CN107022562A - 利用CRISPR/Cas9系统对玉米基因定点突变的方法 - Google Patents
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Abstract
本发明提供利用CRISPR/Cas9系统对玉米基因定点突变的方法,针对玉米中的目标基因设计基于CRISPR/Cas9的sgRNA序列,将含有编码所述sgRNA序列的DNA片段连接到携带CRISPR/Cas的载体中,转化玉米,实现对玉米中特定基因的定点突变。进一步地,通过遗传转化的方法将含有CRISPR/Cas9的载体转入携带目标基因的受体材料中,获得该目标基因定点突变的再生植株。利用本方法可实现对玉米基因组的定点突变,本方法具有实验周期短、操作简便等特点,利用不同靶向的CRISPR/Cas9系统可对不同目标基因进行定点定向改造,为玉米改良育种提供了新方法,对于改良玉米性状具有重大实际意义。
Description
技术领域
本发明涉及植物转基因技术领域和作物遗传育种领域,具体地说,涉及一种利用CRISPR/Cas9系统对玉米基因定点突变的方法。
背景技术
随着生物技术的不断发展,越来越多的新型育种技术不断涌现。传统育种技术周期长,难以对目标形状作定点定向改良。近些年兴起的生物技术育种很大程度上缩短了育种周期,可有目的地引入新性状或改良固有性状。
CRISPR(clustered regularly interspersed short palindromic repeats)/Cas是源于细菌及古细菌中的一种后天免疫系统。它利用靶位点特异性的RNA指引Cas蛋白对靶位点序列进行修饰,进而在靶标序列上形成各种类型的突变。自2013年以来,CRISPR/Cas系统已经成功应用于人类、小鼠、斑马鱼、家蚕、果蝇、酵母、拟南芥及水稻、大豆等多个物种中。
在医学上,该系统可用于某些疾病的治疗。在基础生物学领域,该方法可用于基因功能研究。近年来有学者开始应用该系统对作物内源基因进行改良,用于产生新的育种材料。CRISPR/Cas系统由于其可对靶标位点进行特异性改变而受到高度关注,且定点改变后的作物在第二代即可获得突变基因纯合体,大大缩短了育种周期。玉米是世界上最重要的粮食和饲料作物,目前尚未发现在玉米育种中应用CRISPR/Cas系统的报道。在玉米种质改良上应用该技术具有重要价值。
发明内容
本发明的目的是提供利用CRISPR/Cas9系统对玉米基因定点突变的方法。
本发明的另一目的是提供上述方法在玉米基因定点突变育种中的应用。
为了实现本发明目的,本发明提供利用CRISPR/Cas9系统对玉米基因定点突变的方法。为此,针对玉米中的目标基因设计基于CRISPR/Cas9的sgRNA序列,将含有编码所述sgRNA序列的DNA片段连接到携带CRISPR/Cas的载体中,转化玉米,实现对玉米中特定基因的定点突变。
本发明中所述玉米包括自交种、杂交种、普通玉米、甜玉米、糯玉米等。优选地,所述玉米为祥249自交系。本发明中涉及的目标基因可以是本领域技术人员感兴趣的基因或有生物学功能的任何DNA序列,包括玉米基因组中任何有生物学功能的基因或DNA序列,或者是希望定点改良的任何基因,例如GFP基因。
针对玉米祥249自交系转基因植株中GFP基因设计基于CRISPR/Cas9的sgRNA序列,其中,sgRNA作用位点的核苷酸序列为5’-ACCGGGGTGGTGCCCATCC-3’。
前述的方法,将含有编码所述sgRNA序列的DNA片段连接到携带CRISPR/Cas的载体中,构建得到载体Cas9-GFP-gRNA,用载体Cas9-GFP-gRNA转化玉米,实现对玉米GFP基因的定点突变/敲除。其中,载体Cas9-GFP-gRNA的全序列如SEQ ID NO:2所示。
本发明还提供所述方法在玉米基因定点突变育种中的应用。所述玉米包括但不限于祥249自交系。
所述应用包括以下步骤:
(1)将玉米的幼胚浸入携带CRISPR/Cas9和选择标记基因(例如CP4)的农杆菌菌液中侵染;
(2)将幼胚移至共培养培养基上培养;
(3)将幼胚移至愈伤诱导培养基上培养,诱导初级愈伤组织;
(4)将初级愈伤组织移至筛选培养基上培养,诱导抗性愈伤组织,再转移到分化培养基上,分化形成再生幼苗;
(5)再生幼苗在生根培养基上生根后炼苗、移栽,得到转基因玉米;
(6)根据sgRNA作用位点的核苷酸序列设计引物,通过PCR法鉴定玉米植株突变位点。
其中,所述共培养培养基的组成如下:1/2MS+蔗糖15-30g/L+葡萄糖8-15g/L+脯氨酸0.1-0.3g/L+盐酸硫胺素0.1-1.0mg/L+AgNO315-25μM+L-半胱氨酸100-300mg/L+2,4-D(2,4-二氯苯氧乙酸)0.3-1.0mg/L+毒莠定0.8-3.0mg/L+KT(6-糠基氨基嘌呤)0.01-1mg/L+乙酰丁香酮100-300μM+植物凝胶3-8g/L;优选地,共培养培养基的组成如下:1/2MS+蔗糖20g/L+葡萄糖10g/L+脯氨酸0.115g/L+盐酸硫胺素0.5mg/L+AgNO320μM+L-半胱氨酸200mg/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+KT 0.01-1mg/L+乙酰丁香酮200μM+植物凝胶8g/L;
所述愈伤诱导培养基的组成如下:MS+蔗糖15-30g/L+脯氨酸0.1-0.3g/L+盐酸硫胺素0.1-1.0mg/L+AgNO315-25μM+水解酪蛋白0.1-1.0g/L+2,4-D 0.3-1.0mg/L+毒莠定0.8-3.0mg/L+KT 0.01-1mg/L+特美汀100-300mg/L+植物凝胶3-8g/L;优选地,愈伤诱导培养基的组成如下:MS+蔗糖30g/L+脯氨酸1.38g/L+盐酸硫胺素0.5mg/L+AgNO320μM+水解酪蛋白0.5g/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+KT0.01-1mg/L+特美汀200mg/L+植物凝胶8g/L;
所述筛选培养基的组成如下:MS+蔗糖15-30g/L+脯氨酸0.1-0.3g/L+盐酸硫胺素0.1-1.0mg/L+AgNO315-25μM+水解酪蛋白0.1-1.0g/L+2,4-D 0.3-1.0mg/L+毒莠定0.8-3.0mg/L+特美汀100-300mg/L+草甘膦100-300mg/L+植物凝胶3-8g/L;优选地,筛选培养基的组成如下:MS+蔗糖30g/L+脯氨酸1.38g/L+盐酸硫胺素0.5mg/L+AgNO320μM+水解酪蛋白0.5g/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+特美汀200mg/L+草甘膦200mg/L+植物凝胶8g/L;
所述分化培养基包括分化培养基I和分化培养基II:
MS+蔗糖15-30g/L+硫酸铜3-15μM+MES(2-吗啉乙磺酸)0.3-0.8g/L+6-BA(6-苄氨基嘌呤)1.0-4.0mg/L+特美汀100-300mg/L+草甘膦3-15mg/L+植物凝胶3-8g/L;优选地,分化培养基I:MS+蔗糖20g/L+硫酸铜10μM+MES 0.5g/L+6-BA 3.5mg/L+特美汀200mg/L+草甘膦10mg/L+植物凝胶8g/L;
分化培养基II:MS+蔗糖15-30g/L+硫酸铜3-15μM+MES0.3-0.8g/L+特美汀100-300mg/L+草甘膦3-15mg/L+植物凝胶3-8g/L;优选地,分化培养基II:MS+蔗糖20g/L+硫酸铜10μM+MES 0.5g/L+特美汀200mg/L+草甘膦10mg/L+植物凝胶8g/L;
所述生根培养基的组成如下:MS+蔗糖15-30g/L+MES0.3-0.8g/L+IBA(吲哚丁酸)0.1-0.3mg/L+植物凝胶3-8g/L;优选地,生根培养基的组成如下:MS+蔗糖20g/L+MES 0.5g/L+IBA 0.2mg/L+植物凝胶8g/L。
前述的应用,步骤(1)中玉米的幼胚是从授粉后6-15天,待玉米幼胚长至0.5-2.0mm时,从玉米幼穗上剥取获得。
前述的应用,步骤(1)中将玉米的幼胚浸入如下侵染液中侵染5-15分钟。
其中,侵染液组成为:1/2MS+蔗糖40-80g/L+葡萄糖20-40g/L+L-脯氨酸0.1-0.3g/L+乙酰丁香酮100-300μM+OD600值0.1-0.5携带目的基因和选择标记基因(CP4基因)的农杆菌菌液;优选使用农杆菌菌株为EHA105;
前述的应用,步骤(2)中培养条件为:23℃黑暗培养3-5天;
前述的应用,步骤(3)中培养条件为:26-34℃黑暗培养5-14天。
本发明进一步提供利用上述方法获得的转化玉米细胞、植株部分和转基因植株及其种子和后代。
本发明提供利用CRISPR/Cas9系统对玉米进行定点突变的方法,通过遗传转化的方法将含有CRISPR/Cas9的载体转入携带目标基因的受体材料中,获得该目标基因定点突变的再生植株。利用该方法成功实现了对玉米基因组的定点突变,该方法具有实验周期短、操作简便等特点,利用不同靶向的CRISPR/Cas9系统可对不同目标基因进行定点定向改造,为玉米改良育种提供了新方法,对于改良玉米性状具有重大实际意义。
附图说明
图1为本发明实施例3中经两轮筛选培养结束后得到的两块玉米愈伤组织中GFP的表达情况;其中,A和C为白光灯下愈伤状态,B和D为紫外光下愈伤状态,圆圈标注处为抗性愈伤组织。从图中标注处可以看出两块抗性愈伤处均无GFP表达,而非抗性愈伤处有GFP表达。说明未转化细胞中的GFP基因依旧表达,而转化细胞中GFP基因不再表达,表明转化细胞中GFP基因已失活。
图2为本发明实施例3中GFP基因突变结果序列比对;其中,阳性对照为GFP基因部分序列,样品1-2为转化后植株GFP基因部分序列,从图中可以看出,样品1-2的GFP基因序列与阳性对照相比,在同一位置发生了一个腺嘌呤(A)碱基缺失,从而导致GFP基因功阅读框位移和失活。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
以下实施例中所用玉米材料为祥249自交系,由中国种子集团有限公司提供。
实施例1靶向玉米祥249自交系中GFP基因的CRISPR/Cas9系统的构建
1、针对玉米祥249自交系中GFP基因设计基于CRISPR/Cas9的sgRNA序列。其中,sgRNA作用位点的核苷酸序列为5’-ACCGGGGTGGTGCCCATCC-3’。
2、将含有编码所述sgRNA序列的DNA片段连接到携带CRISPR/Cas的载体中,构建得到载体Cas9-GFP-gRNA,用于转化玉米。其中,载体Cas9-GFP-gRNA的全序列如SEQ ID NO:2所示。
实施例2基因定点突变玉米植株的制备方法
1、玉米果穗处理和幼胚的分离
(1)携带外源GFP基因的玉米祥249自交系T1代转基因玉米植株种于温室,授粉后约10天,待幼胚长至0.8-2.0mm时,收获玉米穗子,去除苞叶,准备灭菌;
(2)将浓度为6-15%的次氯酸钠母液用灭菌水按体积比稀释到15%-20%,按1滴(20μL)/3L加入Tween-20%混匀制成灭菌液;
(3)将玉米穗子放入灭菌液中浸泡15分钟,无菌水冲洗3-5次备用;
(4)用无菌的手术刀片削去种子顶端,用无菌刮铲掘取胚乳使幼胚从种子中暴露出来,剥取幼胚。将分离出来的幼胚放入含有1.8mL悬浮液的2mL塑料离心管中。
2、侵染与共培养
(1)吸去离心管中的悬浮液,加入200μL新鲜的悬浮液,4000rpm离心15秒,45℃水浴热击3分钟,然后转入0℃冰浴1分钟;
(2)用移液枪吸去离心管中的悬浮液,加入1.0mL OD600值为0.1-0.5的农杆菌EHA105侵染液,侵染10分钟。该农杆菌含Cas9-GFP-gRNA;
(3)将离心管中的幼胚悬浮后倒入共培养培养基中,并用移液枪吸去表面多余的农杆菌侵染液,将幼胚盾片朝上放置,于23℃黑暗共培养3-5天。
3、诱导与筛选
(1)共培养后,将幼胚转移到愈伤诱导培养基中,于32℃黑暗诱导培养5-14天;
(2)愈伤诱导培养结束后,将幼胚转入含200mg/L草甘膦的筛选培养基上,于28℃黑暗培养。筛选2-3轮,一轮为2周。
4、植株再生与移栽
(1)筛选培养结束后,将抗性愈伤组织转移至分化培养基I(含10mg/L草甘膦)中,25℃,5000lx,光照培养1周;
(2)将出现绿点的愈伤组织转移至分化培养基II(含10mg/L草甘膦)中,光照培养2周;
(3)将分化出的幼苗转移至生根培养基上,25℃,5000lx,光照培养直至生根;
(4)将转基因再生苗转入专用穴盘中生长,炼苗后移栽于温室中,3-4个月后即可收获后代种子。
转化过程中所用到的试剂和培养基配方如下:
悬浮液:1/2MS+蔗糖68.5g/L+葡萄糖36g/L+L-脯氨酸0.115g/L;
侵染液:1/2MS+蔗糖68.5g/L+葡萄糖36g/L+L-脯氨酸0.115g/L+乙酰丁香酮200μM+OD600值0.3农杆菌液;
共培养培养基的组成如下:1/2MS+蔗糖20g/L+葡萄糖10g/L+脯氨酸0.115g/L+盐酸硫胺素0.5mg/L+AgNO320μM+L-半胱氨酸200mg/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+KT0.01-1mg/L+乙酰丁香酮200μM+植物凝胶8g/L;
愈伤诱导培养基的组成如下:MS+蔗糖30g/L+脯氨酸1.38g/L+盐酸硫胺素0.5mg/L+AgNO320μM+水解酪蛋白0.5g/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+KT 0.01-1mg/L+特美汀200mg/L+植物凝胶8g/L;
筛选培养基的组成如下:MS+蔗糖30g/L+脯氨酸1.38g/L+盐酸硫胺素0.5mg/L+AgNO320μM+水解酪蛋白0.5g/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+特美汀200mg/L+草甘膦200mg/L+植物凝胶8g/L;
分化培养基I:MS+蔗糖20g/L+硫酸铜10μM+MES 0.5g/L+6-BA3.5mg/L+特美汀200mg/L+草甘膦10mg/L+植物凝胶8g/L;
分化培养基II:MS+蔗糖20g/L+硫酸铜10μM+MES 0.5g/L+特美汀200mg/L+草甘膦10mg/L+植物凝胶8g/L;
生根培养基的组成如下:MS+蔗糖20g/L+MES 0.5g/L+IBA0.2mg/L+植物凝胶8g/L。
实施例3基因定点突变玉米的检测
1、GFP基因在定点突变玉米组织中的表达观察
取实施例2的GFP基因定点突变后的抗性愈伤组织,观察GFP基因在愈伤组织中的表达,结果发现,在紫外光下见绿色荧光,说明这部分愈伤中GFP基因未被定点突变,若紫外光下未见绿色荧光则为GFP基因已失活,结果见图1。从图1圆圈标注处可以看出两块新生的草甘膦抗性愈伤处均无GFP表达,而非抗性愈伤处有GFP表达(图1B、D)。说明未转化细胞中的GFP基因正常表达,而转化细胞中GFP基因不再表达,表明转化细胞中GFP基因已被成功编辑失活。
2、基因测序
(1)DNA提取:用购自天根生化科技(北京)有限公司的DNA提取试剂盒抽提实施例2获得的转基因玉米基因组DNA。
(2)GFP基因PCR
将下列试剂从-20℃冰箱中取出解冻:5×PCR Buffer(NEB)、d NTP Mix(10mM,Sigma)、正、反向引物混合液(F:5’-GGATGATGGCATATGCAGCAGC-3’,R:5’-GAAGTCGTGCTGCTTCATGTGG-3’),以及DNA模板。
所有试剂解冻后,简短离心数秒,置于冰上待用;配制PCR反应体系的混合液,混匀,简短离心数秒;将混合液分装至200μL的PCR管中,再加入2μL模板DNA,;将PCR反应管放入Thermo 9700型PCR扩增仪;选择预设PCR扩增程序,开始运行反应。
PCR反应体系:5×PCR Buffer(NEB)10μL,dNTP Mix(10mM,Sigma)1μL,正反向引物混合液(5μM)4μL,Phusion超保真DNA聚合酶(1U,NEB)0.5μL,DNA模板2μL。
PCR反应程序为:98℃预变性30秒;98℃变性10秒,58℃退火10秒,72℃延伸20秒,30个循环;最后72℃延伸5分钟。
(3)样品测序
将所得的PCR产物经纯化后用购自ABI公司的Big Dye3.1试剂盒处理后,用3730×L测序仪进行测序,测序后用Vevtor NTI Advance11.5软件对测序结果进行分析。
图2结果显示,样品中的GFP基因序列与阳性对照相比,在同一位置发生了一个腺嘌呤(A)碱基缺失,从而导致GFP基因功能失活。表明利用本发明提供方法成功实现了对特定基因的定位定点突变。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.利用CRISPR/Cas9系统对玉米基因定点突变的方法,其特征在于,针对玉米中的目标基因设计基于CRISPR/Cas9的sgRNA序列,将含有编码所述sgRNA序列的DNA片段连接到携带CRISPR/Cas的载体中,转化玉米,实现对玉米中特定基因的定点突变。
2.根据权利要求1所述的方法,其特征在于,所述玉米包括自交种、杂交种、普通玉米、甜玉米、糯玉米;优选地,所述玉米为祥249自交系。
3.根据权利要求2所述的方法,其特征在于,所述目标基因包括玉米基因组中任何有生物学功能的基因或DNA序列。
4.根据权利要求2所述的方法,其特征在于,所述目标基因包括GFP;其sgRNA作用位点的核苷酸序列为5’-ACCGGGGTGGTGCCCATCC-3’。
5.根据权利要求4所述的方法,其特征在于,用载体Cas9-GFP-gRNA转化玉米,载体Cas9-GFP-gRNA的全序列如SEQ ID NO:2所示。
6.权利要求1-5任一项所述方法在玉米基因定点突变育种中的应用,所述玉米包括祥249自交系。
7.根据权利要求6所述的应用,其特征在于,包括以下步骤:
(1)将玉米的幼胚浸入携带CRISPR/Cas9和选择标记基因的农杆菌菌液中侵染;优选地,所述选择标记基因为CP4;
(2)将幼胚移至共培养培养基上培养;
(3)将幼胚移至愈伤诱导培养基上培养,诱导初级愈伤组织;
(4)将初级愈伤组织移至筛选培养基上培养,诱导抗性愈伤组织,再转移到分化培养基上,分化形成再生幼苗;
(5)再生幼苗在生根培养基上生根后炼苗、移栽,得到转基因玉米;
(6)根据sgRNA作用位点的核苷酸序列设计引物,通过PCR法鉴定玉米植株突变位点。
8.根据权利要求7所述的应用,其特征在于,
所述共培养培养基的组成如下:1/2MS+蔗糖15-30g/L+葡萄糖8-15g/L+脯氨酸0.1-0.3g/L+盐酸硫胺素0.1-1.0mg/L+AgNO3 15-25μM+L-半胱氨酸100-300mg/L+2,4-D 0.3-1.0mg/L+毒莠定0.8-3.0mg/L+KT 0.01-1mg/L+乙酰丁香酮100-300μM+植物凝胶3-8g/L;优选地,共培养培养基的组成如下:1/2MS+蔗糖20g/L+葡萄糖10g/L+脯氨酸0.115g/L+盐酸硫胺素0.5mg/L+AgNO3 20μM+L-半胱氨酸200mg/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+KT 0.01-1mg/L+乙酰丁香酮200μM+植物凝胶8g/L;
所述愈伤诱导培养基的组成如下:MS+蔗糖15-30g/L+脯氨酸0.1-0.3g/L+盐酸硫胺素0.1-1.0mg/L+AgNO3 15-25μM+水解酪蛋白0.1-1.0g/L+2,4-D 0.3-1.0mg/L+毒莠定0.8-3.0mg/L+KT 0.01-1mg/L+特美汀100-300mg/L+植物凝胶3-8g/L;优选地,愈伤诱导培养基的组成如下:MS+蔗糖30g/L+脯氨酸1.38g/L+盐酸硫胺素0.5mg/L+AgNO3 20μM+水解酪蛋白0.5g/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+KT0.01-1mg/L+特美汀200mg/L+植物凝胶8g/L;
所述筛选培养基的组成如下:MS+蔗糖15-30g/L+脯氨酸0.1-0.3g/L+盐酸硫胺素0.1-1.0mg/L+AgNO3 15-25μM+水解酪蛋白0.1-1.0g/L+2,4-D 0.3-1.0mg/L+毒莠定0.8-3.0mg/L+特美汀100-300mg/L+草甘膦100-300mg/L+植物凝胶3-8g/L;优选地,筛选培养基的组成如下:MS+蔗糖30g/L+脯氨酸1.38g/L+盐酸硫胺素0.5mg/L+AgNO3 20μM+水解酪蛋白0.5g/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+特美汀200mg/L+草甘膦200mg/L+植物凝胶8g/L;
所述分化培养基包括分化培养基I和分化培养基II:
分化培养基I:MS+蔗糖15-30g/L+硫酸铜3-15μM+MES 0.3-0.8g/L+6-BA 1.0-4.0mg/L+特美汀100-300mg/L+草甘膦3-15mg/L+植物凝胶3-8g/L;优选地,分化培养基I:MS+蔗糖20g/L+硫酸铜10μM+MES 0.5g/L+6-BA 3.5mg/L+特美汀200mg/L+草甘膦10mg/L+植物凝胶8g/L;
分化培养基II:MS+蔗糖15-30g/L+硫酸铜3-15μM+MES 0.3-0.8g/L+特美汀100-300mg/L+草甘膦3-15mg/L+植物凝胶3-8g/L;优选地,分化培养基II:MS+蔗糖20g/L+硫酸铜10μM+MES 0.5g/L+特美汀200mg/L+草甘膦10mg/L+植物凝胶8g/L;
所述生根培养基的组成如下:MS+蔗糖15-30g/L+MES 0.3-0.8g/L+IBA 0.1-0.3mg/L+植物凝胶3-8g/L;优选地,生根培养基的组成如下:MS+蔗糖20g/L+MES 0.5g/L+IBA 0.2mg/L+植物凝胶8g/L。
9.根据权利要求7或8所述的应用,其特征在于,步骤(1)中玉米的幼胚是从授粉后6-15天,待玉米幼胚长至0.5-2.0mm时,从玉米幼穗上剥取获得。
10.根据权利要求7或8所述的应用,其特征在于,步骤(1)中将玉米的幼胚浸入如下侵染液中侵染5-15分钟;
侵染液组成为:1/2MS+蔗糖40-80g/L+葡萄糖20-40g/L+L-脯氨酸0.1-0.3g/L+乙酰丁香酮100-300μM+OD600值0.1-0.5携带目的基因和选择标记基因的农杆菌菌液;优选使用农杆菌菌株为EHA105;
步骤(2)中培养条件为:23℃黑暗培养3-5天;
步骤(3)中培养条件为:26-34℃黑暗培养5-14天。
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