CN114075555B - Aba代谢关键酶及其编码基因在改善小麦愈伤再生能力中的应用 - Google Patents
Aba代谢关键酶及其编码基因在改善小麦愈伤再生能力中的应用 Download PDFInfo
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Abstract
本发明公开了ABA代谢关键酶及其编码基因在改善小麦愈伤再生能力中的应用。本发明的实验证明:敲除小麦中TaABA8OH2基因后得到的基因编辑植物的愈伤再生出芽能力显著提高,说明TaABA8OH2基因可以调控小麦愈伤再生出芽能力,其中TaABA8OH2基因中的靶序列1(GACCTTCCAGCTCTACTCCC)是调控小麦愈伤出芽能力的理想位点。本发明有效解决了现有技术中小麦组织培养方法中再生效率过低的问题,对小麦遗传转化及小麦品种改良具有重要的理论意义和使用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及ABA代谢关键酶及其编码基因在改善小麦愈伤再生能力中的应用。
背景技术
小麦是我国最重要的粮食作物之一,为了确保粮食生产安全,满足持续增长人口的需求,需要不断对小麦的产量、品质、抗病性和抗逆性进行改良。分子育种是改良小麦品种快速有效的方法,而建立高效的小麦遗传转化体系是小麦中顺利开展分子育种的前提条件。目前小麦组织培养的植株再生效率过低,成为小麦遗传转化的限速步骤,严重限制生物技术在小麦品种改良中的应用。
小麦组织培养一直是国内外学者研究的重点。通过对外植体、培养基成份、激素、培养条件、材料的生理状态等调整可以提高小麦愈伤再生效率,但一直没有达到令人满意的水平。
ABA 8’-羟化酶(ABA 8'-hydroxylase,简称ABA8ox)是ABA代谢途径中的关键酶,催化ABA成为8’-羟基-ABA,发生异构化失活,从而调控内源ABA水平,ABA作为一种激素在植物抵御低温、干旱、高温、高盐等外界非生物胁迫的过程中起到至关重要的作用。
发明内容
本发明的目的是提供ABA代谢关键酶及其编码基因在改善植物愈伤再生能力中的应用,以提高植物愈伤的再生效率。
为了实现上述目的,本发明首先提供了ABA 8’-羟化酶或与ABA 8’-羟化酶相关的生物材料的新用途。
本发明提供了ABA 8’-羟化酶或与ABA 8’-羟化酶相关的生物材料在如下X1)-X2)中任一种中的应用:
X1)调控或改善植物愈伤再生能力;
X2)调控或改善植物愈伤出芽能力。
为了实现上述目的,本发明还提供了抑制植物中ABA 8’-羟化酶活性的物质或抑制或沉默植物中ABA 8’-羟化酶编码基因表达的物质或敲除植物中ABA 8’-羟化酶编码基因的物质的新用途。
本发明提供了抑制植物中ABA 8’-羟化酶活性的物质或抑制或沉默植物中ABA8’-羟化酶编码基因表达的物质或敲除植物中ABA 8’-羟化酶编码基因的物质在如下Y1)-Y8)中任一种中的应用:
Y1)提高植物愈伤再生能力;
Y2)提高植物愈伤出芽能力;
Y3)培育愈伤再生能力提高的植物;
Y4)培育愈伤出芽能力提高的植物;
Y5)植物组织培养;
Y6)植物遗传转化;
Y7)研究目的基因功能:
Y8)植物育种。
为了实现上述目的,本发明还提供了一种提高植物愈伤再生能力或提高植物愈伤出芽能力或培育愈伤再生或出芽能力提高的植物的方法。
本发明提供的提高植物愈伤再生能力或提高植物愈伤出芽能力或培育愈伤再生或出芽能力提高的植物的方法为方法一或方法二:
所述方法一包括如下步骤:抑制植物中ABA 8’-羟化酶活性,得到愈伤再生或出芽能力提高的植物;
所述方法二包括如下步骤:抑制或沉默目的植物中ABA 8’-羟化酶编码基因表达或敲除植物中ABA 8’-羟化酶编码基因,得到愈伤再生或出芽能力提高的植物。
为了实现上述目的,本发明还提供了一种植物组织培养的方法。
本发明提供的植物组织培养的方法包括如下步骤:
M1)取上述愈伤再生或出芽能力提高的植物种子的胚,置于胚性愈伤诱导培养基上培养,得到愈伤组织;
M2)将所述愈伤组织在分化培养基中培养,得到再生苗;
M3)将所述再生苗在生根壮苗培养基中培养,得到再生植株。
上述M1)中,所述培养方法为用手术镊子将胚挑出,盾面向上置于胚性愈伤诱导培养基上,25℃黑暗培养4-5个月(每2周继代一次,继代时均采用胚性愈伤增殖培养基),得到颗粒较小、致密易碎、浅黄鲜亮的愈伤组织。
所述胚性愈伤诱导培养基的制备方法:在MS培养基中加入2,4-二氯苯氧乙酸(俗称2,4-D)、谷氨酰胺和水解酪蛋白,使2,4-二氯苯氧乙酸的浓度为2mg/L、谷氨酰胺的浓度为500mg/L、水解酪蛋白的浓度为500mg/L。
所述胚性愈伤增殖培养基的制备方法:在MS培养基中加入2,4-二氯苯氧乙酸、谷氨酰胺和水解酪蛋白,使2,4-二氯苯氧乙酸的浓度为1mg/L、谷氨酰胺的浓度为500mg/L、水解酪蛋白的浓度为500mg/L。
所述M2)中,所述培养方法为先将愈伤组织在分化培养基(1)中培养,得到产生芽点的胚性愈伤组织,然后将所述产生芽点的胚性愈伤组织转移至分化培养基(2)上培养,得到分化绿芽的愈伤组织(即再生苗)。
所述分化培养基(1)的制备方法:在MS培养基中加入6-糖基氨基嘌呤(俗称激动素KT)、水解酪蛋白和潮霉素,使6-糖基氨基嘌呤的浓度为2mg/L、水解酪蛋白的浓度为500mg/L、潮霉素的浓度为20mg/L。
所述分化培养基(2)的制备方法:在MS培养基中加入6-糖基氨基嘌呤(俗称激动素KT)、水解酪蛋白和潮霉素,使6-糖基氨基嘌呤的浓度为2mg/L、水解酪蛋白的浓度为500mg/L、潮霉素的浓度为40mg/L。
所述M3)中,所述生根壮苗培养基的制备方法:在MS培养基中加入α-萘乙酸(俗称生长素NAA),使α-萘乙酸的浓度为0.5mg/L。
为了实现上述目的,本发明还提供了一种植物遗传转化的方法。
本发明提供的植物遗传转化的方法包括如下步骤:
N1)用含有目的基因的载体的农杆菌侵染上述M1)获得的愈伤组织,得到侵染后愈伤组织;
N2)将所述侵染后愈伤组织在筛选培养基中培养,得到抗性愈伤组织;
N3)将所述抗性愈伤组织在分化培养基中培养,得到再生苗;
N4)将所述再生苗在生根壮苗培养基中培养,得到再生植株。
上述植物遗传转化的方法中,所述含有目的基因的载体的农杆菌为含有重组载体pCAMBIA1301-UBI-WCSP3的农杆菌EHA105。
为了实现上述目的,本发明还提供了一种研究目的基因功能的方法。
本发明提供的研究目的基因功能的方法包括如下步骤:将目的基因按照上述遗传转化方法导入目的植物中,得到转目的基因植物;将所述转目的基因植物与野生型目的植物进行杂交,得到杂交后代,在所述杂交后代中筛选含有目的基因且ABA 8’-羟化酶基因与所述野生型目的植物中的ABA 8’-羟化酶基因的基因型相同的植株,利用该植株研究所述目的基因的功能。
上述研究目的基因功能的方法中,所述目的基因具体可为WCSP3基因。
上述应用或方法中,所述ABA 8’-羟化酶具体为TaABA8OH2蛋白质;所述TaABA8OH2蛋白质为如下A1)或A2)或A3)或A4)任一所示蛋白质:
A1)由序列表中序列3所示的氨基酸序列组成的蛋白质;
A2)在序列表中序列3所示的蛋白质的N端或/和C端连接标签得到的融合蛋白;
A3)将序列表中序列3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且功能相同的蛋白质;
A4)与A1)-A3)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
所述生物材料具体为下述B1)-B10)中的任一种:
B1)编码TaABA8OH2蛋白质的核酸分子;
B2)含有B1)所述核酸分子的表达盒;
B3)含有B1)所述核酸分子的重组载体;
B4)含有B2)所述表达盒的重组载体;
B5)含有B1)所述核酸分子的重组微生物;
B6)含有B2)所述表达盒的重组微生物;
B7)含有B3)所述重组载体的重组微生物;
B8)含有B4)所述重组载体的重组微生物;
B9)含有B1)所述核酸分子的转基因细胞系;
B10)含有B2)所述表达盒的转基因细胞系。
进一步的,B1)所述核酸分子为如下C1)-C4)中任一所示DNA分子:
C1)序列表中序列1所示的基因组DNA分子;
C2)序列表中序列2所示的cDNA分子;
C3)与C1)或C2)限定的DNA分子序列至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%同源性,且编码TaABA8OH2蛋白质的DNA分子;
C4)在严格条件下与C1)或C2)或C3)限定的DNA分子杂交,且编码TaABA8OH2蛋白质的DNA分子。
上述应用或方法中,所述敲除植物中ABA 8’-羟化酶编码基因的物质为CRISPR/Cas9系统,所述CRISPR/Cas9系统中的sgRNA的靶序列为序列表中的序列4、序列5或序列6。
所述敲除植物中ABA 8’-羟化酶编码基因通过所述CRISPR/Cas9系统基因编辑所述目的植物实现。
为了实现上述目的,本发明最后提供了一种CRISPR/Cas9系统。
所述CRISPR/Cas9系统中的sgRNA的靶序列为序列表中的序列4、序列5或序列6;
所述CRISPR/Cas9系统的功能为如下Y1)-Y8)中任一种:
Y1)提高植物愈伤再生能力;
Y2)提高植物愈伤出芽能力;
Y3)培育愈伤再生能力提高的植物;
Y4)培育愈伤出芽能力提高的植物;
Y5)植物组织培养;
Y6)植物遗传转化;
Y7)研究目的基因功能:
Y8)植物育种。
上述任一所述应用或方法或系统中,所述植物可为双子叶植物或单子叶植物;所述单子叶植物可为小麦;所述小麦的品种具体可为京花9号。
本发明的实验证明:敲除小麦中TaABA8OH2基因后得到的基因编辑植物的愈伤再生出芽能力显著提高,说明TaABA8OH2基因可以调控小麦愈伤再生出芽能力,其中TaABA8OH2基因中的靶序列1(GACCTTCCAGCTCTACTCCC)是调控小麦愈伤出芽能力的理想位点。本发明有效解决了现有技术中小麦组织培养方法中再生效率过低的问题,对小麦遗传转化及小麦品种改良具有重要的理论意义和使用价值。
附图说明
图1为敲除CRISPR-Cas9编辑TaABA8OH2基因的靶序列所在位置。
图2为pCRISPR-Cas9-TaABA8OH2载体结构图。RB:T-DNA右边界;UBI:UBI启动子;LB:T-DNA左边界;35S:CaMV 35S启动子;U6:U6启动子;Cas9:优化的Cas9基因;ABA:TaABA8OH2的标靶序列;Hygro:潮霉素筛选标记基因;NOS Ter:NOS终止子;polyA Ter:polyA终止子。
图3为pCRISPR-Cas9-TaABA8OH2转基因植株TaABA8OH2基因的测序结果。A为靶点1测序结果。B为靶点2测序结果。C为靶点3测序结果。
图4为不同类型小麦愈伤诱导再生后的发芽率统计。A为利用标靶序列1敲除TaABA8OH2基因的小麦愈伤。B为利用标靶序列2敲除TaABA8OH2基因的小麦愈伤。C为利用标靶序列3敲除TaABA8OH2基因的小麦愈伤。D为野生型小麦(京花9)愈伤。
图5为L88-9的表型及分子鉴定结果。A为L88-9株系的表型。B为小麦杂交子代的分子鉴定,1:DL2000;2-5:小麦杂交后代;6:阴性对照。C为重组载体的结构示意图。D为L88-9株系中靶序列1的分子鉴定,1:100bp ladder;2:野生型小麦京花9号;3:L88-9。E为L88-9株系中靶序列1的测序结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的京花9号(TriticumaestivumL.cv Jinghua 9)记载于文献“刘建平,张立全,田立平,苏青,赵克勇,单福华,张立平,赵昌平,高产优质早熟冬小麦新品种-京花9号,麦类作物学报,2008年第5期第921页”中,公众可从申请人处获得,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
下述实施例中的CRISP-Cas载体是杭州百格生物技术有限公司(百格基因)(http://www.biogle.cn/)的产品,型号为BGK03。
下述实施例中的MS培养基的溶剂为水,每升MS培养基中的溶质及其加入量见表1。MS培养基的制备方法如下:按照表1的加入量将各个组分溶于水并用水定容至1L,调pH值至5.8,120℃高压灭菌20分钟。
表1、每升MS培养基中的溶质组成及各个溶质的含量
溶质 | 加入量(g) |
硝酸钾(KNO3) | 1.90 |
硝酸铵(NH4NO3) | 1.65 |
磷酸二氢钾(KH2PO4) | 0.17 |
七水硫酸镁(MgSO4·7H2O) | 0.37 |
二水氯化钙(CaCl2·2H2O) | 0.44 |
碘化钾(KI) | 0.00083 |
硼酸(H3BO3) | 0.0062 |
四水硫酸锰(MnSO4·4H2O) | 0.0223 |
七水硫酸锌(ZnSO4·7H2O) | 0.0086 |
二水钼酸钠(Na2MoO4·2H2O) | 0.00025 |
五水硫酸铜(CuSO4·5H2O) | 0.000025 |
六水氯化钴(CoCl2·6H2O) | 0.000025 |
乙二胺四乙酸二钠(Na2·EDTA) | 0.0373 |
七水硫酸亚铁(FeSO4·7H2O) | 0.0278 |
甘氨酸(Glycine) | 0.002 |
盐酸硫胺素(VB1) | 0.0001 |
盐酸吡哆醇(VB6) | 0.0005 |
烟酸(VB5) | 0.0005 |
麦芽糖(Maltose) | 30 |
植物凝胶GelzanTMCM(Sigma公司) | 2.5 |
实施例1、TaABA8OH2蛋白及其编码基因在提高小麦愈伤再生出芽率中的应用
本发明中的TaABA8OH2基因来源于小麦品种京花9号,位于小麦染色体5A上,其基因组序列如序列表中序列1所示,其CDS序列如序列表中序列2所示,TaABA8OH2基因编码的TaABA8OH2蛋白的氨基酸序列如序列表中序列3所示。
一、转基因小麦的获得
1、sgRNA靶序列的设计
根据TaABA8OH2基因序列,本发明设计了三条sgRNA靶序列,分别记作sgRNA1、sgRNA2和sgRNA3。靶序列所在位置见图1。sgRNA靶序列分别如下:
sgRNA1:GACCTTCCAGCTCTACTCCC(序列4);
sgRNA2:GCCCCTGGCGGACCTGGCGG(序列5);
sgRNA3:GACGATCCAGGTGAGCAACGC(序列6)。
2、Oligo二聚体的合成
将正向引物F:TGTGTGGACCTTCCAGCTCTACTCCC和反向引物R:AAACGGGAGTAGAGCTGGAGGGTCCA合成Oligo二聚体1。
将正向引物F:TGTGTGGCCCCTGGCGGACCTGGCGG和反向引物R:AAACGAACGTGAAGGAGATGATGCCA合成Oligo二聚体2。
将正向引物F:TGTGTGGACGATCCAGGTGAGCAACGC和反向引物R:AAACGCGTGCTCACCTGGATCGTCCA合成Oligo二聚体3。
加粗序列为靶序列;下划线所示序列为构建载体所需附加序列。
Oligo二聚体合成体系如下:Buffer Anneal 18μl、UP Oligo(10μM)1μl、LowOligo(10μM)1μl、H2O补足体系至20μl。
Oligo二聚体合成条件如下:95℃加热3分钟,然后以约0.2℃秒缓慢降至20℃。
3、pCRISPR-Cas9-TaABA8OH2的构建
将Oligo二聚体1构建至CRISP-Cas载体U6启动子后,得到pCRISPR-Cas9-TaABA8OH2-1重组载体。pCRISPR-Cas9-TaABA8OH2-1重组载体含有sgRNA1靶序列。
将Oligo二聚体2构建至CRISP-Cas载体U6启动子后,得到pCRISPR-Cas9-TaABA8OH2-2重组载体。pCRISPR-Cas9-TaABA8OH2-2重组载体含有sgRNA2靶序列。
将Oligo二聚体3构建至CRISP-Cas载体U6启动子后,得到pCRISPR-Cas9-TaABA8OH2-3重组载体。pCRISPR-Cas9-TaABA8OH2-3重组载体含有sgRNA3靶序列。
pCRISPR-Cas9-TaABA8OH2载体结构图见图2。
具体构建方法如下:按下列反应体系在冰上混合各个组分:CRISPR-Cas载体2μl、Oligo二聚体1μl、Enzyme Mix 1μl、H2O补足体系至10μl,混匀后室温(20℃)反应1小时。
4、重组菌的构建
参照电激仪(EasyJecT Plus电激仪,英国EquiBio公司)操作指南,分别将重组载体pCRISPR-Cas9-TaABA8OH2-1、重组载体pCRISPR-Cas9-TaABA8OH2-2和重组载体pCRISPR-Cas9-TaABA8OH2-3用电击法导入农杆菌EHA105(Biovector Co.,LTD公司,目录号为Biovec-11)中,分别经含卡那霉素的抗性平板筛选得到重组菌,将该重组菌分别命名为重组菌pCRISPR-Cas9-TaABA8OH2-1/EHA105、重组菌pCRISPR-Cas9-TaABA8OH2-2/EHA105和重组菌pCRISPR-Cas9-TaABA8OH2-3/EHA105。
按照上述方法,将重组载体替换为pCRISP-Cas9载体,其他步骤均不变,得到重组菌pCRISPR-Cas9/EHA105。
5、转基因小麦的构建
取小麦品种京花9号(Triticum aestivuml L.cv.Jinghua9)开花后2周的未成熟种子,剥去稃片,置于70%(体积比)酒精水溶液中浸泡30秒,用无菌蒸馏水冲洗2次,然后置于转入0.1g/100mL升汞水溶液中浸泡10分钟(消毒),用无菌蒸馏水冲洗4-5次后,然后用手术镊子将胚挑出,盾面向上置于胚性愈伤诱导培养基上,25℃黑暗培养4-5个月(每2周继代一次,继代时均采用胚性愈伤增殖培养基),得到颗粒较小、致密易碎、浅黄鲜亮的愈伤组织。
将重组菌pCRISPR-Cas9-TaABA8OH2-1/EHA105、重组菌pCRISPR-Cas9-TaABA8OH2-2/EHA105和重组菌pCRISPR-Cas9-TaABA8OH2-3/EHA105导入京花9(Triticum aestivumlL.cv.Jinghua9)的愈伤组织中,再用含300mg/L头孢霉素的无菌水洗涤4-5遍,无菌滤纸吸干后转至MSD1S1培养基上,筛选一代;两周后,转移至MSD1S2培养基上,筛选二代(2周/代);取出经过三代筛选生长旺盛的抗性愈伤组织,转移至分化培养基(1)上,在光照培养箱(光周期设定为12小时光照,12小时黑暗,白天28℃,夜晚25℃)中培养14天;然后转移至分化培养基(2)上培养(2周/代),在分化培养箱中培养至产生再生苗。再生苗在生根壮苗培养基上生根壮苗;待小苗长至10厘米左右时,打开容器封口膜,炼苗2-3天,然后将小苗移入人工气候室栽培,获得T0代转pCRISPR-Cas9-TaABA8OH2-1小麦、T0代转pCRISPR-Cas9-TaABA8OH2-2小麦和T0代转pCRISPR-Cas9-TaABA8OH2-3小麦。
按照上述方法,将pCRISPR-Cas9-TaABA8OH2/EHA105替换为重组菌pCRISPR-Cas9/EHA105,其他步骤均不变,得到T0代转空载体小麦。
上述方法中,所用培养基是以表1制备的MS培养基作为基础培养基如下所示的配方进行配制的。
胚性愈伤诱导培养基的制备方法:在MS培养基中加入2,4-二氯苯氧乙酸(俗称2,4-D)、谷氨酰胺和水解酪蛋白,使2,4-二氯苯氧乙酸的浓度为2mg/L、谷氨酰胺的浓度为500mg/L、水解酪蛋白的浓度为500mg/L。
胚性愈伤增殖培养基的制备方法:在MS培养基中加入2,4-二氯苯氧乙酸、谷氨酰胺和水解酪蛋白,使2,4-二氯苯氧乙酸的浓度为1mg/L、谷氨酰胺的浓度为500mg/L、水解酪蛋白的浓度为500mg/L。
MSD1S1培养基的制备方法:在MS培养基中加入2,4-二氯苯氧乙酸、谷氨酰胺、水解酪蛋白、头孢霉素(Cefotaxime)和潮霉素(Hygromycine),使2,4-二氯苯氧乙酸的浓度为1mg/L、谷氨酰胺的浓度为500mg/L、水解酪蛋白的浓度为500mg/L、头孢霉素的浓度为300mg/L、潮霉素的浓度为20mg/L。
MSD1S2培养基的制备方法:在MS培养基中加入2,4-二氯苯氧乙酸、谷氨酰胺、水解酪蛋白、头孢霉素和潮霉素,使2,4-二氯苯氧乙酸的浓度为1mg/L、谷氨酰胺的浓度为500mg/L、水解酪蛋白的浓度为500mg/L、头孢霉素的浓度为300mg/L、潮霉素的浓度为40mg/L。
分化培养基(1)的制备方法:在MS培养基中加入6-糖基氨基嘌呤(俗称激动素KT)、水解酪蛋白和潮霉素,使6-糖基氨基嘌呤的浓度为2mg/L、水解酪蛋白的浓度为500mg/L、潮霉素的浓度为20mg/L。
分化培养基(2)的制备方法:在MS培养基中加入6-糖基氨基嘌呤(俗称激动素KT)、水解酪蛋白和潮霉素,使6-糖基氨基嘌呤的浓度为2mg/L、水解酪蛋白的浓度为500mg/L、潮霉素的浓度为40mg/L。
生根壮苗培养基的制备方法:在MS培养基中加入α-萘乙酸(俗称生长素NAA),使α-萘乙酸的浓度为0.5mg/L。
6、测序鉴定转基因小麦中TaABA8OH2的突变
对T0代转pCRISPR-Cas9-TaABA8OH2-1小麦、T0代转pCRISPR-Cas9-TaABA8OH2-2小麦和T0代转pCRISPR-Cas9-TaABA8OH2-3小麦基因组中TaABA8OH2序列进行PCR鉴定。PCR鉴定引物序列如下:
pCRISPR-Cas9-TaABA8OH2-1:
5'-CCTACGCCCACCACGTAATC-3';
5'-GCCTCTTCATGGCGTGGAAA-3';
pCRISPR-Cas9-TaABA8OH2-2:
5'-CTCTTCAAGCCGACGTACCC-3';
5'-GTAGCCTTTCTCCACGACGG-3';
pCRISPR-Cas9-TaABA8OH2-3:
5'-CCGTCGTGGAGAAAGGCTAC-3';
5'-CTCCAAAATCACCCTGTGCG3'。
实验重复三次,每次每个株系随机取3棵小麦幼苗的全苗进行基因组DNA的提取、PCR鉴定及测序。
通过测序鉴定,从T0代转pCRISPR-Cas9-TaABA8OH2-1小麦中筛选得到一个纯合突变型植株(即两条染色体发生的突变一致),命名为T0代转pCRISPR-Cas9-TaABA8OH2-1小麦L88株系。
通过测序鉴定,从T0代转pCRISPR-Cas9-TaABA8OH2-2小麦中筛选得到一个纯合突变型植株(即两条染色体发生的突变一致),命名为T0代转pCRISPR-Cas9-TaABA8OH2-2小麦L142株系。
通过测序鉴定,从T0代转pCRISPR-Cas9-TaABA8OH2-3小麦中筛选得到一个纯合突变型植株(即两条染色体发生的突变一致),命名为T0代转pCRISPR-Cas9-TaABA8OH2-3小麦L149株系。
T0代转pCRISPR-Cas9-TaABA8OH2小麦L88、L142、L149株系的测序结果见图3。结果表明:T0代转pCRISPR-Cas9-TaABA8OH2-1小麦L88株系、T0代转pCRISPR-Cas9-TaABA8OH2-2小麦L142株系和T0代转pCRISPR-Cas9-TaABA8OH2-3小麦L149株系中的TaABA8OH2序列与野生型小麦京花9号(JH9)相比在标靶区域均有预期改变,说明pCRISPR-Cas9已经成功编辑目标区段。
经测序鉴定,与野生型小麦京花9号(JH9)基因组DNA相比,T0代转pCRISPR-Cas9-TaABA8OH2-1小麦L88株系的差异仅在于对应于序列1所示的TaABA8OH2基因序列第217位的碱基C突变为碱基T,该突变位点及其周边核苷酸的测序结果见图3A。
经测序鉴定,与野生型小麦京花9号(JH9)基因组DNA相比,T0代转pCRISPR-Cas9-TaABA8OH2-2小麦L142株系的差异仅在于对应于序列1所示的TaABA8OH2基因序列的第490位的碱基G突变为碱基C,且第497位的碱基A缺失,且第500位由碱基A突变为碱基G,且第501位由碱基G突变为A,且第504位和第505位之间插入碱基G。突变位点及其周边核苷酸的测序结果见图3B。
经测序鉴定,与野生型小麦京花9号(JH9)基因组DNA相比,T0代转pCRISPR-Cas9-TaABA8OH2-3小麦L149株系的差异仅在于对应于序列1所示的TaABA8OH2基因序列的第750位和第751位之间插入碱基A,该突变位点及其周边核苷酸的测序结果见图3C。
二、转基因小麦愈伤的再生出芽率检测
实验重复三次,每次重复实验的具体步骤如下:
1、将野生型京花9号(JH9)、T0代转pCRISPR-Cas9-TaABA8OH2-1小麦L88株系、T0代转pCRISPR-Cas9-TaABA8OH2-2小麦L142株系和T0代转pCRISPR-Cas9-TaABA8OH2-3小麦L149株系的种子萌发后置于在光照培养箱中(光强为10000μmol/m2/s,光照时间为16h/d,温度为30℃)培养至穗期。
2、取不同小麦材料开花后2周的未成熟种子,剥去稃片,置于70%(体积比)酒精水溶液中浸泡30秒,用无菌蒸馏水冲洗2次,然后置于0.1g/100mL升汞水溶液中浸泡10分钟(消毒),用无菌蒸馏水冲洗4-5次后,然后用手术镊子将胚挑出,盾面向上置于胚性愈伤诱导培养基上,25℃黑暗培养4-5个月(每2周继代一次,继代时均采用胚性愈伤增殖培养基),得到颗粒较小、致密易碎、浅黄鲜亮的愈伤组织。
3、将愈伤组织转移至分化培养基(1)上,在光照培养箱(光周期设定为12小时光照,12小时黑暗,白天28℃,夜晚25℃)中培养14天,得到产生芽点的胚性愈伤组织,统计出芽率(%)。计算公式如下:出芽率(%)=(产生芽的愈伤组织数/愈伤组织数)×100%。
4、将产生芽点的胚性愈伤组织转移至分化培养基(2)上,在光照培养箱(光周期设定为12小时光照,12小时黑暗,白天28℃,夜晚25℃)中培养14天,得到分化绿芽的愈伤组织,统计绿芽分化率。计算公式如下:绿芽分化率(%)=(分化绿芽的愈伤组织数/愈伤组织数)×100%。
5、将分化绿芽的愈伤组织置于生根壮苗培养基上生根壮苗,待小苗长至10厘米左右时,打开容器封口膜,炼苗2-3天,再将小苗移入人工气候室栽培,培养14天,培养条件:光周期设定为10小时光照,14小时黑暗,白天28℃,夜晚25℃,得到小麦植株,统计移栽成活率(%)。计算公式如下:移栽成活率(%)=(成活苗数/移栽苗总数)×100%。
结果如图4所示:图4中的愈伤组织照片为上述步骤4中获得的愈伤组织,其中,图4A为利用标靶序列1敲除TaABA8OH2基因的转pCRISPR-Cas9-TaABA8OH2-1小麦L88株系愈伤;图4B为利用标靶序列2敲除TaABA8OH2基因的转pCRISPR-Cas9-TaABA8OH2-2小麦L142株系愈伤;图4C为利用标靶序列3敲除TaABA8OH2基因的转pCRISPR-Cas9-TaABA8OH2-3小麦L149株系愈伤;图4D为野生型JH9的小麦愈伤。从图中可以看出:L88株系所得愈伤的出芽率显著增强,愈伤所长出的芽形态正常,无白化及其它异常现象,均能长成正常小麦植株,命名为“超级愈伤”,从表2中的出芽率(%)、绿芽分化率(%)和移栽成活率(%)统计结果也可以看出:L88株系所得愈伤的出芽率(%)、绿芽分化率(%)和移栽成活率(%)也均高于L142株系、L149株系及野生型小麦JH9所得愈伤。L142株系所得愈伤的出芽率(%)虽然与野生型小麦JH9相比有明显升高,但愈伤所长出的芽存在白化及其它类型的生长发育异常现象,导致绿芽分化率(%)降低,最终移栽成活率(%)明显低于L88株系。L149株系所得愈伤的出芽率(%)与野生型小麦JH9相比仅仅略有增强,愈伤所长出的芽存在白化及其它类型的生长发育异常现象,移栽后仅有其中一部分可以长为正常小麦植株,移栽成活率(%)虽然高于野生型小麦JH9所得愈伤,但明显低于L142和L149。L88、L142、L149的移栽成活率(%)均明显高于野生型小麦JH9。T0代转空载体小麦所得愈伤的出芽率(%)、绿芽分化率(%)和移栽成活率(%)与野生型小麦JH9无显著性差异。
由此可见,TaABA8OH2基因可以调控小麦愈伤出芽能力,其中TaABA8OH2基因中的靶序列1是调控小麦愈伤出芽能力的理想位点。
表2、出芽率(%)、绿芽分化率(%)和移栽成活率(%)统计结果
实施例2、超级愈伤在小麦遗传转化中的应用
一、重组载体的制备
将序列7所示的WCSP3基因的CDS序列插入pCAMBIA1301-UBI载体(上海柯雷生物科技有限公司,货号为kl-zl-0827)的KpnI和PstI酶切位点间,得到重组载体pCAMBIA1301-UBI-WCSP3。该载体的结构示意图如图5C所示。
二、重组菌的制备
将步骤1中的重组载体用电击法导入农杆菌EHA105中,经含卡那霉素的抗性平板筛选得到重组菌。
三、转WCSP3小麦的获得
按照实施例1步骤5中的方法,将步骤2制备的重组菌导入实施例1步骤二中的“超级愈伤”中,经鉴定,得到转WCSP3小麦。转WCSP3小麦株系愈伤的出芽率为79%。
四、杂交及筛选
将步骤3获得的阳性转WCSP3小麦与野生型小麦京花9号杂交,得到F1代。F1代自交,得到F2代。在37份F2代小麦植株中通过PCR鉴定和测序筛选含有WCSP3目的基因且TaABA8OH2基因与野生型小麦京花9号中的TaABA8OH2基因的基因型相同的植株。
最终得到含有WCSP3目的基因且TaABA8OH2基因与野生型小麦京花9号中的TaABA8OH2基因的基因型相同的小麦,命名为L88-9株系。
WCSP3目的基因的PCR鉴定引物序列如下:5’-CAGATCTGAGTGCCCCGTTT-3’;5’-ACACCAAACTTGACTTCGATAACT-3’,扩增产物大小为245bp的植株为含有WCSP3目的基因的植株,PCR鉴定结果如图5B所示。
针对TaABA8OH2基因靶序列1的PCR鉴定引物序列如下:5’-atctgcaggcatgcctgcagtgcagcgtgacc-3’;5’-cctcctcctggtgcggtgacgtcggaggccttg-3’。
L88-9株系生长发育正常,其表型如图5A所示,与野生型小麦京花9号表型相同。L88-9株系中TaABA8OH2基因靶序列1的PCR鉴定和测序结果如图5D和图5E所示,结果表明,L88-9株系中的TaABA8OH2基因已经恢复野生型状态。L88-9导入外源目的基因WCSP3后,其基因组学序列和表型状态都恢复野生型状态,可利用其分析导入外源目的基因WCSP3的功能机制。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国科学院植物研究所
<120> ABA代谢关键酶及其编码基因在改善小麦愈伤再生能力中的应用
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1601
<212> DNA
<213> Artificial Sequence
<400> 1
cctcgctggt ctaagtgatc cttctcaagg atcatttgga tggccttctt cctcctcctc 60
ctgtgcatcc tcatttctgt ggccatcgtg tcctacgccc accacgtaat ccggcggcaa 120
cgccggggcc cgcagggcag cgctcgtggc cgccatgaga aagccgccct caagctgccc 180
cctggctcca tgggcctgcc ttacatcggc gagaccctcc agctctactc ccaggacccc 240
agcgtcttcc tctcctccaa gcagaagcgg tacggcgaga tcttcaagac gcacctcctc 300
ggctgcccgt gcgtgatgct ggcgagcccg gaggcggcgc gcttcgtgct ggtgtcgcgg 360
gcgcacctct tcaagccgac gtacccgcgg agcaaggagc gcctcatcgg cccctcggcg 420
ctcttcttcc accagggcga ctaccacctc cgcctccgca ggctcgtcca gggcccgctc 480
ggccccgagg ccctggacga gcctgcggcg gacatcgagg acgccgtgcg gtccacgctc 540
gcggcctggg cggacggcga cgccgccagc actttccacg ccatgaagag gctctcgttc 600
gacgtcggca tcgtgacgat cttcggcggg cggctggacg agcggcggaa ggaggagctg 660
aggcggaact acgccgtcgt ggagaaaggc tacaactcct tccccaacgg cttccccggg 720
acgctatact acaagacgat ccaggtgagc acgcggctga acggcgtgct gagcgacatc 780
ctgcacgagc ggagggagcg tggggagccc ggcgacgacc tgctcggctg cctcatgcgg 840
tcacgggcag gcggcggcga cgacgacgac gaggaggagg gcgcgctgct gacggacgag 900
caggtcgccg acaacgtcat cggcgtgctg ttcgcggcgc aggacacgac ggccagcgtg 960
ctcacctgga tcgtcaagta cctccacgac cggccgaagc tgctggaggc cgtccgggcg 1020
gagcacgcgg cgatccacga ggccaacgac ggcgggaggc ggccgctgac atgggcgcag 1080
acgaggagca tgacgctgac gcacagggtg attttggaga gcctgaggat ggcgagcatc 1140
atctccttca cgttcaggga ggccgtggcc gacgtggagt acaaagggtt tcttatcccc 1200
aaggggtgga aggtgatgcc gctattcagg aacatccatc acagcccgga ctacttccag 1260
gatccacaga agttcgaccc ttcgagattc aaggtggcgc cgcggccgag caccttcacg 1320
ccgttcggga gcggggtgca cgcgtgcccc gggaacgagc tggccaagct cgagatgctg 1380
gtgctcatcc accacctcgt caccggctac aggtgggagg ttgttggatc gagcgacgac 1440
gtcgagtaca gcccattccc tgttccccgc catggcctac tcgccaggtt acggcgggat 1500
gacagcgtct gcgtgggtag gaaggggtgc ccgactgatg acgactacga cgacgaagac 1560
gaagtgatag tgtgattagt caacgtagat agctagaggg c 1601
<210> 2
<211> 1536
<212> DNA
<213> Artificial Sequence
<400> 2
atggccttct tcctcctcct cctgtgcatc ctcatttctg tggccatcgt gtcctacgcc 60
caccacgtaa tccggcggca acgccggggc ccgcagggca gcgctcgtgg ccgccatgag 120
aaagccgccc tcaagctgcc ccctggctcc atgggcctgc cttacatcgg cgagaccctc 180
cagctctact cccaggaccc cagcgtcttc ctctcctcca agcagaagcg gtacggcgag 240
atcttcaaga cgcacctcct cggctgcccg tgcgtgatgc tggcgagccc ggaggcggcg 300
cgcttcgtgc tggtgtcgcg ggcgcacctc ttcaagccga cgtacccgcg gagcaaggag 360
cgcctcatcg gcccctcggc gctcttcttc caccagggcg actaccacct ccgcctccgc 420
aggctcgtcc agggcccgct cggccccgag gccctggacg agcctgcggc ggacatcgag 480
gacgccgtgc ggtccacgct cgcggcctgg gcggacggcg acgccgccag cactttccac 540
gccatgaaga ggctctcgtt cgacgtcggc atcgtgacga tcttcggcgg gcggctggac 600
gagcggcgga aggaggagct gaggcggaac tacgccgtcg tggagaaagg ctacaactcc 660
ttccccaacg gcttccccgg gacgctatac tacaagacga tccaggtgag cacgcggctg 720
aacggcgtgc tgagcgacat cctgcacgag cggagggagc gtggggagcc cggcgacgac 780
ctgctcggct gcctcatgcg gtcacgggca ggcggcggcg acgacgacga cgaggaggag 840
ggcgcgctgc tgacggacga gcaggtcgcc gacaacgtca tcggcgtgct gttcgcggcg 900
caggacacga cggccagcgt gctcacctgg atcgtcaagt acctccacga ccggccgaag 960
ctgctggagg ccgtccgggc ggagcacgcg gcgatccacg aggccaacga cggcgggagg 1020
cggccgctga catgggcgca gacgaggagc atgacgctga cgcacagggt gattttggag 1080
agcctgagga tggcgagcat catctccttc acgttcaggg aggccgtggc cgacgtggag 1140
tacaaagggt ttcttatccc caaggggtgg aaggtgatgc cgctattcag gaacatccat 1200
cacagcccgg actacttcca ggatccacag aagttcgacc cttcgagatt caaggtggcg 1260
ccgcggccga gcaccttcac gccgttcggg agcggggtgc acgcgtgccc cgggaacgag 1320
ctggccaagc tcgagatgct ggtgctcatc caccacctcg tcaccggcta caggtgggag 1380
gttgttggat cgagcgacga cgtcgagtac agcccattcc ctgttccccg ccatggccta 1440
ctcgccaggt tacggcggga tgacagcgtc tgcgtgggta ggaaggggtg cccgactgat 1500
gacgactacg acgacgaaga cgaagtgata gtgtga 1536
<210> 3
<211> 511
<212> PRT
<213> Artificial Sequence
<400> 3
Met Ala Phe Phe Leu Leu Leu Leu Cys Ile Leu Ile Ser Val Ala Ile
1 5 10 15
Val Ser Tyr Ala His His Val Ile Arg Arg Gln Arg Arg Gly Pro Gln
20 25 30
Gly Ser Ala Arg Gly Arg His Glu Lys Ala Ala Leu Lys Leu Pro Pro
35 40 45
Gly Ser Met Gly Leu Pro Tyr Ile Gly Glu Thr Leu Gln Leu Tyr Ser
50 55 60
Gln Asp Pro Ser Val Phe Leu Ser Ser Lys Gln Lys Arg Tyr Gly Glu
65 70 75 80
Ile Phe Lys Thr His Leu Leu Gly Cys Pro Cys Val Met Leu Ala Ser
85 90 95
Pro Glu Ala Ala Arg Phe Val Leu Val Ser Arg Ala His Leu Phe Lys
100 105 110
Pro Thr Tyr Pro Arg Ser Lys Glu Arg Leu Ile Gly Pro Ser Ala Leu
115 120 125
Phe Phe His Gln Gly Asp Tyr His Leu Arg Leu Arg Arg Leu Val Gln
130 135 140
Gly Pro Leu Gly Pro Glu Ala Leu Arg Lys Leu Val Pro Asp Ile Glu
145 150 155 160
Asp Ala Val Arg Ser Thr Leu Ala Ala Trp Ala Asp Gly Asp Ala Ala
165 170 175
Ser Thr Phe His Ala Met Lys Arg Leu Ser Phe Asp Val Gly Ile Val
180 185 190
Thr Ile Phe Gly Gly Arg Leu Asp Glu Arg Arg Lys Glu Glu Leu Arg
195 200 205
Arg Asn Tyr Ala Val Val Glu Lys Gly Tyr Asn Ser Phe Pro Asn Gly
210 215 220
Phe Pro Gly Thr Leu Tyr Tyr Lys Ala Ile Gln Ala Arg Arg Arg Leu
225 230 235 240
Asn Gly Val Leu Ser Asp Ile Leu His Glu Arg Arg Glu Arg Gly Glu
245 250 255
Pro Gly Asp Asp Leu Leu Gly Cys Leu Met Arg Ser Arg Ala Gly Gly
260 265 270
Gly Asp Asp Asp Asp Glu Glu Glu Gly Ala Leu Leu Thr Asp Glu Gln
275 280 285
Val Ala Asp Asn Val Ile Gly Val Leu Phe Ala Ala Gln Asp Thr Thr
290 295 300
Ala Ser Val Leu Thr Trp Ile Val Lys Tyr Leu His Asp Arg Pro Lys
305 310 315 320
Leu Leu Glu Ala Val Arg Ala Glu His Ala Ala Ile His Glu Ala Asn
325 330 335
Asp Gly Gly Arg Arg Pro Leu Thr Trp Ala Gln Thr Arg Ser Met Thr
340 345 350
Leu Thr His Arg Val Ile Leu Glu Ser Leu Arg Met Ala Ser Ile Ile
355 360 365
Ser Phe Thr Phe Arg Glu Ala Val Ala Asp Val Glu Tyr Lys Gly Phe
370 375 380
Leu Ile Pro Lys Gly Trp Lys Val Met Pro Leu Phe Arg Asn Ile His
385 390 395 400
His Ser Pro Asp Tyr Phe Gln Asp Pro Gln Lys Phe Asp Pro Ser Arg
405 410 415
Phe Lys Val Ala Pro Arg Pro Ser Thr Phe Thr Pro Phe Gly Ser Gly
420 425 430
Val His Ala Cys Pro Gly Asn Glu Leu Ala Lys Leu Glu Met Leu Val
435 440 445
Leu Ile His His Leu Val Thr Gly Tyr Arg Trp Glu Val Val Gly Ser
450 455 460
Ser Asp Asp Val Glu Tyr Ser Pro Phe Pro Val Pro Arg His Gly Leu
465 470 475 480
Leu Ala Arg Leu Arg Arg Asp Asp Ser Val Cys Val Gly Arg Lys Gly
485 490 495
Cys Pro Thr Asp Asp Asp Tyr Asp Asp Glu Asp Glu Val Ile Val
500 505 510
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
gaccttccag ctctactccc 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 5
gcccctggcg gacctggcgg 20
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 6
gacgatccag gtgagcaacg c 21
<210> 7
<211> 696
<212> DNA
<213> Artificial Sequence
<400> 7
atgggggaga gggtcaaggg aaccgtgaag tggttcaacg tcaccaaggg gttcggcttc 60
atctccccgg acgacggcgg cgaggacctt ttcgtccacc agtccgccat caagtccgac 120
ggctaccgca gcctcaacga gaacgacgcc gtcgagttcg agataatcac cggcgacgac 180
ggacgcacca aggcctccga cgtcaccgca ccaggaggag gggcgctctc cggcggctcc 240
cgccctggcg aaggcggtgg tgaccgcggg ggccgcggcg gctatggagg cggcggcggt 300
ggctacggcg gtggcggtgg tggctacgga ggcggtggtg gcggctacgg cggcggtggt 360
ggcggttacg gtggcggtgg ctatggaggc ggcggtggcg gcggccgtgg gtgctacaag 420
tgcggcgaag atggccacat ctccagggac tgcccccagg gcggcggtgg cggcggcggc 480
tacggcggtg gtggctacgg aggtggtggc ggcggcggcc gtgagtgcta caagtgcggc 540
gaggagggcc acatctccag ggactgcccc cagggcggcg gcggcggcgg ctacggaggc 600
ggtggcggcc gtggcggtgg cggcggcggt ggcggctgct tctcctgcgg cgagtccggc 660
cacttctccc gcgagtgccc caacaaggcc cactag 696
Claims (5)
1.抑制或沉默植物中ABA 8’-羟化酶编码基因表达的物质或敲除植物中ABA 8’-羟化酶编码基因的物质在如下Y1)-Y8)中任一种中的应用:
Y1)提高植物愈伤再生能力;
Y2)提高植物愈伤出芽能力;
Y3)培育愈伤再生能力提高的植物;
Y4)培育愈伤出芽能力提高的植物;
所述ABA 8’-羟化酶为TaABA8OH2蛋白质;所述TaABA8OH2蛋白质为如下A1)或A2)所示蛋白质:
A1)由序列表中序列3所示的氨基酸序列组成的蛋白质;
A2)在序列表中序列3所示的蛋白质的N端或/和C端连接标签得到的融合蛋白;
所述敲除植物中ABA 8’-羟化酶编码基因的物质为CRISPR/Cas9系统,所述CRISPR/Cas9系统中的sgRNA的靶序列为序列表中的序列4、序列5或序列6;
所述植物为小麦。
2.一种提高植物愈伤再生能力或提高植物愈伤出芽能力或培育愈伤再生或出芽能力提高的植物的方法,包括如下步骤:抑制或沉默目的植物中ABA 8’-羟化酶编码基因表达或敲除植物中ABA 8’-羟化酶编码基因,得到愈伤再生或出芽能力提高的植物;
所述ABA 8’-羟化酶为TaABA8OH2蛋白质;所述TaABA8OH2蛋白质为如下A1)或A2)所示蛋白质:
A1)由序列表中序列3所示的氨基酸序列组成的蛋白质;
A2)在序列表中序列3所示的蛋白质的N端或/和C端连接标签得到的融合蛋白;
所述敲除植物中ABA 8’-羟化酶编码基因通过CRISPR/Cas9系统基因编辑所述植物实现;
所述植物为小麦。
3.一种植物组织培养的方法,包括如下步骤:
M1)取权利要求2中的愈伤再生或出芽能力提高的植物种子的胚,置于胚性愈伤诱导培养基上培养,得到愈伤组织;
M2)将所述愈伤组织在分化培养基中培养,得到再生苗;
M3)将所述再生苗在生根壮苗培养基中培养,得到再生植株。
4.一种植物遗传转化方法,包括如下步骤:
N1)用含有目的基因的载体的农杆菌侵染权利要求3中所述M1)获得的愈伤组织,得到侵染后愈伤组织;
N2)将所述侵染后愈伤组织在筛选培养基中培养,得到抗性愈伤组织;
N3)将所述抗性愈伤组织在分化培养基中培养,得到再生苗;
N4)将所述再生苗在生根壮苗培养基中培养,得到再生植株。
5.一种研究目的基因功能的方法,包括如下步骤:将目的基因按照权利要求4所述的方法导入目的植物中,得到转目的基因植物;将所述转目的基因植物与野生型目的植物进行杂交,得到杂交后代,在所述杂交后代中筛选含有目的基因且ABA 8’-羟化酶基因与所述野生型目的植物中的ABA 8’-羟化酶基因的基因型相同的植株,利用该植株研究所述目的基因的功能。
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CN110577938A (zh) * | 2019-11-11 | 2019-12-17 | 中国农业科学院生物技术研究所 | ABA 8’-羟化酶基因OsABA8ox2在植物光形态建成和根发育中的应用 |
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CN110577938A (zh) * | 2019-11-11 | 2019-12-17 | 中国农业科学院生物技术研究所 | ABA 8’-羟化酶基因OsABA8ox2在植物光形态建成和根发育中的应用 |
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