CN115160420B - 盔形毕赤酵母scp类分泌蛋白及其应用 - Google Patents
盔形毕赤酵母scp类分泌蛋白及其应用 Download PDFInfo
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Abstract
本发明公开了氨基酸序列如SEQ ID No.1或SEQ ID No.2所示的盔形毕赤酵母SCP类分泌蛋白及其在制备诱导柑橘果实抗病性的保鲜剂中的应用,研究结果证实,农杆菌介导的遗传转化在柑橘果实上瞬时表达SCP类分泌蛋白,能一定程度抑制柑橘果实绿霉病的发病和病斑直径的增长;本发明从盔形毕赤酵母中挖掘出具有诱导柑橘果实抗病性的SCP类分泌蛋白,不仅丰富了盔形毕赤酵母诱导柑橘果实抗病性的机理,拓宽了拮抗酵母在果实保鲜领域的应用方式,也为柑橘果实采后绿霉病的防治提供了新的解决方法。
Description
技术领域
本发明属于基因工程技术和果实采后病害生物防治技术领域,涉及盔形毕赤酵母(Pichia galeiformis)SCP类分泌蛋白及其应用。
背景技术
拮抗酵母是果实采后病害防治中常用的生物防治拮抗菌。已有大量文献报道拮抗酵母能诱导果实抗病性,涉及果实多种抗病基因的表达、激素合成和信号转导途径、抗病相关物质的合成等。然而,拮抗酵母分泌蛋白对果实抗病性的作用还不清楚。
柑橘是芸香科柑橘亚科柑橘属植物,是世界和中国产量最多的水果。新鲜柑橘果实在采后运输、贮藏等过程中极易受微生物侵染,损失可达总产量的30%以上。由真菌指状青霉(Penicillium digitatum)引起的绿霉病是柑橘果实贮藏过程中主要的侵染性病害。目前产业上主要采用咪鲜胺等化学杀菌剂控制果实采后病害,但存在杀菌剂残留、微生物产生耐药性等问题。
发明内容
本发明的目的在于探究和明确拮抗酵母分泌蛋白在诱导果实抗病性中的作用,以期为果实采后病害的防治提供新的解决方案。
经研究,本发明提供如下技术方案:
1.盔形毕赤酵母SCP类分泌蛋白,氨基酸序列如SEQ ID No.1或SEQ ID No.2所示。
2.盔形毕赤酵母SCP类分泌蛋白的编码基因。
进一步,所述编码基因的核苷酸序列如SEQ ID No.3或SEQ ID No.4所示。
3.含有盔形毕赤酵母SCP类分泌蛋白编码基因的重组表达载体。
进一步,所述重组表达载体是将核苷酸序列如SEQ ID No.3或SEQ ID No.4所示的盔形毕赤酵母SCP类分泌蛋白编码基因克隆入植物表达载体pCAMBIA2300的多克隆位点KpnI和PstI之间而得到。
4.含有上述重组表达载体的工程菌。
进一步,所述工程菌是将含有盔形毕赤酵母SCP类分泌蛋白编码基因的重组表达载体转入农杆菌GV3101中而得到。
5.盔形毕赤酵母SCP类分泌蛋白在制备诱导柑橘果实抗病性的保鲜剂中的应用。
进一步,所述诱导柑橘果实抗病性为诱导柑橘果实绿霉病抗病性。
6.所述工程菌在制备诱导柑橘果实抗病性的保鲜剂中的应用。
进一步,所述诱导柑橘果实抗病性为诱导柑橘果实绿霉病抗病性。
本发明的有益效果在于:本发明提供了盔形毕赤酵母SCP类分泌蛋白及其在制备诱导柑橘果实抗病性的保鲜剂中的应用,研究结果证实,农杆菌介导的遗传转化在柑橘果实上瞬时表达SCP类分泌蛋白,能一定程度抑制柑橘果实绿霉病的发病和病斑直径的增长。本发明从盔形毕赤酵母中挖掘出具有诱导柑橘果实抗病性的SCP类分泌蛋白,不仅丰富了盔形毕赤酵母诱导柑橘果实抗病性的机理,拓宽了拮抗酵母在果实保鲜领域的应用方式,也为柑橘果实采后绿霉病的防治提供了新的解决方法。
附图说明
图1为盔形毕赤酵母SCP类分泌蛋白PgSCP1、PgSCP2编码基因的电泳图,其中M为DNA分子量标准。
图2为重组载体pCAMBIA2300-PgSCP1、pCAMBIA2300-PgSCP2的电泳图,其中M为DNA分子量标准。
图3为含有PgSCP1或PgSCP2编码基因的农杆菌工程菌的电泳图,其中M为DNA分子量标准。
图4为盔形毕赤酵母SCP类分泌蛋白PgSCP1、PgSCP2对柑橘果实采后绿霉病的诱导效果;A和C为发病率,B和D为病斑直径,*表示与对照组(Control)相比差异显著(P<0.05);E为柑橘果实在接种指状青霉后于25℃贮藏6天后的绿霉病发病症状。
具体实施方式
为了使本发明的目的、技术方案和有益效果更加清楚,下面对本发明的优选实施例进行详细的描述。
本实施例所用盔形毕赤酵母分离于柑橘果园的柠檬果实表面。
1.引物设计
根据盔形毕赤酵母的基因序列和pCAMBIA2300载体上的多克隆位点KpnI和PstI,设计以下引物,委托生工生物工程(上海)股份有限公司进行合成。
PgSCP1-F:caatttactattctagggtaccATGCAATTATCTAGTCTAGCAGTTC(SEQ ID No.5)
PgSCP1-R:gtatgggtatctagactgcagTCAAGCGAGAGGCAAGACTTC(SEQ ID No.6)
PgSCP2-F:caatttactattctagggtaccATGAAGTTTTCAAAATCAGTGATAT(SEQ ID No.7)
PgSCP2-R:gtatgggtatctagactgcagTCAGAGAGAAAAGTTACCCTCT(SEQ ID No.8)
2.盔形毕赤酵母总RNA提取及cDNA合成
用北京酷来搏科技有限公司的真菌RNA提取试剂盒(RE781-50T)提取盔形毕赤酵母总RNA,具体步骤按照试剂盒说明书进行。
所得盔形毕赤酵母总RNA用PrimeScriptTM RT regent Kit试剂盒(Takara)反转录合成cDNA,具体步骤按照试剂盒说明书进行,用酶标仪Take3检测所得cDNA的浓度和质量,-20℃保存备用。
3.盔形毕赤酵母SCP类分泌蛋白编码基因序列克隆
以反转录合成的cDNA为模板,使用引物PgSCP1-F和PgSCP1-R,使用诺唯赞高保真酶P505试剂盒,PCR扩增核苷酸序列如SEQ ID No.3所示的PgSCP1编码基因序列,具体步骤按照试剂盒说明书进行。
以反转录合成的cDNA为模板,使用引物PgSCP2-F和PgSCP2-R,使用诺唯赞高保真酶P505试剂盒,PCR扩增核苷酸序列如SEQ ID No.4所示的PgSCP2编码基因序列,具体步骤按照试剂盒说明书进行。
PCR产物进行1%琼脂糖凝胶电泳(150V,30min),结果如图1所示,用无菌刀片切下目的条带,用胶回收试剂盒进行回收,具体步骤按照试剂盒说明书进行,回收产物-20℃保存备用。
4.质粒pCAMBIA2300的KpnI和PstI双酶切
将质粒pCAMBIA2300进行KpnI和PstI双酶切,酶切反应液置37℃金属浴中30min。
酶切产物进行琼脂糖凝胶电泳,用无菌刀片切下目的条带,用胶回收试剂盒进行回收,具体步骤按照试剂盒说明书进行,回收产物-20℃保存备用。
5.重组载体的构建
将回收的线性化质粒pCAMBIA2300与PgSCP1编码基因序列使用诺唯赞同源重组酶进行连接,具体步骤按照试剂说明书进行,连接反应液置37℃金属浴中30min。同样的方法将PgSCP2编码基因序列与线性化质粒pCAMBIA2300连接。
连接产物转化大肠杆菌感受态细胞。用质粒测序引物对转化子进行菌液PCR验证。验证结果为阳性的转化子委托生工生物工程(上海)股份有限公司进行测序。测序结果比对正确的重组质粒即为成功构建的重组载体pCAMBIA2300-PgSCP1、pCAMBIA2300-PgSCP2。
6.农杆菌工程菌的构建
提取重组载体pCAMBIA2300-PgSCP1、pCAMBIA2300-PgSCP2(电泳结果如图2所示,以质粒pCAMBIA2300为对照),分别加入农杆菌GV3101感受态细胞中,轻弹混匀,置冰上5min,液氮5min,37℃5min,冰上5min;加无抗LB液体培养基600μL,28℃、200rpm培育2~3h,4000rpm离心2min,弃上清,将余液均匀涂布于LB平板(含有25μg/L利福平和50μg/L卡那霉素);挑取单菌落至500μL LB液体培养基(含有25μg/L利福平和50μg/L卡那霉素)中,28℃、200rpm摇菌培养16h后检测目的片段。结果如图3所示,含有PgSCP1、PgSCP2编码基因的农杆菌即为成功构建的PgSCP1、PgSCP2农杆菌工程菌。
7.农杆菌工程菌在柑橘果实上瞬时表达分泌蛋白诱导柑橘果实绿霉病抗病性的效果
分别将PgSCP1、PgSCP2的农杆菌工程菌在LB培养基(含有25μg/L利福平和50μg/L卡那霉素)中培养过夜,当菌液OD600约为1时,4000rpm离心5min,将菌体用孵育液(1L孵育液含有5g葡萄糖,1.0663g MES(使用浓度5mM),0.760g Na3PO4·12H2O(使用浓度2mM),0.785mL 127.4mM AS母液(使用浓度0.1mM))重悬并调整OD600=0.8,等量混合基因沉默抑制子P19,28℃避光孵育2~3h,得农杆菌工程菌液;挑选大小均匀一致、色泽均匀、无机械伤和疤痕的柑橘果实,用2%次氯酸钠浸泡2min后用清水冲洗干净,自然风干,用75%酒精擦果实赤道部位,待酒精风干后,用1mL无菌枪头在果实赤道部位对立面各打一个孔,用1mL注射器(去掉针头)向孔中注射约0.5mL农杆菌工程菌液,以含有pCAMBIA2300质粒的农杆菌为对照(control);接种农杆菌工程菌1d后,在每个孔的右方1cm处再打一个孔,用移液枪接种10μL 1×104CFU/mL的指状青霉(Penicillium digitatum)孢子悬浮液,待菌液吸收后,单果套袋,避光放置于25℃的环境中,从第3天开始统计发病率和病斑直径。
结果如图4所示,含有PgSCP1、PgSCP2编码基因的农杆菌工程菌在柑橘果实上瞬时表达分泌蛋白PgSCP1(氨基酸序列如SEQ ID No.1所示)、PgSCP2(氨基酸序列如SEQ IDNo.2所示),能一定程度抑制柑橘果实绿霉病的发病和病斑直径的增长。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
序列表
<110> 西南大学
<120> 盔形毕赤酵母SCP类分泌蛋白及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 262
<212> PRT
<213> 盔形毕赤酵母(Pichia galeiformis)
<400> 1
Met Gln Leu Ser Ser Leu Ala Val Leu Ser Leu Leu Ser Ser Ile Ala
1 5 10 15
Ser Ala Ala Pro Val Val Glu Thr Val Thr Ala Val Val Thr Thr Thr
20 25 30
Val Ile Val Gly Gly Gln Thr Ser Ala Ile Asn Gln Ala Pro Val Phe
35 40 45
Thr Ala Ser Ala Ser Pro Val Glu Tyr Ala Ala Val Ala Asp Asp Ala
50 55 60
Thr Ser Ser Thr Ala Ala Ala Ala Ala Ala Val Thr Pro Ser Pro Thr
65 70 75 80
Thr Phe Glu Thr Ser Tyr Val Ala Ala Ala Thr Thr Asp Ala Ser Glu
85 90 95
Thr Ser Ser Thr Ser Ser Ala Asp Glu Gln Ala Thr Thr Ser Ser Thr
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Ser Thr Thr Ser Ala Ala Ala Ala Thr Thr Ser Ala Ser Ser Asp Phe
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Ala Ser Gln Ile Leu Ala Glu His Asn Ala Lys Arg Ala Leu His Gly
130 135 140
Val Ser Asn Leu Ser Trp Asp Asp Thr Leu Ala Thr Tyr Ala Gln Asn
145 150 155 160
Tyr Ala Asp Lys Tyr Asp Cys Ser Gly Ser Leu Thr His Ser Gly Gly
165 170 175
Gln Tyr Gly Glu Asn Leu Ala Leu Gly Tyr Thr Val Thr Gly Ser Val
180 185 190
Asp Ala Trp Tyr Ser Glu Gly Asp Ser Tyr Thr Tyr Gly Ser Gly Cys
195 200 205
Ser Val Tyr Asp His Phe Thr Gln Val Ile Trp Lys Asp Thr Thr Lys
210 215 220
Val Gly Cys Gly Tyr Lys Gln Cys Asp Ser Tyr Trp Gly Thr Tyr Ile
225 230 235 240
Ile Cys Ser Tyr Asp Pro Ala Gly Asn Tyr Val Gly Glu Cys Asp Ser
245 250 255
Glu Val Leu Pro Leu Ala
260
<210> 2
<211> 537
<212> PRT
<213> 盔形毕赤酵母(Pichia galeiformis)
<400> 2
Met Lys Phe Ser Lys Ser Val Ile Ser Leu Ile Ile Cys Thr Leu Ala
1 5 10 15
Ser Ala Glu Ala Ala Pro Thr Ile Pro Gln Asp Val Cys Ser Asp Ala
20 25 30
Ser Thr Val Thr Val Tyr Ser Ser Thr Phe Leu Pro Phe Pro Asn Ala
35 40 45
Val Ala Ser Pro Thr Ser Ile Glu Ser Leu Ser Thr Val Ser Thr Pro
50 55 60
Thr Thr Ser Ser Ser Thr Val Thr Ser Ser Ser Glu Thr Ser Ser Ser
65 70 75 80
Ala Ser Ser Ser Ser Ser Ser Leu Ser Thr Glu Phe Ser Val Ser Ile
85 90 95
Pro Thr Ser Ser Ser Ser Ser Thr Ser Ser Ser Ser Ser Thr Ser Ser
100 105 110
Ser Ser Ser Thr Ser Ser Ser Ser Ser Thr Ser Ser Ser Ser Ser Thr
115 120 125
Ser Phe Ser Ser Ser Thr Ser Ser Thr Ser Ser Ser Ser Tyr Asp Asp
130 135 140
Ser Thr Ser Thr Ser Gly Phe Ser Ile Val Gly Pro Ala Ser Pro Val
145 150 155 160
Ile Thr Ser Ser Ser Val Ser Leu Asp Ser Ser Thr Gly Gly Phe Ile
165 170 175
Phe Thr Thr Ser Ile Asn Ser Pro Asp Ser Ser Ser Thr Ser Ser Ile
180 185 190
Asp Thr Leu Thr Thr Ser Thr Ser Gly Glu Ser Ser Ala Thr Thr Gly
195 200 205
Thr Ala Asn Asn Ser Ser Lys Lys Ala Thr Ser Thr Ser Thr Ser Ile
210 215 220
Ala Ser Leu Thr Glu Ser Ser Ser Ser Ser Thr Ser Ser Thr Ser Ser
225 230 235 240
Thr Glu Ser Ser Ser Ser Ser Thr Ser Ser Thr Ser Ser Thr Glu Ser
245 250 255
Ser Ser Ser Ser Thr Ser Ser Thr Ser Ser Thr Glu Ser Ser Ser Ser
260 265 270
Ser Thr Ser Ser Thr Ser Ser Thr Lys Pro Thr Ser Thr Ser Thr Ser
275 280 285
Ser Thr Ser Thr Thr Ser Ser Thr Glu Pro Thr Ser Thr Ser Thr Ser
290 295 300
Ser Thr Ser Thr Thr Ser Ser Thr Glu Pro Thr Ser Thr Ser Thr Ser
305 310 315 320
Ser Thr Ser Thr Thr Ser Ser Thr Glu Pro Thr Ser Thr Ser Thr Ser
325 330 335
Ser Thr Ser Thr Thr Ser Ser Thr Glu Pro Thr Ser Thr Ser Thr Ser
340 345 350
Ala Thr Ser Thr Thr Thr Ser Ala Thr Thr Ala Ala Thr Thr Ser Ser
355 360 365
Ser Ser Thr Phe Asp Phe Ala Thr Ala Tyr Leu Asp Lys His Asn Tyr
370 375 380
Phe Arg Ser Leu Met Lys Asp Thr Asn Asp Val Thr Trp Asn Ser Asp
385 390 395 400
Ile Ala Ala Ile Ala Gln Ser Tyr Ala Asp Glu Tyr Thr Cys Gly Ala
405 410 415
Asp Leu Glu His Ser Gly Asn Ser Tyr Asn Gly Gly Ala Leu Gly Glu
420 425 430
Asn Leu Ala Arg Gly Phe Asn Phe Gln Lys Ala Ser Gly Val Thr Ala
435 440 445
Trp Phe Asn Glu Ile Lys Tyr Tyr Asn Tyr Ser Glu Pro Gly Phe Thr
450 455 460
Glu Asp Thr Gly His Phe Thr Gln Leu Val Trp Ser Glu Thr Thr Glu
465 470 475 480
Ile Gly Cys Gly Tyr Lys Asn Cys Gly Ser Tyr Trp Gly Tyr Tyr Val
485 490 495
Val Cys Asn Tyr Ser Pro Pro Gly Asn Val Gly Leu Val Gly Gly Ser
500 505 510
Asp Pro Asn Tyr Phe Tyr Glu Arg Asp Val His Glu Pro Ile Asp Pro
515 520 525
Ala Thr Ala Glu Gly Asn Phe Ser Leu
530 535
<210> 3
<211> 789
<212> DNA
<213> 盔形毕赤酵母(Pichia galeiformis)
<400> 3
atgcaattat ctagtctagc agttctctcc ttactctcct cgatcgccag cgctgcgcct 60
gtcgtggaaa cagtcacagc cgtggtcacc acgacggtga ttgtcggcgg ccagacgtcg 120
gctatcaacc aggcaccggt cttcacggcg tccgcctccc ctgtcgaata cgcagcagtc 180
gccgacgacg caacttccag cacggctgca gccgcagccg ccgtcactcc ttctccaacc 240
acgttcgaaa cctcgtacgt cgccgccgca accaccgacg cgtccgaaac ctcgtccacg 300
tcttctgccg acgaacaagc aaccacctcc tccacctcca ccacctccgc agctgccgcc 360
actacgtcgg cctcctcgga cttcgcttcg cagatcttgg ctgaacacaa cgccaagaga 420
gcgttgcacg gcgtctccaa cttgtcttgg gacgacacct tggccaccta cgcgcaaaac 480
tacgcggaca agtacgactg ctccggctcc ttgacccact ccggcggcca gtacggtgaa 540
aacttggccc tcggatacac cgtcacaggc tctgtcgacg catggtactc cgaaggtgac 600
agctacacct acggctccgg ctgctctgtc tacgaccatt tcacacaggt catctggaaa 660
gacaccacca aggtcggatg cggttacaag caatgtgact cctactgggg aacatacatc 720
atctgctcct acgacccagc aggtaactat gtcggtgaat gtgactccga agtcttgcct 780
ctcgcttga 789
<210> 4
<211> 1614
<212> DNA
<213> 盔形毕赤酵母(Pichia galeiformis)
<400> 4
atgaagtttt caaaatcagt gatatcccta atcatctgca ctttggcatc cgccgaagct 60
gcgcctacaa ttccccagga cgtctgctcc gatgcttcga cagtcaccgt gtattcgtcc 120
actttcttac cattcccgaa tgctgttgct tcccctactt ctattgaatc gttgtccacc 180
gtttctacgc cgacaacttc ctcaagcact gtgactagtt cttctgagac ctcatcctct 240
gcttccagct cgagttcatc gctatctacg gaattctcag tctcaattcc aaccagctct 300
tcaagctcaa ccagctcttc aagctcaacc agctcttcaa gctcaaccag ctcttcaagc 360
tcaaccagct cttcaagctc aaccagcttt tcaagctcaa ccagctcaac cagctcatcc 420
agctacgacg attccacgag cacctccggg ttctcaatcg taggccctgc ttctccggta 480
ataaccagct ctagtgttag cttggatagt tcaaccggcg ggttcatctt taccaccagc 540
attaacagtc ctgattcaag ctctacgagc tcgatcgaca ccttgacgac ttcgacctcc 600
ggtgaatcta gtgcaactac tgggactgcc aacaattctt cgaagaaggc aacaagcacg 660
tccacttcta tcgcttcctt gacggagtca tcgagctcat ctacttctag tacttcctcg 720
acggaatcat cgagctcatc tacttctagt acttcctcga cggaatcatc gagctcatct 780
acttctagta cttcctcgac ggaatcatca agctcatcta cttctagtac ctcatcgaca 840
aagccaacaa gcacatctac ttctagtact tctaccactt cctcgacgga gccaacaagc 900
acatctactt ctagtacttc taccacttcc tcgacggagc caacaagcac atctacttct 960
agtacttcta ccacttcctc gacggagcca acaagcacat ctacttctag tacttctacc 1020
acttcctcga cggagccaac aagcacgtcc acctctgcca cttccacgac gacgagtgct 1080
actactgctg ccaccacatc ctcatccagc acgtttgact ttgccaccgc ctacttggac 1140
aaacacaatt atttcagaag cctcatgaag gacacaaatg acgtcacttg gaactcagac 1200
attgccgcta tcgcacaaag ctatgccgat gagtacacct gcggtgccga ccttgagcac 1260
tccgggaact cttacaacgg cggggccttg ggagaaaact tagcacgtgg cttcaacttc 1320
caaaaggcca gcggcgtcac tgcatggttc aacgaaatca agtactacaa ctacagcgag 1380
cctggtttca ccgaggacac cggtcacttc acgcagctcg tgtggagtga gacaaccgag 1440
atcggttgtg gatacaagaa ctgtggcagc tactggggtt actacgtcgt gtgcaactac 1500
agcccaccag ggaacgttgg gctggttggc ggcagtgacc ctaactactt ctacgagagg 1560
gacgtccacg aacctatcga cccggcaact gcagagggta acttttctct ctga 1614
<210> 5
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
caatttacta ttctagggta ccatgcaatt atctagtcta gcagttc 47
<210> 6
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtatgggtat ctagactgca gtcaagcgag aggcaagact tc 42
<210> 7
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
caatttacta ttctagggta ccatgaagtt ttcaaaatca gtgatat 47
<210> 8
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gtatgggtat ctagactgca gtcagagaga aaagttaccc tct 43
Claims (8)
1.盔形毕赤酵母SCP类分泌蛋白,其特征在于,氨基酸序列如SEQ ID No.1所示。
2.权利要求1所述盔形毕赤酵母SCP类分泌蛋白的编码基因。
3.如权利要求2所述的编码基因,其特征在于,核苷酸序列如SEQ ID No.3所示。
4.含有权利要求2所述编码基因的重组表达载体。
5.如权利要求4所述的重组表达载体,其特征在于:是将核苷酸序列如SEQ ID No.3所示的盔形毕赤酵母SCP类分泌蛋白的编码基因克隆入植物表达载体pCAMBIA2300的多克隆位点KpnI和PstI之间而得到。
6.含有权利要求4所述重组表达载体的工程菌。
7.如权利要求6所述的工程菌,其特征在于,是将所述重组表达载体转入农杆菌GV3101中而得到。
8.权利要求1所述的盔形毕赤酵母SCP类分泌蛋白或权利要求6所述的工程菌在制备诱导柑橘果实绿霉病抗病性的保鲜剂中的应用。
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