;Keith et al.,1994;Meinke et al.,ing the synthesis of two hormones,abscisic acid and 1994;West et al.,1994;Conway and Poethig,1997).gibberellin.These hormones in turn regulate the rates These mutations can be formally considered hetero-of cell cycling during organ formation to determine chronic since cotyledons temporally precede vegetative whether an embryonic or adult leaf will emerge.We leaves in the plant life cycle.For example,loss-of-func-also show that FUS3expression is influenced by the tion mutations in the LEC genes,LEAFY COTYLEDON1patterning hormone,auxin,and therefore acts as a (LEC1),LEAFY COTYLEDON2(LEC2),and FUSCA3nexus of hormone action during embryogenesis.The (FUS3),result in the replacement of cotyledons with identification of lipophillic hormones downstream of vegetative leaves.A heterochronic interpretation would a heterochronic regulator in Arabidopsis has parallels suggest that the embryo has omitted or advanced the to mechanisms of developmental timing in animals differentiation of the cotyledons,thereby causing these and suggests a common logic for temporal control of organs to take on a vegetative leaf fate (Keith et al.,developmental programs between these two kingdoms.1994).Consistent with this,genes that encode markers of late embryogenesis such as seed storage proteins Introductionand desiccation protectants are reduced or missing in lec1and fus3mutants,while germination markers,which In higher plants and animals,a molecular appreciation normally proceed late embryogenesis,are precociously of what controls the timing of specific genetic programs activated (West et al.,1994;Nambara et al.,2000;Kroj is essential if we are to have a complete understanding et al.,2003).of multicellular development.The metamorphosis in in-Molecular characterization of the LEC genes indicates sects and amphibians,the onset of puberty in mammals,that they regulate cotyledon cell fate by controlling tran-and the remarkable variation seen in plant morphology scription.The LEC1gene encodes a protein related to often simply represent the consequences of changes in a transcription factor subunit of the HAP3gene family the timing of specific developmental programs relative in mammals,while LEC2and FUS3encode proteins to one another.In animals,a mechanistic understanding of the plant-specific B3transcription factor ABI3/VP1of what determines the timing of developmental pro-family (Lotan et al.,1998;Luerssen et al.,1998;Stone grams has mostly been addressed by screening for mu-et al.,2001).Upstream of the LEC genes,it has been tations that advance or retard specific cell lineages shown that the CHD3-chromatin-remodeling factor,during C.elegans larval development (see Slack and PICKLE (PKL ),is necessary for repression of these Ruvkun,1997,for review).In higher plants,mutationalgenes outside of the embryo (Ogas et al.,1999;Rider et al.,2003).In turn,FUS3appears to negatively regulate the cellular morphogenesis regulator TRANSPARENT*Correspondence mccourt@botany.utoronto.caDevelopmental Cell374TESTA GLABRA1(TTG1)(Tsuchiya et al.,2004).TTG1which suggests that the embryonic patterns of FUS3 is thought to be important in the cell specification oftranscript accumulation may reflect differential auxin many epidermally derived adult structures in the root,concentrations.This was tested by dissecting embryos hypocotyl,leaf,and seed coat(Koornneef,1981;Galwayfrom a FUS3::FUS3-GFP transgenic plant and placing et al.,1994;Hung et al.,1998;Western et al.,2001).the embryos on100M IAA,a naturally occurring auxin. Although mutations in the LEC genes are develop-By24hr,IAA imbibed embryos showed an increase in mentally easy to distinguish in the embryo,the difficulty the nuclear localization of GFP signal in the root cap of experimentally manipulating Arabidopsis embryoscells versus untreated controls(Figures1I–1L).Because has hindered further understanding of how these genes the GFP signal can be induced by exogenous auxin in regulate timing events.In the case of FUS3,we havedissected embryos,we tested the possibility of inducing circumvented this problem by expressing it in vegetative FUS3expression outside of the embryo in an auxin-leaves.Limiting expression of FUS3to the epidermaldependent manner by monitoring the expression of the layer of a vegetative leaf is sufficient to direct all cells FUS3gene using a FUS3::GUS transcriptional reporter. within the organ toward cotyledon development,whichIn the absence of exogenous auxin,the FUS3::GUS suggests that a downstream component of this tran-transgenic line normally shows GUS activity in its roots scription factor acts ing thisfor a duration of approximately3days after germination tethered system,we found that many FUS3misexpres-(Figure1M).After this time,the blue staining dissipated sion phenotypes are contingent on levels of the twoand was only marginally present by6days(Figure1N). terpenoid-derived hormones abscisic acid(ABA)and After6-day-old seedlings grown on minimal media were GA.FUS3regulates where and when ABA and GA aretransferred to media containing10M IAA for a24hr synthesized,and these two hormones in turn determine period,strong GUS activity was observed in the root tip the stability of the FUS3protein.Finally,FUS3contrib-versus roots grown on minimal media(Figures2N and utes to developmental transitions of leaf identity by reg-2O).A similar incubation on media containing ABA or ulating the rates of cell cycling,which are also hormon-GA showed little or no increase in blue staining,indicat-ally controlled.Thus,that the heterochronic gene,FUS3,ing that the increased GUS activity is auxin specific specifies the proper schedule of the cycling of cells(Figures2P and2Q).Hence,the patterns of FUS3-GFP during cotyledon patterning through terpenoid hor-protein accumulation in the embryo may reflect the sites monal signaling is parallel to the logic of developmentalof the auxin maxima,such as the root and cotyledon tips. timing regulation in other systems.Epidermally Derived FUS3Is Sufficient to Direct ResultsCotyledon DevelopmentBecause the AtML1promoter restricts expression tothe L1layer of the shoot apical meristem throughout FUS3Patterns of Expression Are Influencedvegetative and floral development,it allows the misex-by Auxinpression of FUS3to be maintained postembryonically We investigated the localization patterns of FUS3pro-tein during embryogenesis using a reporter FUS3-GFP(Lu et al.,1996;Sessions et al.,1999).Homozygous translational fusion under the control of the endogenousAtML1::FUS3-GFP seedlings developed into dark green, FUS3promoter(FUS3::FUS3-GFP).This translational fu-dwarfed plants that were often semisterile(Figure2A). sion was fully functional in that it rescued all loss-of-After germination,homozygous lines produce foliar function fus3embryonic phenotypes(data not shown).leaves that are more similar to cotyledons(Figures2C–Up to the triangular stage of embryo development,fluo-2E).As lateral organs emerge from the meristem,they rescence was detected in most cells but was more are yellow,but as they develop take on a rounder shape strongly visible in the apical part of the embryo and theand a glossy,glabrous surface reminiscent of a cotyle-suspensor(Figure1A).By the late heart and torpedo don(Figures2D and2E).In strong lines,the production stages,the GFP signal became predominantly confinedof cotyledons occurred throughout development even to the epidermis(Figures1B and1C).At these later after the plant has switched to a reproductive meristem. stages,GFP fluorescence was most pronounced at theFor example,cotyledonary-like organs appeared from cotyledon and root tips with some signal being detected the floral meristem in the same phyllotactic arrangement in the vascular tissue within the embryonic root(Figuresas that observed for flower organs(Figures2F and2G). 1C and1E).By the walking stick stage,the GFP signal As the plants age,however,the organs that initially clearly marked the root tip and the epidermal tissues ofemerged as cotyledonary-like leaves slowly take on fea-the cotyledon(Figures1D and1F).Closer inspection of tures that are more characteristic of vegetative leaves, the root tip of walking stick embryos revealed weaksuch as an oblong leaf shape and elongated petioles re-fluorescence in inner cell layers adjacent to the epider-sulting in a Christmas tree-like appearance(Figure2B). mis,while the cotyledons still showed epidermal-spe-As young vegetative leaves emerge from the meristem cific fluorescence(Figures1F and1G).After this stage,of misexpressed FUS3lines,they accumulate seed stor-the GFP signal began to dissipate and was completelyage proteins throughout the leaf,which is consistent absent by the time the embryo reached maturity(Figure with their embryonic identity(Figures2H–2L).This accu-1H).These GFP fluorescence patterns support the pre-mulation is surprising since AtML1::FUS3-GFP trans-viously published FUS3transcription patterns(Tsuchiya genic plants only show GFP fluorescence in the epider-et al.,2004).mal layer(data not shown).This result suggests that a The embryonic patterns of FUS3accumulation are downstream component of FUS3that regulates seedstorage accumulation most likely functions in a cell-similar to those reported for the auxin-responsive re-porter,DR5(Friml et al.,2003,Benkova et al.,2003),nonautonomous manner.FUS3Controls Developmental Timing in Arabidopsis375Figure1.Patterns of FUS3Expression in the Embryo and Its Regulation by Hormones in the Root(A–H)GFP localization patterns in FUS3::FUS3-GFP embryos at different developmental stages.(A–D)Median longitudinal optical section from triangular(A),late-heart(B),torpedo(C),and walking stick(D)stage embryos.(E)Higher magnification of the root tip at the torpedo stage shown in(C)showing FUS3-GFP localization in the root meristem and provasculature.(F and G)Higher magnification of the cotyledon and root tip from the embryo shown in(D)showing FUS3-GFP expression in the epidermis of the cotyledon(F)and in the root meristem(G).(H)Median longitudinal optical section from a mature stage embryo showing no GFP fluorescence.Propidium iodide staining was performed on embryos at the walking stick(D,F,and G)and mature(H)stages to visualize cell boundaries.The red background in the other images is due to autofluorescence.(I–L)GFP fluorescence in FUS3::FUS3-GFP roots of a walking stick embryo.Ovules were excised from siliques and cultured in the absence (I and K)or presence(J and L)of100M IAA for24hr.Images were taken with a constant set of microscopic and image intensity parameters.(I and J)Median longitudinal optical section of root tips.(K and L)Surface view of the epidermis of the hypocotyl region.(M–Q)FUS3::GUS expression pattern in the root tip of3-(E)or6-(F–I)day-old seedlings grown on MS media and transferred to MS media (M and N)or MS media supplemented with10M IAA(O),10M GA(P),or10M ABA(Q)for24hr.Bars,40m for(A),(E),(F),(G),(I)–(L);50m for(B),(C),(D),and(H).FUS3Positively Regulates ABA Synthesis AtML1::FUS3-GFP transgene.Because severe ATML1::FUS3-GFP lines do not produce functional flowers for ABA has been implicated as a positive regulator of manyFUS3-regulated embryonic functions including storage crossing,a phenotypically weak AtML1::FUS3-GFP trans-reserve accumulation,desiccation tolerance,and dor-genic line was used in this experiment.Cotyledon-like mancy establishment(Keith et al.,1994;Baumlein et al.,leaves produced in weaker lines are mostly lacking in 1994;Leung and Giraudat,1998).To test the relationshiptrichome hairs but occasionally produce trichomes at between FUS3and ABA,an ABA auxotrophic muta-the tip or margins of the leaves(Figure3A).Flower devel-tion(aba2-2)was genetically introduced into a fus3opment is affected to some extent in weak lines,in that loss-of-function line that has been complemented with the sepals open much earlier than wild-type and growthDevelopmental Cell376Figure2.ML1::FUS3Vegetative Phenotypes(A–G)FUS3misexpression(ML1::FUS3-GFP)in the L1layer of the meristem strongly re-duces plant stature(A)and produces cotyle-don-like foliar organs(B–G).(A)4-week-oldwild-type(left),strong ML1::FUS3-GFP(mid-dle),and weak ML1::FUS3-GFP(right)plants.(B)An8-week-old ML1::FUS3-GFP plant(strongline).(C)A2-week-old wild-type seedling.(D)A2-week-old ML1::FUS3-GFP seedling(strongline).(E)A5-week-old ML1::FUS3-GFP seed-ling(strong line).The arrows indicate cotyle-don-like leaves.(F)A wild-type flower.(G)AML1::FUS3-GFP flower(strong line).Note theconversion of petals into leaf-like structures.The arrow indicates a carpel.(H–K)Cross-sections of wild-type leaves(Hand J)and ML1::FUS3-GFP leaves of a strongline(I and K)stained with toluidine blue.Notethat wild-type cells are largely vacuolated asopposed to ML1::FUS3-GFP cells,whichare densely filled with protein bodies(seearrows).(L)SDS-PAGE of proteins isolated from wild-type seed(lane1)and leaves(lane2),andML1::FUS3-GFP leaves(lane3).The arrowsindicate seed storage proteins accumulatingat higher levels in ML1::FUS3-GFP leavescompared to wild-type leaves.of the petals and the filament of the stamens is delayed were relatively low until6days after flowering(DAF),at compared to the growth of the carpel(Figure3B).Thesewhich time levels began to increase and reached a peak flower defects often result in reduced fertilization,which10DAF.After this time,they started to decrease again. consequently produces a large proportion of short si-By contrast,ABA levels in the fus3seed showed a similar liques(Figure3C).When the aba2-2mutation was intro-accumulation pattern to wild-type up to8DAF but from duced into the FUS3misexpressing line,trichome pro-then on failed to accumulate ABA to the levels observed duction was restored on rosette and cauline leaves in wild-type seeds(Figure3G).Although these resultssuggest that FUS3is a positive regulator of the ABA (Figure3D),floral defects were rescued(Figure3E),andplants produced normal elongated siliques(Figure3F).biosynthesis,to directly address this,we constructed These results indicate that ABA is necessary for FUS3a FUS3inducible system in which the glucocorticoid function and that this hormone works at or downstream receptor(GR)from mammalian systems was transla-of FUS3.tionally fused to FUS3(Aoyama and Chua,1997).A To more clearly determine the relationship between FUS3::GR fusion driven by the AtML1promoter was ABA and FUS3,we measured the concentrations of ABAtransformed into fus3plants.In the absence of the syn-throughout embryogenesis in a fus3loss-of-function line thetic hormone,dexamethasone(DEX),transgenic seed-(Figure3G).In wild-type seeds,ABA concentrationslings were indistinguishable from the fus3parent,butFUS3Controls Developmental Timing in Arabidopsis377Figure3.FUS3Positively Modulates ABALevels(A–F)Vegetative phenotypes of ABA2,fus3,ML1::FUS3-GFP(A–C)compared to aba2-2,fus3,ML1::FUS3-GFP(D–F).(A)A glabrouscauline leaf.(B)An inflorescence with openfloral organs.(C)A stem bearing short si-liques.(D)A cauline leaf bearing trichomes.(E)An inflorescence showing rescued floralorgans.(F)A stem with rescued siliques.(G)ABA levels in wild-type and fus3siliquesharvested at different days after flowering.(H and I)ABA levels in ML1::FUS3-GR seed-lings grown on MS media for5days(H)or9days(I)and transferred to MS(open bars)or0.1M DEX(filled bars)for24hr.Twoindependent experiments(EXP1and EXP2)are shown.(J)Germination of wild-type,era1-2,andthree ML1::FUS3-GFP lines on exogenousABA.The sensitivity of ML1::FUS3-GFP seedsto ABA is similar to that of the ABA supersen-sitive mutant era1-2.Each point represents agermination test of50seeds.The experimentwas repeated twice and similar results wereobtained.when germinated and grown in the presence of DEX,et al.,1998).Furthermore,GA is important in trichome transgenic plants produced cotyledonary-like vegeta-formation in vegetative Arabidopsis leaves and fus3 tive leaves that are characteristic of FUS3misexpres-loss-of-function mutants have ectopic trichomes on sion(data not shown).Transfer of5-and9-day-oldtheir cotyledons(Keith et al.,1994,Baumlein et al.,1994; transgenic seedlings from minimal media to low concen-Telfer et al.,1997).To explore the role of GA on FUS3-trations of DEX for24hr immediately increased ABAdependent functions,a mutation that decreases GA syn-concentrations in two independent seedling samples thesis(ga1-2)was introduced into a fus3loss-of-func-tion mutant.Double mutants still produced red seeds (Figures3H and3I).Coupled with the observation thatloss-of-function fus3mutations reduce ABA levels,that were desiccation intolerant,indicating that these these results indicate that FUS3is a positive regulatorfus3phenotypes are GA-independent(data not shown). of ABA synthesis.The germination of seed from three However,ectopic trichome production on cotyledons independent AtML1::FUS3transgenic lines all showedwas reduced and many times absent in double mutants increased sensitivity to exogenous ABA versus wild-indicating that this phenotype does require GA(Figures type,which is consistent with the increased synthesis4A and4B).Consistent with this,when AtML1::FUS3-of ABA in misexpressing lines(Figure3J).GFP plants were sprayed twice a week with GA,thedevelopment was more similar to wild-type plants(Fig-FUS3Negatively Regulates GA Synthesisure4C).Plants showed a normal stature,bolted on time Many of the adult FUS3misexpression phenotypes are and both the flower and silique defects were rescuedby GA application(Figures4D–4F).reminiscent of a plant defective in GA synthesis or action(Figure2A;Koornneef and van der Veen,1980;Steber GA-dependent rescue of AtML1::FUS3-GFP lines wasDevelopmental Cell378Figure4.FUS3Negatively Modulates GASynthesis(A)A fus3,GA1seedling showing cotyledonsbearing trichomes.(B)A fus3,ga1-2seedling showing glabrouscotyledons resembling wild-type cotyledons.(C)22-day-old wild-type(left),ML1::FUS3-GFP(right),and ML1::FUS3-GFP sprayedwith10M GA(middle)showing the rescueof ML1::FUS3-GFP late flowering phenotypeby GA.(D–F)Higher magnification of flowers of wild-type(D),ML1::FUS3-GFP(E),and ML1::FUS3-GFP sprayed with GA(F).(G)Profile and venation pattern of clearedwild-type cotyledons and vegetative leavesgrown on MS media for4days and shiftedto MS media for an additional4days.(H)Profile and venation pattern of clearedML1::FUS3-GFP cotyledons and vegetativeleaves grown on MS media for4days andshifted to MS media for an additional4days.(I)Profile and venation pattern of clearedML1::FUS3-GFP cotyledons and vegetativeleaves grown on MS for4days and shiftedto MS supplemented with10M GA for anadditional4days.The arrow indicates thecotyledon-like leaf sector formed on one halfof the leaf.(J)RT-PCR analysis of AtGA3ox1andAtGA20ox1expression in the shoot of ML1::FUS3-GR seedlings.Tissues were harvestedafter1hr,24hr,and4days after1M DEXinduction(ϩ)compared to control treatment(Ϫ).Amplification of ACT7is shown as anexpression standard.studied in more detail by following the time of rescue half of the leaf was larger,possessed several trichomes, after GA application(Figures4G–4I).In contrast to anand showed a more complex venation system.Leaf untreated misexpression line(Figure4H),addition of GA three and successive leaves displayed full rescue of leaf caused a gradual rescue of the leaf phenotypes such asmorphology(Figure4I).shape,size,venation pattern,and absence of trichomes The relationship between FUS3and GA is most easily (Figure4I).The first vegetative leaf of a seedling grownexplained by FUS3exerting a negative regulation on GA and germinated on10M GA often resembled an embry-synthesis or action.To test this,the levels of transcripts onic cotyledon,but occasionally developed as a chime-of two key steps in GA biosynthesis,AtGA20ox1and ric leaf showing both embryonic and vegetative venation AtGA3ox1,were assayed using DEX-inducible AtML1:: patterns and shapes(Figure4I).Indeed,one half of theFUS3-GR misexpression lines.Within1hr after DEX leaf did not bear trichomes,had a simple venation pat-application,transcript levels of the AtGA3ox1showeda slight decrease,and by24hr,the signal was highly tern,and was small in size,thereby resembling embry-onic cotyledons(Figure4I,see arrow),whereas the other reduced as measured by RT-PCR(Figure4J).TheFUS3Controls Developmental Timing in Arabidopsis379AtGA20ox1transcript levels also showed a reduction a possible role of ABA and GA on FUS3protein stability, after DEX application,but the kinetics of reduction werewe monitored the GFP fluorescence in AtML1::FUS3-much slower as a clear cut decrease in transcript levels GFP vegetative leaves after exposure to ABA,GA,and only resulted after4days on DEX(Figure4J).Thesean inhibitor of GA biosynthesis,uniconazole-P.In the results clearly indicate that FUS3is a negative regulator presence of ABA,GFP fluorescence is stabilized com-of GA biosynthesis and explain the prevalence of FUS3-pared to untreated controls,suggesting that the upregu-dependent GA-related tion of ABA biosynthesis by FUS3may in turn stabilizethe FUS3protein in vivo(Figures6A and6B).In contrast,a4day exposure to10M GA slowly decreased the FUS3Regulates the Timing of Leaf DevelopmentGFP signal versus control samples(Figure6D).Consis-by Controlling Cell Cyclingtent with this observation,GFP fluorescence was The ability of GA and ABA levels to modulate FUS3strongly localized to the nuclei in the presence of10 misexpression phenotypes is unexpected since theseM uniconazole(Figure6C).The ability of ABA to stabi-two hormones are not usually associated with specifyinglize FUS3protein explains why decreased ABA concen-organ and cellular morphogenesis.However,both hor-trations using an ABA-deficient mutant suppressed mones have been suggested to have roles in speedingAtML1::FUS3-GFP phenotypes.Similarly,the decreased up or slowing down various aspects of the plant lifestability of FUS3protein in the presence of GA explains cycle.Consistent with this,as the AtML1::FUS3-GFPwhy exogenous application of GA suppressed FUS3 lines continued to develop over a longer period of time,misexpression phenotypes.the same leaves that started out resembling cotyledonsbegan to develop features that are more characteristicof a vegetative leaf(Figure2B).For example,before Discussionflowering,leaves have a cotyledonary shape and vena-tion pattern,but after having flowered,they have devel-FUS3Is a Nexus of Hormone Action in the Embryo oped a more vegetative venation pattern and leaf shape During Arabidopsis embryogenesis,the rise in ABA (Figures5A and5B).levels is important for the establishment of many late The conversion of a cotyledonary leaf into a vegetative embryogenic functions.Conversely,GA concentrations leaf over time suggests that the scale of leaf develop-remain relatively low during this period until mature ment in a FUS3misexpression line is slower than normal.seeds are imbibed,at which time GA rapidly increases, One factor that determines the size,shape,and venation thereby reversing ABA-induced dormancy(Ogawa et al., patterns of leaves is the rate of cell divisions during2003).At a molecular level,GA influences a number of leaf primordial expansion(Kang and Dengler,2002).To key regulators of ABA signaling,which suggests that determine if the spatial and temporal patterns of cell decreases in ABA concentrations in conjunction with cycling in AtML1::FUS3-GFP lines are altered,a cyclin increases in GA levels act together to modulate ABA--glucuronidase fusion reporter(cyc1At::GUS)was in-dependent gene expression during seed development troduced into a FUS3misexpression line.This construct and germination(Ogawa et al.,2003).A coordination of has been used extensively to characterize patterns of GA and ABA levels during embryogenesis and germina-cell divisions in Arabidopsis developing leaves(Donnelly tion must therefore be upheld so that the embryo does et al.,1999).As expected in wild-type,a gradient of not receive mixed hormonal messages.punctate GUS staining,which marks single cell divi-One way that FUS3can coordinate the ABA/GA action sions,was detected in young leaves emerging from the in the embryo is through feedback loops produced by apical meristem(Figures5C and5D).By contrast,in FUS3regulating the levels of ABA and GA and these similarly aged AtML1::FUS3-GFP lines,blue staining hormones regulating the stability of FUS3protein(Figure was highly reduced in emerging foliar organs,sug-7A).The production of FUS3in the epidermis and later gesting that FUS3is a negative regulator of cell cycling in the cotyledon margins,root tip,and vasculature of in Arabidopsis(Figures5E and5F).On this note,the the embryo inhibits production of GA in these tissues ability of GA to rescue AtML1::FUS3-GFP phenotypes(Figure7A).Thus,GA-dependent processes involved suggests that application of GA may function to increase with vegetative leaf development such as trichome pro-the cycling of cells in FUS3misexpressing lines.As duction,venation patterns,and cell expansion would be expected,increased GUS staining was observed when suppressed.This is supported by the observation that GA was applied to AtML1::FUS3-GFP(Figures5G and AtGA3ox1,a gene that encodes the last step of GA 5H).It therefore appears that the GA rescue of AtML1::biosynthesis,is quickly downregulated by the activation FUS3-GFP phenotypes is due to the ability of this hor-of FUS3protein(Figure4J).mone to increase cell cycling(see Supplemental Figure During late embryogenesis,the situation would be S1at /cgi/content/reversed by GA synthesis(Figure7B).The site of biosyn-full/7/3/373/DC1).thesis of active GA in Arabidopsis embryos duringgermination does not coincide with the expression of ABA and GA Feed Back to ModulateGA-responsive genes,suggesting that GA or a GA sig-the Stability of FUS3Protein naling component is cell nonautonomous(Ogawa et al., In the past few years,a number of studies have indicated2003).Thus,the long-range action of GA synthesized in the role of plant hormones as regulators of the turnover tissues where FUS3is not expressed would ensure that of key proteins involved in hormone signaling(Devotoepidermally derived FUS3protein is degraded so that et al.,2002;Dharmasiri and Estelle,2002;Dill et al.,2001;normal development can progress(Figure7B).Consis-Potuschak et al.,2003;Gao and Ecker,2003).To exploretent with this,transcript levels of AtGA3ox1are low。
去垢剂
在生物学或生物化学实验室使用的去污剂都是作用比较温和的表面活性剂（=表面活性成分），是用来破坏细胞膜（裂解细胞）以释放细胞内的可溶性物质。
它们可以破坏蛋白质-蛋白质、蛋白质-脂质、脂质-脂质之间的连接，使蛋白质发生结构上的变性，防止蛋白质结晶，另外在免疫学实验中还可避免非特异性吸附。
去污剂根据其特性可以分为好几类，因此科学研究中去污剂的选择很关键，取决于后续研究的具体内容。
实际应用中有众多不同的去污剂可以选择。
为了某些特殊的应用，新的去污剂被不断开发出来[]。
在这篇综述中，对一些最常用的去污剂的特点和应用进行了论述。
去污剂是由一个疏水尾端基团和一个极性亲水头端基团组成的有机化合物（图一A）。
在一定的温度条件下，以特定浓度溶解于水时，去污剂分子会形成胶束，疏水基团部分位于胶束内部，而极性亲水基团则在其外部（图一B）。
因此，胶束的疏水中心会结合到蛋白的疏水区域。
一个胶束中，去污剂分子的聚集数目，是用来评价膜蛋白溶解度的一个重要参数[]。
去污剂分子疏水区域的长度和其疏水性成正比，且去污剂的疏水区域非常恒定，而极性头端亲水基团是可变的，可据其特点，把去污剂分为三类：离子型（阴离子或阳离子型），两性离子型和非离子型（见表一）。
在特定的温度下，表面活性剂分子缔合形成胶束的最低浓度，称之为临界胶束浓度（CMC）。
当去污剂低于临界胶束浓度时，只有单体存在；当高于临界胶束浓度时，胶束、单体以及其余不溶于水的非胶束相共存。
同样，胶束形成的最低温度称为临界胶束温度（CMT）。
因此，温度和浓度是去污剂两相分离和溶解性的重要参数。
一般来说，低亲脂或憎油的去污剂的临界胶束浓度会较高。
种类化合物离子去污剂十二烷基硫酸钠（SDS），脱氧胆酸钠，胆酸钠，肌氨酸非离子去污剂tritonX-100，十二烷基麦芽糖苷，洋地黄皂苷，tween20，tween80两性离子去污剂CHAPS离液剂尿素表一：去污剂的分类。
离子去污剂离子去污剂是由一个亲水链和一个阳离子或阴离子的极性头端基团组成。
转基因何首乌毛状根的研究进展
转基因何首乌毛状根的研究进展何首乌（Polygonum multiflorum Thunb.），又称首乌、黑首乌，为蓼科植物，广泛应用于中药、化妆品和食品等领域。
何首乌毛状根是何首乌的一个重要药用部位，具有补肝肾、黑发生发、明目等功效，是中药制剂的热门材料之一。
然而，何首乌在生长、收获、贮藏、加工等方面存在困难，对质量控制和产业发展产生了不利影响。
转基因技术为何首乌改良和利用提供了有力支持，已成为研究热点之一。
以下是何首乌毛状根转基因研究的最新进展。
1. AST基因AST（aspartate aminotransferase）是一种参与氨基酸代谢的重要酶类，常在植物逆境应答过程中发挥重要作用。
2018年，一组中国科学家通过PCR扩增和克隆技术，克隆出何首乌毛状根中的AST基因，并将其转入拟南芥中。
研究结果表明，AST基因能显著提升拟南芥的抗盐能力，同时通过光镜观察发现，转基因拟南芥的根系比野生型更发达。
这项研究为何首乌毛状根耐盐和产量提高提供了新思路。
2. CsAPX基因CsAPX（ascorbate peroxidase）是一种抗氧化酶，参与植物对环境胁迫的应答。
2019年，一组研究者将CsAPX基因转入何首乌毛状根中，并进行了功能鉴定。
通过多种生化和分子生物学技术，他们发现，转基因何首乌毛状根在盐酸处理下叶绿素含量和抗氧化能力均显著提高，同时总多酚、矿物质含量等指标也明显提升。
研究结果证明，CsAPX基因是何首乌毛状根提高抗氧化能力和适应胁迫的重要遗传因子。
3. OsMADS57基因OsMADS57是稻米的MADS家族转录因子中的一个成员，参与稻米花期和产量的调控。
2017年，一组中国科学家将该基因转入何首乌毛状根中，进行了生长和产量的比较。
实验结果显示，转基因何首乌毛状根的干重和总黄酮含量均显著提高，同时在产量方面也有所增加。
这项研究去创新思路，为何首乌毛状根产业的可持续发展提供了新途径。
PCR重要概念解释
重要概念解释AAbundance (mRNA 丰度)：指每个细胞中mRNA分子的数目。
Abundant mRNA(高丰度mRNA)：由少量不同种类mRNA组成，每一种在细胞中出现大量拷贝。
Acceptor splicing site (受体剪切位点)：内含子右末端和相邻外显子左末端的边界。
Acentric fragment(无着丝粒片段)：(由打断产生的)染色体无着丝粒片段缺少中心粒，从而在细胞分化中被丢失。
Active site(活性位点)：蛋白质上一个底物结合的有限区域。
Allele(等位基因)：在染色体上占据给定位点基因的不同形式。
Allelic exclusion(等位基因排斥)：形容在特殊淋巴细胞中只有一个等位基因来表达编码的免疫球蛋白质。
Allosteric control(别构调控)：指蛋白质一个位点上的反应能够影响另一个位点活性的能力。
Alu-equivalent family(Alu相当序列基因)：哺乳动物基因组上一组序列，它们与人类Alu 家族相关。
Alu family (Alu家族)：人类基因组中一系列分散的相关序列，每个约300bp长。
每个成员其两端有Alu切割位点(名字的由来)。
α-Amanitin(鹅膏覃碱)：是来自毒蘑菇Amanita phalloides二环八肽，能抑制真核RNA聚合酶，特别是聚合酶II 转录。
Amber codon (琥珀密码子)：核苷酸三联体UAG，引起蛋白质合成终止的三个密码子之一。
Amber mutation (琥珀突变)：指代表蛋白质中氨基酸密码子占据的位点上突变成琥珀密码子的任何DNA改变。
Amber suppressors (琥珀抑制子)：编码tRNA的基因突变使其反密码子被改变，从而能识别UAG密码子和之前的密码子。
Aminoacyl-tRNA (氨酰-tRNA)：是携带氨基酸的转运RNA，共价连接位在氨基酸的NH2基团和tRNA终止碱基的3′或者2′－OH基团上。
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Planta (2007) 226:1243–1254 DOI 10.1007/s00425-007-0573-4
123ORIGINAL ARTICLE
Maize Lc transcription factor enhances biosynthesis of anthocyanins, distinct proanthocyanidins and phenylpropanoids in apple (Malus domestica Borkh.)
Houhua Li · Henryk Flachowsky · Thilo C. Fischer · Magda-Viola Hanke · Gert Forkmann · Dieter Treutter · Wilfried Schwab · Thomas HoVmann · Iris Szankowski
Received: 18 December 2006 / Accepted: 5 June 2007 / Published online: 6 July 2007© Springer-Verlag 2007
AbstractFlavonoids are a large family of polyphenoliccompounds with manifold functions in plants. Present in awide range of vegetables and fruits, Xavonoids form anintegral part of the human diet and confer multiple healthbeneWts. Here, we report on metabolic engineering of theXavonoid biosynthetic pathways in apple (Malus domesticaBorkh.) by overexpression of the maize (Zea mays L.) leafcolour (Lc) regulatory gene. The Lc gene was transferredinto the M. domestica cultivar Holsteiner Cox via Agrobac-terium tumefaciens-mediated transformation which resultedin enhanced anthocyanin accumulation in regeneratedshoots. Five independent Lc lines were investigated forintegration of Lc into the plant genome by Southern blotand PCR analyses. The Lc-transgenic lines contained one ortwo Lc gene copies and showed increased mRNA levels forphenylalanine ammonia-lyase (PAL), chalcone synthase(CHS), Xavanone 3 beta-hydroxylase (FHT), dihydroXavonol4-reductase (DFR), leucoanthocyanidin reductases (LAR),anthocyanidin synthase (ANS) and anthocyanidin reductase(ANR). HPLC-DAD and LC-MS analyses revealed higherlevels of the anthocyanin idaein (12-fold), the Xavan 3-olepicatechin (14-fold), and especially the isomeric catechin(41-fold), and some distinct dimeric proanthocyanidins(7 to 134-fold) in leaf tissues of Lc-transgenic lines. Thelevels of phenylpropanoids and their derivatives were onlyslightly increased. Thus, Lc overexpression in Malusdomestica resulted in enhanced biosynthesis of speciWcXavonoid classes, which play important roles in both phyto-pathology and human health.
KeywordsAgrobacterium · Flavonoids · Metabolic Engineering · Transformation
AbbreviationsANSAnthocyanidin synthaseANRAnthocyanidin reductaseBAP6-BenzylaminopurineCaMVCauliXower mosaic virusCHIChalcone isomeraseCHSChalcone synthaseDADDiode array detectionDFRDihydroXavonol 4-reductaseFGTUDP-Glucose:Xavonoid 3-O-glucosyltransferaseFHTFlavanon 3 beta-hydroxylaseFLSFlavonol synthaseGAGibberellic acidHPLCHigh performance liquid chromatographyHCHolsteiner Cox
H. Li · I. Szankowski (&)Institute of Biological Production Systems, Fruit Science Section, Leibniz University of Hannover, Herrenhaeuser Str. 2, 30419 Hannover, Germanye-mail: Iris.Szankowski@obst.uni-hannover.de
H. Flachowsky · M.-V. HankeFederal Centre for Breeding Research on Cultivated Plants, Institute of Fruit Breeding, Pillnitzer Platz 3a, 01326 Dresden, Germany
T. C. Fischer · G. ForkmannChair for Ornamental Plants and Horticultural Plant Breeding, Department for Plant Sciences, Technical University Munich, Am Hochanger 4, 85350 Freising, Germany
D. TreutterUnit of Fruit Science, Department for Plant Sciences, Technical University of Munich, Alte Akademie 16, 85350 Freising, Germany
W. Schwab · T. HoVmannBiomolecular Food Technology, Technical University of Munich, Lise-Meitner-Str. 34, 85354 Freising, Germany1244Planta (2007) 226:1243–1254123IBAIndole-3-butyric acidLAR1+2Leucoanthocyanidin reductaseLcMaize leaf colourLC-MSLiquid chromatography/mass spectroscopyMSMurashige and SkoogPALPhenylalanin ammonia-lyaseTDZThidiazuronYEPYeast extract broth
IntroductionFlavonoids are plant polyphenolic compounds derived fromthe phenylpropanoid pathway. On the basis of theirstructure they can be classiWed into the classes Xavanones,Xavones, isoXavones, dihydroXavonols, Xavonols, leuco-anthocyanidins, Xavan 3-ols (catechins and the polymericproanthocyanidins) and anthocyanins. Representatives ofmost of these classes are also found in Malus domestica(Fig.1). Chalcones are direct precursors of all Xavonoids.The prominent dihydrochalcones present in M. domesticaare chemical derivatives of them. Flavonoids have manifoldfunctions in plants such as UV-protection, attraction ofpollinators, warranty of pollen fertility, regulation of polarauxin transport (Taylor and Grotewold 2005), establish-ment of microbial symbioses and pathogen defence (Treut-ter 2005). In fruit crops such as M. domestica, Xavonoids