CN114561410A - 极端嗜盐曲霉Hog1基因及其在提高植物耐盐性中的应用 - Google Patents
极端嗜盐曲霉Hog1基因及其在提高植物耐盐性中的应用 Download PDFInfo
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- CN114561410A CN114561410A CN202210325886.1A CN202210325886A CN114561410A CN 114561410 A CN114561410 A CN 114561410A CN 202210325886 A CN202210325886 A CN 202210325886A CN 114561410 A CN114561410 A CN 114561410A
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- aspergillus
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- hog1 gene
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Abstract
本发明提供一种极端嗜盐曲霉Hog1基因及其在提高植物耐盐性中的应用,本发明将极端嗜盐曲霉Hog1基因转入植物,经模拟盐胁迫试验表明,转基因植物在盐度胁迫下生长状态、生物量、种子重量方面明显优于野生型植物,耐盐性显著增强,表明本发明提供的Hog1基因在提高植物耐盐碱方面的具有重要应用价值,为植物抗逆改良提供了重要基因资源。
Description
技术领域
本发明属于植物基因工程技术领域,更具体地说,涉及一种极端嗜盐曲霉Hog1基因及其在提高植物耐盐胁迫能力中的应用。
背景技术
土壤盐碱化会导致土壤中含盐过量、渗透潜力下降、土壤结构变化、妨碍作物对水肥的吸收。因此土壤盐碱化是限制农作物生长、造成粮食减产的主要非生物胁迫因子之一。近年来,我国土壤盐碱化面积逐年增加,造成大量土地闲置,由于盐碱地无法经过短期改造进行他用,对我国粮食安全和土地资源合理利用影响巨大。利用基因工程技术提高植物耐盐性,是维持盐碱地农业生产、生态修复盐碱地的有效途径之一。
目前利用基因工程技术提高植物的耐盐性的研究主要集中在烟草、拟南芥等模式植物。例如刘珂采用叶盘法将蜡样芽孢杆菌HK012的acdS基因转入烟草,发现该基因在烟草中的表达能够有效缓解盐胁迫对植株的伤害。张超利用农杆菌介导法将从疏绵状嗜热丝孢菌和嗜热子囊菌光孢变种中分离出的MnSOD酶基因转入烟草,提高了烟草在种子萌发及小苗生长阶段的盐耐受性。目前转基因技术所采用的基因大多来自非耐盐物种,这使得转基因植株仅对一定范围的盐胁迫具有耐受性。
油菜是我国重要的油料作物,土壤盐碱化制约着油菜在盐碱地上的种植。提高油菜耐盐性对于提高盐碱地油菜的种植面积和产量以及盐碱地改良利用具有重要意义。
发明内容
本发明所要解决的技术问题在于提供一种极端嗜盐曲霉Hog1基因及其在提高植物耐盐性中的应用,极端嗜盐曲霉Hog1基因转入植物,提高了植物耐盐胁迫能力。
本发明是通过以下技术方案来实现:
一种极端嗜盐曲霉Hog1基因,其核苷酸序列如SEQ ID NO.1所示。
一种权利要求1所述的极端嗜盐曲霉Hog1基因编码的蛋白,其氨基酸序列如SEQID NO.2所示。
一种含权利要求1所述的极端嗜盐曲霉Hog1基因的表达载体。
优选的,所述表达载体由极端嗜盐曲霉Hog1基因和pBWA(V)HS连接得到。
一种含所述的表达载体的细胞。
一种含所述的极端嗜盐曲霉Hog1基因的宿主菌。
优选的,所述宿主菌为农杆菌。
所述的极端嗜盐曲霉Hog1基因在提高植物耐盐性中的应用。
优选的,所述应用包括以下步骤:
1)构建含极端嗜盐曲霉Hog1基因的表达载体;
2)将所构建的含极端嗜盐曲霉Hog1基因的表达载体转化到植物或植物细胞中;
3)培育步骤2)所得植物或植物细胞,筛选得到耐盐性提高的转基因植物。
优选的,所述的植物为油菜。
与现有技术相比,本发明具有以下有益的技术效果:
本发明从极端嗜盐生物基因组中挖掘新的耐盐基因,并利用基因工程手段提高作物耐盐性,具体是将极端嗜盐曲霉(Aspergillus montevidensis)的Hog1基因转入植物,经模拟盐胁迫试验表明,转基因植物在盐度胁迫下生长状态、生物量、种子重量方面明显优于野生型植物,耐盐性显著增强,表明本发明提供的Hog1基因在提高植物耐盐碱方面的具有重要应用价值,为植物抗逆改良提供了重要基因资源。
附图说明
图1为过表达载体pBWA(V)HS-Hog1-osgfp图谱。
图2为基于cDNA序列构建的系统发育树。
图3为基于氨基酸序列构建的系统发育树。
图4为转基因植株Hog1基因PCR检测电泳照片。其中,a为PCR反应扩增Hog1基因的电泳照片,M为DL6000DNA marker,1为Hog1基因PCR产物;b是Hog1基因转入载体后菌落PCR的电泳照片,M为DL6000DNA marker。
图5为Hog1基因在转基因油菜与野生型油菜植株的根部分布对比图;
图6为转基因油菜植株的获得。
图7为不同盐浓度下转基因油菜植株株高分析图。
图8为不同盐浓度下转基因油菜植株生物量分析图。
图9为转基因油菜种子干重分析图。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。
本发明以极端嗜盐曲霉(A.montevidensis)为材料,使用引物经PCR扩增基因片段,目的cDNA片段长度为822bp,如SEQ ID NO.1所示,编码273个氨基酸的蛋白,如SEQ IDNO.2所示。
由生物信息学分析得知,极端嗜盐曲霉Hog1基因对应目标蛋白质的长度应为273,分子量为35KDa,预测等电点为6.30。
将极端嗜盐曲霉Hog1基因的cDNA序列提交到GenBank进行数据比对,分析显示,该基因序列依次与Aspergillus glaucus CBS 516.65、Aspergillus ruber CBS 135680、Aspergillus steynii IBT 23096、Aspergillus chevalieri等菌株Hog1基因的同源性在87.6%~71.72%之间。下载相似性高的Hog1基因的cDNA序列构建最大简约树,参见图2,其中与所研究的目标菌株A.montevidensis的Hog1基因同源性较高的是A.glaucus CBS516.65,两者形成一个独立分支,步长值87%。同时A.ruber CBS 135680形成的亲缘关系较近,与两者形成的亲缘关系较近,三者共同形成一个较大分支。
将Hog1基因编码的氨基酸序列同GenBank数据库数据进行比较分析显示,所推测的氨基酸序列同Aspergillus glaucus(登录号:ABB16294)、Eurotium herbariorum(登录号:ABB16294)、A.niger(登录号:EHA18150)、A.kawachii(登录号:GAA88530)、A.oryzae(登录号:EIT78792)、A.clavatus(登录号:EAW07538)、Blumeria graminis(登录号:Q8TGA9)及Talaromyces stipitatus(登录号:XP_002478549)等Hog1 MAPK同源性在88%–97%之间。下载相似性高的Hog1 MAPK氨基酸序列构建最大简约树,参见图3。阿姆斯特丹曲霉M–70同A.glaucus(登录号:ABB16294)和E.herbariorum(登录号:ABB16294)聚于一个分枝上,分枝自展支持率达到99%,说明该分枝可靠性较高。尽管阿姆斯特丹曲霉Hog1基因DNA序列同数据库中真菌Hog1同源基因序列相似性在69%–87%之间,然而不同真菌HOG1 MAPK氨基酸序列相似性很高,该基因在进化过程中具有结构功能的保守性。
本发明将获得的Hog1基因序列,如SEQ ID NO.1所示的核苷酸序列,与表达载体pBWA(V)HS连接构建得到原核表达载体pBWA(V)HS-HOG1-osgfp,转入大肠杆菌中,经过筛选验证,再转化过表达载体到农杆菌GV3101中,后侵染油菜外植体。
本发明对过表达Hog1基因的油菜在不同盐浓度(0mM、60mM、120mM、240mM)处理下,通过对油菜长度、重量及种子干重的测定,结果表明,Hog1基因过表达可显著提高油菜的耐盐胁迫能力,分别如图6-8所示。
实施例1Hog1基因过表达载体pBWA(V)HS-Hog1-osgfp的构建
1.极端嗜盐曲霉(A.montevidensis ZYD4)的培养及诱导
将实验室保存的极端嗜盐曲霉(A.montevidensis ZYD4)的菌种接种于沙氏培养基(蛋白胨10g、琼脂20g、葡萄糖40g,加蒸馏水定容至1L,115℃,20min高压蒸汽灭菌),28℃恒温培养168h,观察记录。加浓度为3M NaCl诱导培养30分钟,镜检观察。将菌丝用滤纸过滤,去离子水冲洗三次,液氮冷冻保存。
2.Hog1基因cDNA序列的获得
取收集的菌丝体,先用去离子无菌水清洗三次,再经75%乙醇消毒,将菌体置于研钵中,加入液氮,缓慢研磨,直至变为粉末状。以获得的菌丝破碎物为原料结合Trizol总DNA提取试剂盒说明书对菌体总DNA进行提取,在-70℃环境下储藏收集到的DNA,备用。
3.设计并合成引物
(1)设计PCR扩增片段引物,并在引物末端引入酶切位点保护碱基,使得扩增产物5’和3’最末端序列分别具有相应的酶切位点。
(2)将设计好的引物序列送武汉伯远生物科技有限公司进行合成。
引物序列如下:
HOG1-F:
5’-CAGTGGTCTCACAACATGAAGCGTACGTTCAGAGA-3’
HOG1-R:
5’-CAGTGGTCTCATACACATTCTTTTCTTCCACTCCT-3’
4.扩增目的片段
(1)将合成的引物稀释成终浓度为10μmol/L的储藏液。
(2)利用稀释的引物及模板进行PCR扩增。体系如下:
将上述材料加入薄壁管内混匀并点离后放入PCR仪内,选择好合适的退火温度和延伸温度,即可开始PCR扩增。
(3)PCR结束后进行琼脂糖凝胶电泳,电泳结果参见图4(a),显示条带为800bp左右的片段。回收目的基因,纯化产物标记为rDNAG1。
5.Hog1目的片段和载体pBWA(V)HS的双酶切
(1)用限制性内切酶BsaI和Eco31I进行双酶切。
载体pBWA(V)HS酶切体系如下:
载体 | 4ul |
Buffer | 2ul |
BsaI | 1ul |
Eco31I | 1ul |
H<sub>2</sub>O | 13ul |
Hog1目的片段酶切体系如下:
目的片段 | 4ul |
Buffer | 2ul |
BsaI | 1ul |
Eco31I | 1ul |
H<sub>2</sub>O | 13ul |
于37℃酶切约1h。
(2)将载体酶切物和回收片段酶切产物合并一起用PCR纯化试剂盒纯化,纯化产物标记为P-rDNAG1。
6.Hog1目的片段和载体pBWA(V)HS的连接
连接体系如下:
P-rDNAG1 | 2.5ul |
Buffer | 1ul |
T4-ligase | 1ul |
H<sub>2</sub>O | 5.5ul |
于37℃连接1h。
7.转化
(1)将感受态细胞DH5α置于冰上待其自然解冻后,取10μLHOG1目的片段和载体pBWA(V)HS的连接产物加入感受态细胞中于冰上放置15min。
(2)之后于42℃水浴中热激45s,热激转化,然后迅速置于冰上(4℃)放置5min。
(3)加入300μL不含抗生素的SOC培养基,于37℃、200rpm振荡培养45min。
(4)将活化培养处理后的感受态细胞,接种于含卡那霉素(30μg/ml)的LB固体培养基平板上,将培养皿倒置于37℃环境中培养12h,得到过表达载体pBWA(V)HS-HOG1-osgfp。
8.菌斑PCR鉴定
挑取10个菌斑同时进行200ul EP管接菌和PCR鉴定
(1)菌斑PCR引物序列
Hog1菌检-F:5’-CAGGAATATAGCTACGCCG-3’
GFP-40R:5’-TCGCCGTCGAGCTCCACGAGG-3’
(2)PCR体系如下:
将上述材料加入薄壁管内混匀并点离后放入PCR仪内,选择好合适的退火温度和延伸温度,即可开始PCR扩增。
(3)PCR结束后进行琼脂糖凝胶电泳,电泳结果参见图4(b),检测目标条带为500bp左右的片段。
(4)取1-3个阳性条带对应的菌液,取100ul送样测序。
实施例2过表达载体pBWA(V)HS-Hog1-osgfp转化至农杆菌
(1)使用的农杆菌菌株为GV3101。采用的是液氮冻融法将构建好的pBWA(V)HS-Hog1-osgfp表达载体(参见图1)转入农杆菌。
具体操作如下:
1)取-80℃保存的农杆菌感受态于冰上融化。
2)每100μL感受态加入0.01-1μg质粒DNA(过表达载体pBWA(V)HS-Hog1-osgfp),用手拨打管底混匀,依次于冰上静置5min、液氮5min、37℃水浴5min、冰浴5min。
3)加入700μL无抗生素的LB液体培养基,于28℃振荡培养2~3h。
4)6000rpm离心一分钟收菌,留取100μL左右上清轻轻吹打重悬菌块涂布于含相应抗生素的LB平板上,倒置放于28℃培养箱培养48h。
5)PCR检测阳性克隆,4℃保存备用。
实施例3农杆菌侵染油菜
1.制备油菜外植体
挑选油菜种子进行消毒,将消毒后的油菜种子接种于MS培养基上,生长7d获得油菜无菌苗,待幼苗下胚轴长到瓶口时,用镊子取出无菌苗,切除子叶柄及子叶尖,下胚轴切成1到2cm的小段段作为外植体,放置于预培培养基上。
2.浸染
将PCR检测的阳性克隆农杆菌,摇菌至OD6000.8时,将预培养2-3d的油菜外植体置于农杆菌悬浮液中侵染10min,将侵染后的油菜外植体放于滤纸板上晾干,置于共培培养基上,共培2d,再外植体转入延筛培养基上,培养7d。
3.愈伤的诱导及筛选
挑选有效愈伤组织转移至含有潮霉素的筛选培养基,筛选15d左右,共筛选2到3次。
4.分化及生根
将生长旺盛的阳性愈伤组织转移到分化培养基上,待其分化出幼苗,将分化出来的幼苗转移到生根培养上生根7-10d,获得阳性植株。
5.检测
将已经长根的幼苗进行标号,各取0.5cm2的油菜叶片,磨样,吸取磨样液作为DNA模板进行PCR扩增,跑琼脂糖凝胶电泳,判断阳性幼苗以及阳性率。
6.荧光显微镜观察
分别剪取阳性幼苗和野生型幼苗的根系,装片后,用荧光显微镜对比观察Hog1基因在植物根部的分布情况。结果如图5所示,野生型幼苗在显微镜下几乎无绿色显现,而阳性幼苗可观察到明显绿色,表明Hog1基因成功转入植物根部并大量扩增。
实施例3转基因油菜耐盐胁迫功能的研究
将转基因油菜与野生型油菜在含不同盐浓度(0mM、60mM、120mM、240mM)进行栽培,测定油菜植株长度(根+茎)、重量及种子干重。
获得成熟的转基因油菜植株,如图6所示,转基因油菜生长情况良好,叶片肥大、根系旺盛。
以不同盐浓度对油菜长度绘制柱形图,结果如图7所示,可以看出,转基因油菜相较于野生型油菜在高盐浓度下植株生长状况更好。
以不同盐浓度对油菜重量绘制柱形图,结果如图8所示,可以看出,转基因油菜相较于野生型油菜在高盐浓度下植株更重。
以不同盐浓度对油菜种子干重绘制柱形图,结果如图9所示,可以看出,转基因油菜相较于野生型油菜种子质量更优。
序列表
<110>陕西科技大学
<120>极端嗜盐曲霉Hog1基因及其在提高植物耐盐性中的应用
<160>6
<170>SIPOSequenceListing 1.0
<210>1
<211>822
<212>DNA
<213>极端嗜盐曲霉(A.montevidensis ZYD4)
<400>1
atgaagcgta cgttcagaga agtgcacttg ttgaatagac tgcgacatga taatcttatc 60
aatatgaatg atatcttcat ctctccgtcg gaagacatat atctggtcac ggattttatg 120
atgacagatc tgcatcaagt tattcgagag acaacgttgg agggccaatt tatccagttc 180
tttacttatc agatcctgcg aggattgaaa ttcatccatt cagccggtgt tatccatcgt 240
gacctgaagc cccagaatct cctcgttaac aacaattgtg atcttaaaat ttgtgatttt 300
ggactcgcgc gggagcaaga ccaccagatg accggctatg tcgtgacaag atattaccgg 360
gccccagaag tcatgctgac atggcaggaa tatagctacg ccgttgacat gtggagcgca 420
ggatgcattt ttgccgagat gctccgggga acgcccctct ttccaggtaa aaaccatatt 480
gaccagttca cgatcatcac gcaagttttg ggaaacccac ctcaggaggt tgttgagagg 540
gtatacagca gaaatacact gaaattcctg gagtcgttac caccgcgcga gccacgtccg 600
ctttcatcgt tcttcacagg tgttgaagag gaagcggttg acctcattga gaaaatgctt 660
caacttgacc cgtacaagag gatcactgct acagacgccc tgtctcatcc atatcttgtg 720
aattttcatg attcagacga tgagcctgtg gctagtcaag aaattgacat gtcatatgac 780
gaagtaaaac tttcgccgga ggagtggaag aaaagaatgt ga 822
<210>2
<211>273
<212>PRT
<213>极端嗜盐曲霉(A.montevidensis ZYD4)
<400>2
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Asp Asn Leu Ile Asn Met Asn Asp Ile Phe Ile Ser Pro Ser Glu Asp
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Ile Tyr Leu Val Thr Asp Phe Met Met Thr Asp Leu His Gln Val Ile
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Arg Glu Thr Thr Leu Glu Gly Gln Phe Ile Gln Phe Phe Thr Tyr Gln
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Ile Leu Arg Gly Leu Lys Phe Ile His Ser Ala Gly Val Ile His Arg
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Asp Leu Lys Pro Gln Asn Leu Leu Val Asn Asn Asn Cys Asp Leu Lys
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Ile Cys Asp Phe Gly Leu Ala Arg Glu Gln Asp His Gln Met Thr Gly
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Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Met Leu Thr Trp
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Gln Glu Tyr Ser Tyr Ala Val Asp Met Trp Ser Ala Gly Cys Ile Phe
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Ala Glu Met Leu Arg Gly Thr Pro Leu Phe Pro Gly Lys Asn His Ile
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Asp Gln Phe Thr Ile Ile Thr Gln Val Leu Gly Asn Pro Pro Gln Glu
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Val Val Glu Arg Val Tyr Ser Arg Asn Thr Leu Lys Phe Leu Glu Ser
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Leu Pro Pro Arg Glu Pro Arg Pro Leu Ser Ser Phe Phe Thr Gly Val
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Glu Glu Glu Ala Val Asp Leu Ile Glu Lys Met Leu Gln Leu Asp Pro
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Tyr Lys Arg Ile Thr Ala Thr Asp Ala Leu Ser His Pro Tyr Leu Val
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Asn Phe His Asp Ser Asp Asp Glu Pro Val Ala Ser Gln Glu Ile Asp
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Met Ser Tyr Asp Glu Val Lys Leu Ser Pro Glu Glu Trp Lys Lys Arg
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Met
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<210> 3
<211> 35
<212> DNA
<213> HOG1-F(Artificial)
<400> 3
cagtggtctc acaacatgaa gcgtacgttc agaga 35
<210> 4
<211> 35
<212> DNA
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<400> 4
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<210> 5
<211> 19
<212> DNA
<213> HOG1菌检-F(Artificial)
<400> 5
caggaatata gctacgccg 19
<210> 6
<211> 21
<212> DNA
<213> GFP-40R (Artificial)
<400> 6
tcgccgtcga gctccacgag g 21
Claims (10)
1.一种极端嗜盐曲霉Hog1基因,其特征在于,其核苷酸序列如SEQ ID NO.1所示。
2.一种权利要求1所述的极端嗜盐曲霉Hog1基因编码的蛋白,其特征在于,其氨基酸序列如SEQ ID NO.2所示。
3.一种含权利要求1所述的极端嗜盐曲霉Hog1基因的表达载体。
4.根据权利要求3所述的含极端嗜盐曲霉Hog1基因的表达载体,其特征在于,所述表达载体由极端嗜盐曲霉Hog1基因和pBWA(V)HS连接得到。
5.一种含权利要求3或4所述的表达载体的细胞。
6.一种含有权利要求1所述的极端嗜盐曲霉Hog1基因的宿主菌。
7.根据权利要求6所述的含极端嗜盐曲霉Hog1基因的宿主菌,其特征在于,所述宿主菌为农杆菌。
8.权利要求1所述的极端嗜盐曲霉Hog1基因在提高植物耐盐性中的应用。
9.根据权利要求8所述的应用,其特征在于,包括以下步骤:
1)构建含极端嗜盐曲霉Hog1基因的表达载体;
2)将所构建的含极端嗜盐曲霉Hog1基因的表达载体转化到植物或植物细胞中;
3)培育步骤2)所得植物或植物细胞,筛选得到耐盐性提高的转基因植物。
10.根据权利要求8或9所述的应用,其特征在于,所述的植物为油菜。
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CN103131718A (zh) * | 2012-12-28 | 2013-06-05 | 江南大学 | 来源产甘油假丝酵母新型耐高渗功能基因CgHog1的克隆及其应用 |
CN103820491A (zh) * | 2014-01-26 | 2014-05-28 | 河北农业大学 | 玉米大斑病菌stk1基因在植物抗盐方面的应用 |
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WO2008095826A1 (en) * | 2007-02-05 | 2008-08-14 | Vib Vzw | Method to obtain salt tolerance in eukaryotic cells |
CN103131718A (zh) * | 2012-12-28 | 2013-06-05 | 江南大学 | 来源产甘油假丝酵母新型耐高渗功能基因CgHog1的克隆及其应用 |
CN103820491A (zh) * | 2014-01-26 | 2014-05-28 | 河北农业大学 | 玉米大斑病菌stk1基因在植物抗盐方面的应用 |
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