CN113957078A - 耐除草剂抗虫转基因大豆京豆323培育及特异性检测方法 - Google Patents

耐除草剂抗虫转基因大豆京豆323培育及特异性检测方法 Download PDF

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CN113957078A
CN113957078A CN202111039551.5A CN202111039551A CN113957078A CN 113957078 A CN113957078 A CN 113957078A CN 202111039551 A CN202111039551 A CN 202111039551A CN 113957078 A CN113957078 A CN 113957078A
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邱丽娟
郭勇
郭兵福
张丽娟
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Abstract

本发明提供了一种耐除草剂抗虫转基因大豆培育及特异性检测方法，为将外源g2m‑epsps，gr79m‑epsps和cry1C基因构建的表达框插入到目标大豆的13号染色体的15122549‑15122594位之间，得到转基因大豆。本发明的耐除草剂抗虫转基因大豆可进一步与其他优良大豆株系杂交，对其他农艺性状如产量、品质等性状进行改良。根据外源基因插入位点的侧翼序列，建立了特异性检测该转化体插入位点的方法，利用这一特异性检测方法可以鉴定插入的T‑DNA和植物基因组DNA的结合区域，从而对这一转基因大豆及其衍生物进行鉴定。

Description

耐除草剂抗虫转基因大豆京豆323培育及特异性检测方法
技术领域
本发明涉及大豆转基因领域，特别涉及耐除草剂抗虫转基因大豆京豆323培育及特异性检测方法。
背景技术
大豆是一年生草本植物，是世界上最重要的豆类。大豆起源于中国，中国学者大多认为原产地是云贵高原一带。也有很多植物学家认为是由原产中国的乌苏里大豆衍生而来。现种植的栽培大豆是从野生大豆通过长期定向选择、改良驯化而成的。由于大豆的先天性的缺点，现在很多会对大豆进行品种改良，转基因就是一个很好的方法。
发明内容
本发明提供了一种耐除草剂抗虫转基因大豆京豆323培育及特异性检测方法，其能够防止害虫侵害，具有好的抗倒伏和抗病能力，方便推广。
为了达到上述发明目的，本发明采用的技术方案为：
一种耐除草剂抗虫转基因大豆京豆323的培育方法，利用农杆菌介导法向目标大豆内引入了抗草甘膦基因g2m-epsps，gr79m-epsps和抗虫基因cry1C得到转基因大豆京豆323，所述方法具体为：
g2m-epsps，gr79m-epsps和cry1C基因以pGR18为载体插入到目标大豆的13号染色体，外源基因插入位点为大豆基因组第13号染色体的15122549-15122594之间；
转基因大豆京豆3235’端侧翼序列位于大豆参考基因组6号染色体的15122061…15122549位，3’端侧翼序列位于大豆参考基因组19号染色体的15122594…15123061位。
作为改进，所述G2M-EPSPS基因表达框位置为6265-8837，包括35S启动子808bp、G2M-EPSPS基因 1335bp和PolyA终止子201bp；所述Cry1C基因表达框位置为9070-12135，35S启动子835bp、CrylC基因1893bp和NOS终止子253bp组成；GR79M-EPSPS基因表达框位置为12370-16167，包括pUbi启动子2036bp、 GR79M-EPSPS基因1338bp和PolyA终止子196bp。
作为改进，所述大豆参考基因组6号染色体的15122061…15122549位的序列具体为： AGTAAGTTTCTTGTTCTTTTTATAATTTTCATATATATATATATATGAAAACTCAAGTGGGCACGAGAAACAATAGAACCCGGGTCCACAA TCACCATTAGACGAGCACTCGTCATTTAGCTGATCAAAGATTAGAAAACAATATGATACAAAGTTAAAAACTAATAATGGACAACTATATT AAGAGAGAAGAACAAAAAGTACAAATTAACCAATAACCAGTTTATGTATGCCCAAAACAGAGATGGATAAAATATTATTTTATAATTCAAT AAAGTTGTATATGAAATTTACATGTGCATATCAAAAACAAAATTGATTGGCTTATGAGTATAATAAATTGATAATAATTAAATAAGATTGT TGTATTGTCTATTCGTTTGAGTTCTCACTTATATTATAAATTAATGATGCAACAATCATATCCATATCCATCAATTCAAGATTCATGGCTG ATCCGTATCTGTACAACAACAAGAAAATGGATGC。
作为改进，所述大豆参考基因组19号染色体的15122594…15123061位的序列具体为：TTCCAGGAAAAAAAAACAAATATTTCAACTATAACAAGTTAATATCAGAAAAGAAGGATTCGAATAATAAATCTTGGATGGCATAATCTAA ACTTTCATAATTAGCAAGTTTTTTACGTATTACATGTATTTTTTATGTTTAAATTAAATTGCGTATCGAAAGTTTAGTTAAAACAGTGTTT TAACCTTTATTTTTTTTTTAAAAAAAAAGACTAAACGTGAAAAAAAAAAAAACACAACGAAATAGCTTACTTACGAGATACATAAAGTATT TAATTATGTTTAAGATGATCTTTCAAGTTGCCTTTATATGCCCTTTTCTTTATAAAAAAAGAAAAAAATATGTTTCAAATGGATTACATTA TAAAAAATGGTCCAGCCATGAATTATGAATTCTAAATTATTAGGTTTAAAAAATGGATGGTAAAGACTTGTGATAGACAGCTTGCTTACGA GATATATAAAGTA。
一种耐除草剂抗虫转基因大豆京豆323的特异性检测方法，所述的检测方法为：
S1：利用来源于京豆323插入位点两侧大豆基因组的引物JD323P-1和JD323P-2和来源于外源基因的引物GR18LB-R和GR18RB-F，分别组成可以特异性检测京豆323大豆中5’插入位置的引物对JD323P-1 和GR18LB-R以及特异性检测3’插入位置的引物对GR18RB-F和JD323P-2，进行进行PCR反应；
S2：设置对照组：无模板对照组和受体Jack对照组；
S3：基因组的整合分析，观察两个引物对及两个对照组是否扩增条带。
作为改进，所述PCR反应的反应条件为：94℃变性4min，94℃变性30s，60℃退火30s，72℃延伸30s， 35个循环。
作为改进，所述引物JD323P-1的序列为GTTGTATTGTCTATTCGTTTGAGTTCT，所述引物JD323P-2的序列为TCGTAAGTAAGCTATTTCGTTGTGTT，所述引物GR18LB-R的序列为TGCCAAGACTTACCCTGATTACT，所述引物 GR18RB-F的序列为GGGTCAAGGTTCCGCACATT。
一种用于检测样品中存在转基因大豆京豆323植物材料的试剂盒，所述的试剂盒包括权利要求1中所述的外源基因、权利要求5中所述的引物对。
本发明的有益效果为：
本发明通过转基因和特异性检测方法表明，本转基因方法能够在大豆中进行稳定遗传和稳定表达，且具有好的防止害虫侵害得能力，同时具有好的抗病能力。
附图说明
图1为pGR18载体图谱；
图2为转基因大豆京豆323T3代材料草甘膦抗性鉴定图；
图3为转基因大豆京豆323T4代材料草甘膦抗性鉴定；
图4为京豆323转基因大豆T3代(A)和T4代(B)植株对靶标害虫的抗性鉴定图；
图5为转基因材料京豆323在各世代、各组织中的表达分析图；
图6为京豆323插入序列结构及在大豆13号染色体上的相对位置图；
图7为特异性检测引物在转基因大豆京豆323基因组中的相对位置图；
图8为京豆323转基因大豆5’端(A)和3’端(B)插入位点特异性PCR检测图。
具体实施方式
下面结合附图来进一步说明本发明的具体实施方式。其中相同的零部件用相同的附图标记表示。
为了使本发明的内容更容易被清楚地理解，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。
如图1-8所示，利用农杆菌介导法引入了抗草甘膦基因g2m-epsps，gr79m-epsps和抗虫基因cry1C。在转基因植物中，植物细胞过量合成G2M-EPSPS和GR79-EPSPS酶，在草甘膦存在条件下它能在植物体内替代本身被草甘膦结合的EPSPS酶执行功能，使植物免受草甘膦的毒害。因此转G2m-EPSPS基因大豆可大幅度提高对除草剂的耐受性。在转基因植物中过量表达抗逆基因Cry1C，能增强植物对鳞翅目害虫的抗性。
转化用的载体g2m-epsps，gr79m-epsps和cry1C基因转化大豆所用载体是pGR18(图1)。所用载体除靶标基因外，无筛选标记基因和报告基因。留下的对细菌的选择标记基因卡那霉素抗性基因kanR不在 T-DNA中，所以不能插入植物基因组，而仅仅作为载体构建时在大肠杆菌及农杆菌生长时的选择标记。载体中各种元件分别来源于大肠杆菌和农杆粒的质粒。载体无致病性，也无演变为致病性的可能性。
转基因方法：
采用农杆菌介导大豆子叶节转化法，农杆菌菌株为Ag10。大豆子叶节消毒后诱导愈伤，然后与农杆菌共培养以感染愈伤，筛选被转化的愈伤，之后在选择培养上进行植株再生。
无菌外植体的获得：选用正常萌发无污染的幼苗制备外植体。在超净工作台中用手术刀沿下胚轴将大豆切下，保留3-4mm的下胚轴，置于无菌培养皿中，皿中添加适量的共培养液以方便剥离种皮，再沿子叶下胚轴垂直将胚轴切开，剔除干净真叶组织，在子叶和子叶下胚轴的连接处轴向作5-7个切口，切口约3-4mm 长。每个外植体是由一片子叶连着一段下胚轴组成，一枚种子可以形成两个外植体。
农杆菌的准备：从超低温冰箱中取出低温保藏的农杆菌菌株于冰上冻融，用接种环或无菌枪头蘸取少量的菌种接种于LB平板上，LB固体培养基含50mg/L对应的抗生素。置于28℃恒温条件下倒置培养1-2d 以获得单克隆。2d后，挑取单克隆接种于5mlYEP培养液中(含对应抗生素)于220rpm、28℃过夜活化培养(约12h)。当菌液第一次活化至饱和状态时，从中抽取1ml菌液接种至含100mlYEP(含对应抗生素) 的三角摇瓶中，在28℃，220rpm条件下第二次活化。待农杆菌充分活化至OD600＝1.0左右时，将菌液于 4000rpm、4℃条件下离心10min，弃上清收集沉淀，用等体积共培养液悬浮沉淀于管底的菌体，此时OD600 约为0.5-0.8左右，备用。
外植体与农杆菌共培养：外植体制备时，每40-60片外植体置于一个100ml的三角瓶中，每瓶添加约 50ml重悬的农杆菌菌液，菌液需漠过外植体。于黑暗或弱光条件下共侵染30-35min，每隔5min轻摇三角瓶一次以使农杆菌和外植体充分接触。侵染完成后小心倒掉多余的农杆菌菌液，在共培养培养基上平铺一层无菌滤纸，将浸染后的外植体向轴一侧朝下平铺于滤纸上，24℃，黑暗或弱光条件下共培养3d。
抗性筛选与再生：共培养3d后，外植体经抗性丛生芽诱导、伸长芽诱导、生根等阶段获得再生植株；在丛生芽诱导和伸长阶段分别添加适宜的抗性筛选剂，生根阶段根据实验时的实际需求，添加适宜浓度的农杆菌抑菌剂及IBA以诱导生根，丛生芽诱导和伸长芽诱导阶段每两周继代外植体一次，继代时在外植体的背面重新制备一新的切口以使外植体能更好地吸收养分。伸长芽伸长至4-6cm，可诱导生根，生根后先揭开培养皿上的封口膜开口炼苗1-3天；再移栽至盆栽或大田中生长，获得转基因植株。
对T3-T4代京豆323转基因大豆及非转基因大豆Jack进行田间草甘膦抗性鉴定。使用41％草甘膦水剂，手持式压力喷雾器为美国Hudson公司。在第一至第三复叶期，对其喷施6L/公顷(400ml/亩)的草甘膦异丙胺盐(Roundup)溶液，2周后调查药害。结果表明在上述喷施剂量下，Jack不具备抗性，2周后植株全部死亡；T3代纯合的转基因大豆和京豆323株系生长不受抑制，叶片不褪绿、不皱缩，无新叶黄化等草甘膦药害反应。这表明转基因大豆京豆323对草甘膦表现为高抗(图2)。同样的，T4代纯合的转基因大豆京豆323株系也表现出生长不受抑制，叶片不褪绿、不皱缩，无新叶黄化等草甘膦药害反应(图3)。说明京豆323转基因大豆中草甘膦的抗性可以在不同的世代稳定遗传。
采集T3和T4代转基因大豆植株上健康、鲜嫩程度一致的展开叶片，用打孔器将叶片打成2.5cm圆片，选取6片放入铺有海绵培养皿内，以13mL无菌水保湿，按1头/皿的比例接入2龄斜纹夜蛾幼虫,，接虫后每天调察叶片损害情况(拍照)，7天后进行结果调查分析。结果表明，受体对照Jack叶片损伤严重，而京豆323的T3和T4代植株对斜纹夜蛾都表现高抗(图4)。说明京豆323转基因大豆中抗虫性可以在不同的世代稳定遗传。
利用TRIzol试剂盒(Invitrogen)提取大豆不同组织总RNA。将转基因大豆的根、茎、叶和种子分别在液氮中充分研磨至细粉状，取100mg粉末至1.5mL的离心管中，每管加入1mLTRIzol提取液，剧烈振荡混匀，室温静置5min；加入200μL氯仿，剧烈震荡混匀15s，室温放置5min；4℃，12000rpm离心5min，将600μL上清液转移至新的离心管中；每管加入0.5mL异丙醇，混匀，室温静置10min；4℃，12000rpm 离心10min，倒掉上清液；加入1mL75％乙醇洗涤沉淀，4℃，12000rpm离心5min；弃去上清液，RNA沉淀室温干燥5-10min；加入200μLRNasefreeH2O，室温放置20min；加入20μL3mol/L醋酸钠，再加入660μ L乙醇，混匀，-20℃放置至少2h；取出，4℃，12000rpm离心5min；倒掉上清，加入1mL75％乙醇洗涤沉淀，4℃，12000rpm离心5min；RNA沉淀室温干燥5-10min；加入50μLRNasefreeH2O室温放置15-20min；置-80℃保存备用。
提取的RNA分别采用1.0％琼脂糖凝胶电泳和紫外分光光度计法进行检测。琼脂糖凝胶电泳检测时，若分离的总RNA呈现三条清晰的条带且靠近点样孔条带的亮度约为第二条带亮度的2倍时(28S:18S＝2:1)，表示RNA相对完整，没有被降解。
RNA的纯化选用TURBODNA-free试剂盒(Ambion)，所有操作在冰上完成。在1.5mL离心管中加入 10μgRNA、2.5μL10×Buffer、0.5μLDNaseI和0.25μLRNaseInhibitor，再补水至25μL，于37℃水浴1h。消化后加入2.5μLDNaseinactivatingreagent，混匀，室温放置5min；4℃，12000rpm离心3min，转移上清液至新的离心管中，于-80℃保存备用。
1st-strandcDNA的合成反应利用TaKaRa公司生产的 PrimeScriptTMII1stStrandcDNASynthesisKit(6210A)合成cDNA。参照说明书，cDNA的合成反应采用2步法。第一步，每200μL的PCR管中依次加入1μgRNA、1μLOligodT(50μm)、1μLdNTP，补水至10μL后于 65℃反应5min，再迅速转移至冰上。第二步反应在冰上进行，每200μL的PCR管依次加入10μL第一步反应液、4μL5×PrimeScriptIIBuffer、0.5μL(20U)RNaseInhibitor、1μL(200U)PrimeScriptIIRTase，再补水至20μL；离心混匀后依次于30℃保温10min，42℃保温60min，70℃保温15min，反应结束后将cDNA 稀释50倍备用。
PCR反应在Bio-rad的T100TMThermalCycler仪上进行。PCR反应体系25μL，包括ExTaq0.2μL，10 ×ExTaqBuffer2.5μL，dNTP2μL，上游引物(10mM)0.5μL，下游引物(10mM)0.5μL，cDNA5-15μL(根据Actin扩增结果上下调整模板浓度一致)，加水补足至25μL。反应程序采用三步法，包括95℃保温5min；再进行28个循环的扩增(95℃，30sec；58℃，30sec；72℃，30sec)；最后72℃延伸5min。扩增后样品加入5μL6×LoadingBuffer，使用1.5％琼脂糖凝胶电泳(含溴化乙锭)检测，5V/cm电泳30min，紫外灯下观察照相。
相关引物信息表：
Figure RE-GDA0003418654600000051
提取T3、T4代转基因材料的根、茎、叶和种子的RNA，反转录后以ACTIN基因为内参对G2m-EPSPS、 GR79m-EPSPS和Cry1C基因进行RT-PCR检测，结果表明G2m-EPSPS、GR79m-EPSPS和Cry1C基因在京豆323 T3、T4代转基因材料的各个组织中均有表达。而且三个基因均是在根、茎、叶中表达量较高，种子中表达量较低。说明外源基因可以在转基因材料中稳定表达，如图5所示：
A-D：ACTIN(A)、G2m-EPSPS(B)、GR79m-EPSPS(C)和Cry1C(D)基因在转基因材料京豆323中的T3代表达水平情况；E-H：ACTIN(E)、G2m-EPSPS(F)、GR79m-EPSPS(G)和Cry1C(H)基因在转基因材料京豆323 中的T4代表达水平情况；M：DNA marker；1-4：京豆323的根(1)、茎(2)、叶(3)和种子(4)。
插入序列的大小和结构
京豆323大豆中外源基因插入到13号染色体。设计特异性引物对插入位点进行PCR检测和测序分析，明确了外源基因插入位点为大豆基因组第13号染色体15122549-15122594之间(图6)，且转基因大豆京豆323中T-DNA序列与其质粒载体GR18的相应遗传元件的排列顺序是一致的。
通过测序获得京豆323大豆5’端侧翼序列位于大豆参考基因组(Glycine maxWm82.a2.v1)6号染色体的15122061…15122549位；
3’端侧翼序列位于大豆参考基因组(Glycine max Wm82.a2.v1)19号染色体的15122594…15123061 位；
京豆323大豆中插入的T-DNA片段都包括三个基因元件的表达框：G2M-EPSPS基因表达框(位置 6265-8837)，包括35S启动子808bp、G2M-EPSPS基因1335bp和PolyA终止子201bp；Cry1C基因表达框 (位置9070-12135)，35S启动子(835bp)、CrylC基因(1893bp)和NOS终止子(253bp)组成；GR79M-EPSPS 基因表达框(位置12370-16167)，包括pUbi启动子2036bp、GR79M-EPSPS基因1338bp和PolyA终止子 196bp。
确定特性的分析方法
利用来源于京豆323插入位点两侧大豆基因组的引物(JD323P-1和JD323P-2)和来源于外源基因的引物(GR18LB-R和GR18RB-F)，分别组成可以特异性检测京豆323大豆中5’插入位置的引物对1(JD323P-1 和GR18LB-R)以及特异性检测3’插入位置的引物对2(GR18RB-F和JD323P-2)，每个引物的位置和扩增片段大小如图所示，扩增产物的序列如前所述。PCR反应条件：94℃变性4min，94℃变性30s，60℃退火30s，72℃延伸30s，35个循环。如预期，无模板对照(泳道1)和受体Jack(泳道2)无PCR产物产生。京豆323(泳道3-5)利用位于插入位点5’端的引物对1可以扩增出486bp的条带，利用位于插入位点3’端的引物对2可以扩增出712bp的条带(图8)。
图8中的A：引物对1(JD323P-1和GR18LB-R)的扩增结果；B：引物对2(GR18RB-F和JD323P-2) 的扩增结果。M：DNA marker；1：无菌水对照；2：Jack受体对照；3-5：京豆323不同的阳性单株。
基因组的整合分析可采用插入位点特异性PCR和RT-PCR等方法进行分析。此外，G2M-EPSPS基因和 GR79M-EPSPS基因赋予转基因植物除草剂抗性，可以用草甘膦抗性进行检测；直接喷施200ml/亩草甘膦可以杀死全部非转基因大豆，而本项目的转基因大豆无伤害；同时Cry1C基因的抗性可以通过对鳞翅目害虫的接种鉴定试验鉴定出来。
以上所述仅为本发明专利的较佳实施例而已，并不用以限制本发明专利，凡在本发明专利的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明专利的保护范围之内。
Figure RE-GDA0003418654600000071
Figure RE-GDA0003418654600000081
Figure RE-GDA0003418654600000091
Figure RE-GDA0003418654600000101
Figure RE-GDA0003418654600000111
Figure RE-GDA0003418654600000121
Figure RE-GDA0003418654600000131
Figure RE-GDA0003418654600000141
Figure RE-GDA0003418654600000151
Figure RE-GDA0003418654600000161
Figure RE-GDA0003418654600000171
Figure RE-GDA0003418654600000181
Figure RE-GDA0003418654600000191
Figure RE-GDA0003418654600000201
Figure RE-GDA0003418654600000211
Figure RE-GDA0003418654600000221
Figure RE-GDA0003418654600000231
Figure RE-GDA0003418654600000241
Figure RE-GDA0003418654600000251
序列表
<110> 中国农业科学院作物科学研究所
<120> 耐除草剂抗虫转基因大豆京豆323培育及特异性检测方法
<130> 2020.12.10
<160> 6
<170> SIPOSequenceListing 1.0
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cagctcatca acgaaagaat cgctgagttc gctaggaatg ctgctattgc taaccttgaa 300
ggacttggaa acaacttcaa catctacgtg gaggcattca aggaatggga agaagatcct 360
aacaacccag caaccaggac cagagtgatc gataggttcc gtatccttga tggacttctt 420
gaaagggaca ttcctagctt taggatctct ggatttgaag ttccacttct ctctgtttac 480
gctcaagctg ctaatctcca tcttgctatc cttagagatt ctgtgatctt cggagaaaga 540
tggggattga caaccatcaa cgtgaacgag aactacaaca gactcatcag gcacatcgat 600
gagtacgctg atcactgtgc taacacttac aaccgtggac tcaacaacct tcctaagtct 660
acctatcaag attggatcac atacaaccga cttaggagag accttacatt gactgttctt 720
gatatcgctg ctttctttcc aaactatgac aataggagat atccaattca gccagttggt 780
caacttacaa gggaagttta cactgaccca ctcatcaact tcaacccaca gcttcagtct 840
gttgctcagc ttcctacctt caacgttatg gagagcagcg caatcagaaa tcctcacctc 900
ttcgacatct tgaacaacct tacaatcttt accgattggt ttagtgttgg acgtaacttc 960
tactggggag gacatcgagt gatctctagc ctcatcggag gtggtaacat cacatctcct 1020
atctacggaa gagaggctaa ccaggagcct ccaagatcat tcactttcaa cggacctgtg 1080
ttcaggactc tttcaaatcc tactcttcga cttcttcagc aaccttggcc agctccacca 1140
ttcaaccttc gtggtgttga aggagttgag ttctctacac ctacaaacag cttcacctat 1200
cgtggaagag gtactgttga ttctcttact gaacttccac ctgaggacaa cagtgtgcca 1260
cctcgtgaag gatacagtca tcgtctttgt catgcaacct tcgttcaaag atctggaaca 1320
cctttcctta caactggtgt tgtgttctct tggactcatc gtagtgcaac tcttaccaac 1380
acaattgatc cagagaggat caaccagatc cctcttgtga aaggattcag agtttgggga 1440
ggaacctctg tgattacagg accaggattc acaggaggtg atatccttcg aagaaacacc 1500
tttggtgact tcgtttctct tcaagtgaac atcaactcac caatcaccca aagataccgt 1560
cttagatttc gttacgcttc tagtagggat gcacgagtta tcgttcttac aggagctgca 1620
tctacaggag tgggaggtca agttagtgtg aacatgcctc ttcagaaaac tatggagatc 1680
ggagagaacc tcacatctag aacattcaga tacaccgact tcagtaatcc tttctcattc 1740
agagctaatc cagacatcat cggtatcagt gaacaacctc tcttcggtgc aggttctatc 1800
agtagcggtg aactttacat cgacaagatc gagatcatcc ttgcagatgc aacatttgaa 1860
gcagaatctg accttgaaag agcacaaaag tag 1893
<210> 6
<211> 630
<212> PRT
<213> 人工合成(Artificial Sequence)
<400> 6
Met Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser
1 5 10 15
Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn
20 25 30
Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn
35 40 45
Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp
50 55 60
Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu
65 70 75 80
Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile
85 90 95
Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala
100 105 110
Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Ala Thr Arg Thr Arg
115 120 125
Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile
130 135 140
Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr
145 150 155 160
Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile
165 170 175
Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr
180 185 190
Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn
195 200 205
Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp
210 215 220
Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu
225 230 235 240
Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile
245 250 255
Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile
260 265 270
Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn
275 280 285
Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu
290 295 300
Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe
305 310 315 320
Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn
325 330 335
Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg
340 345 350
Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr
355 360 365
Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg
370 375 380
Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr
385 390 395 400
Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Xaa
405 410 415
Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala
420 425 430
Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val
435 440 445
Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro
450 455 460
Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly
465 470 475 480
Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu
485 490 495
Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn
500 505 510
Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser
515 520 525
Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val
530 535 540
Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile
545 550 555 560
Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn
565 570 575
Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln
580 585 590
Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp
595 600 605
Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp
610 615 620
Leu Glu Arg Ala Gln Lys
625 630

Claims (8)

1.一种耐除草剂抗虫转基因大豆京豆323的培育方法，其特征在于，利用农杆菌介导法向目标大豆内引入了抗草甘膦基因g2m-epsps，gr79m-epsps和抗虫基因cry1C得到转基因大豆京豆323，所述方法具体为：
g2m-epsps，gr79m-epsps和cry1C基因以pGR18为载体插入到目标大豆的13号染色体，外源基因插入位点为大豆基因组第13号染色体的15122549-15122594之间；
转基因大豆京豆3235’端侧翼序列位于大豆参考基因组6号染色体的15122061…15122549位，3’端侧翼序列位于大豆参考基因组19号染色体的15122594…15123061位。
2.根据权利要求1所述的一种耐除草剂抗虫转基因大豆京豆323的培育方法，其特征在于，所述G2M-EPSPS基因表达框位置为6265-8837，包括35S启动子808bp、G2M-EPSPS基因1335bp和PolyA终止子201bp；所述Cry1C基因表达框位置为9070-12135，35S启动子835bp、CrylC基因1893bp和NOS终止子253bp组成；GR79M-EPSPS基因表达框位置为12370-16167，包括pUbi启动子2036bp、GR79M-EPSPS基因1338bp和PolyA终止子196bp。
3.根据权利要求1所述的一种耐除草剂抗虫转基因大豆京豆323的培育方法，其特征在于，所述大豆参考基因组6号染色体的15122061…15122549位的序列具体为：
AGTAAGTTTCTTGTTCTTTTTATAATTTTCATATATATATATATATGAAAACTCAAGTGGGCACGAGAAACAATAGAACCCGGGTCCACAATCACCATTAGACGAGCACTCGTCATTTAGCTGATCAAAGATTAGAAAACAATATGATACAAAGTTAAAAACTAATAATGGACAACTATATTAAGAGAGAAGAACAAAAAGTACAAATTAACCAATAACCAGTTTATGTATGCCCAAAACAGAGATGGATAAAATATTATTTTATAATTCAATAAAGTTGTATATGAAATTTACATGTGCATATCAAAAACAAAATTGATTGGCTTATGAGTATAATAAATTGATAATAATTAAATAAGATTGTTGTATTGTCTATTCGTTTGAGTTCTCACTTATATTATAAATTAATGATGCAACAATCATATCCATATCCATCAATTCAAGATTCATGGCTGATCCGTATCTGTACAACAACAAGAAAATGGATGC。
4.根据权利要求1所述的一种耐除草剂抗虫转基因大豆京豆323的培育方法，其特征在于，所述大豆参考基因组19号染色体的15122594…15123061位的序列具体为：
TTCCAGGAAAAAAAAACAAATATTTCAACTATAACAAGTTAATATCAGAAAAGAAGGATTCGAATAATAAATCTTGGATGGCATAATCTAAACTTTCATAATTAGCAAGTTTTTTACGTATTACATGTATTTTTTATGTTTAAATTAAATTGCGTATCGAAAGTTTAGTTAAAACAGTGTTTTAACCTTTATTTTTTTTTTAAAAAAAAAGACTAAACGTGAAAAAAAAAAAAACACAACGAAATAGCTTACTTACGAGATACATAAAGTATTTAATTATGTTTAAGATGATCTTTCAAGTTGCCTTTATATGCCCTTTTCTTTATAAAAAAAGAAAAAAATATGTTTCAAATGGATTACATTATAAAAAATGGTCCAGCCATGAATTATGAATTCTAAATTATTAGGTTTAAAAAATGGATGGTAAAGACTTGTGATAGACAGCTTGCTTACGAGATATATAAAGTA。
5.一种耐除草剂抗虫转基因大豆京豆323的特异性检测方法，其特征在于：所述的检测方法为：
S1：利用来源于京豆323插入位点两侧大豆基因组的引物JD323P-1和JD323P-2和来源于外源基因的引物GR18LB-R和GR18RB-F，分别组成可以特异性检测京豆323大豆中5’插入位置的引物对JD323P-1和GR18LB-R以及特异性检测3’插入位置的引物对GR18RB-F和JD323P-2，进行进行PCR反应；
S2：设置对照组：无模板对照组和受体Jack对照组；
S3：基因组的整合分析，观察两个引物对及两个对照组是否扩增条带。
6.根据权利要求5所述的一种耐除草剂抗虫转基因大豆京豆323的特异性检测方法，其特征在于：所述PCR反应的反应条件为：94℃变性4min，94℃变性30s，60℃退火30s，72℃延伸30s，35个循环。
7.根据权利要求5所述的一种耐除草剂抗虫转基因大豆京豆323的特异性检测方法，其特征在于：所述引物JD323P-1的序列为GTTGTATTGTCTATTCGTTTGAGTTCT，所述引物JD323P-2的序列为TCGTAAGTAAGCTATTTCGTTGTGTT，所述引物GR18LB-R的序列为TGCCAAGACTTACCCTGATTACT，所述引物GR18RB-F的序列为GGGTCAAGGTTCCGCACATT。
8.一种用于检测样品中存在转基因大豆京豆323植物材料的试剂盒，其特征在于：所述的试剂盒包括权利要求1中所述的外源基因、权利要求5中所述的引物对。
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