水稻osgadd45a1osgadd45a2双突变体的构建

摘要水稻（OryzasativaL.）作为三大主要粮食作物之一，全球逾半数人口以其为主食，探究水稻生长发育的分子调控机制是利用分子设计育种开发高产优质水稻新品种的基础。生长阻滞和DNA损伤诱导45蛋白（GrowtharrestandDNAdamage-inducible45;GADD45）是哺乳动物中关键的应激响应因子，由于其在DNA损伤修复、细胞周期控制和细胞凋亡等多个过程中发挥重要作用，在哺乳动物广泛被研究，但其在植物中的同源基因功能研究较少。前期实验室发现水稻中GADD45a的2个同源基因OsGADD45a1与OsGADD45a2能够参与调控水稻生长发育及抗稻瘟病的生理过程，且两个基因编码蛋白具有高度相似的氨基酸序列（仅有3个氨基酸差异），OsGADD45a2的过量表达能够降低全基因组mCG环境的DNA甲基化水平。为了进一步探究二者是否可能存在功能上的冗余，本研究试图分别通过CRISPR/Cas9和人工microRNA（amiRNA）技术构建osgadd45a1osgadd45a2双突变体，为后续对OsGADD45a在水稻生长发育和胁迫响应以及DNA甲基化调节过程中的功能进行进一步探索提供材料支撑。本论文主要研究结果发现：（1）CRISPR/Cas9技术在T0代未能够直接获得双基因纯合移码突变或提前终止的功能缺失性突变体，其中一个基因纯合移码突变、另一个基因杂合或双等位基因突变的植株，最适用于后续进一步筛选；（2）基于amiRNA的基因沉默策略成功实现了OsGADD45a2基因缺失（纯合移码突变）的背景下，OsGADD45a1基因表达抑制的突变体植株。本研究旨在构建osgadd45a1osgadd45a2双突变体，为后续探究OsGADD45a1与OsGADD45a2在水稻中是否存在功能冗余，同时为解析OsGADD45家族调控DNA甲基化奠定了基础。关键词：水稻；突变体；osgadd45a1osgadd45a2；CRISPR/Cas9；amiRNA

1引言1.1水稻种植及粮食安全现状水稻（Oryzasativa

L.）作为全球至关重要的谷类作物之一，主要种植于亚洲，养活了全球一半以上的人口ADDINEN.CITE<EndNote><Cite><Author>Terada</Author><Year>2002</Year><RecNum>1</RecNum><DisplayText><styleface="superscript">[1]</style></DisplayText><record><rec-number>1</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741519203">1</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Terada,R.</author><author>Urawa,H.</author><author>Inagaki,Y.</author><author>Tsugane,K.</author><author>Iida,S.</author></authors></contributors><titles><title>Efficientgenetargetingbyhomologousrecombinationinrice</title><secondary-title>NatureBiotechnology</secondary-title></titles><periodical><full-title>NatureBiotechnology</full-title></periodical><pages>1030-1034</pages><volume>20</volume><number>10</number><dates><year>2002</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>1087-0156</isbn><accession-num>WOS:000178313100021</accession-num><work-type>Article</work-type><urls><related-urls><url><GotoISI>://WOS:000178313100021</url></related-urls></urls><electronic-resource-num>10.1038/nbt737</electronic-resource-num></record></Cite></EndNote>[1]。我国的水稻种植面积约占全球的20%，主要集中在长江流域、珠江流域和东北地区，产量占全球的28%左右。近年来，随着农业技术的进步，我国水稻单产水平显著提高，2021年全国水稻平均单产达到6.8吨/公顷（农业农村部,2022）。然而，由于受耕地面积减少、种植成本上升、极端环境频发等因素影响，水稻生产面临着诸多挑战。稻瘟病（Magnaportheoryzae）属于对农作物危害极大的十大真菌病害之一，对水稻产量造成了极为严重的影响。在全球范围内，由于稻瘟病的侵袭，每年水稻产量会降低10%-30%ADDINEN.CITE<EndNote><Cite><Author>杜轶威</Author><Year>2016</Year><RecNum>2</RecNum><DisplayText><styleface="superscript">[2]</style></DisplayText><record><rec-number>2</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741523319">2</key></foreign-keys><ref-typename="Thesis">32</ref-type><contributors><authors><author>杜轶威</author></authors><tertiary-authors><author>王涛,</author></tertiary-authors></contributors><titles><title>水稻开花相关RING蛋白1(FRRP1)基因的克隆和开花功能分析</title></titles><keywords><keyword>FRRP1</keyword><keyword>H2B</keyword><keyword>RING结构域</keyword><keyword>E3泛素连接酶</keyword><keyword>水稻</keyword></keywords><dates><year>2016</year></dates><work-type>博士</work-type><urls><related-urls><url>/kcms2/article/abstract?v=4fayqqv3WFdJZV5JTSdWH0FnJDCJug_3sn1CD6CWEzB2MDNKtZO3y9SlI9bfVOhwTsxw6EXehyUP_XWg4qoDRdJKDcVCmx7gjznr6hvchp_YrVak7PgUcYenHmIdicmHrwfSRXdZiDqotE2JsJnFBmcwTrenv5-HaS5VO1NmOajCzquzhYgi18nR-2Sp4J74QmPG9-BUEOs=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[2]。在我国，稻瘟病的发病面积大约为500万hm2，受其危害水稻产量普遍下降10%-20%，在一些受灾严重的地区，水稻产量的损失更是高达50%，甚至出现绝收的情况ADDINEN.CITE<EndNote><Cite><Author>易怒安</Author><Year>2015</Year><RecNum>3</RecNum><DisplayText><styleface="superscript">[3]</style></DisplayText><record><rec-number>3</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741523434">3</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>易怒安</author><author>李魏</author><author>戴良英</author></authors></contributors><auth-address>湖南农业大学植物保护学院;作物基因工程湖南省重点实验室;</auth-address><titles><title>水稻抗稻瘟病基因的克隆及其分子育种研究进展</title><secondary-title>分子植物育种</secondary-title></titles><periodical><full-title>分子植物育种</full-title></periodical><pages>1653-1659</pages><volume>13</volume><number>07</number><keywords><keyword>克隆</keyword><keyword>稻瘟病</keyword><keyword>分子育种</keyword><keyword>抗瘟基因</keyword></keywords><dates><year>2015</year></dates><isbn>1672-416X</isbn><call-num>46-1068/S</call-num><urls><related-urls><url>/doi/10.13271/j.mpb.013.001653</url></related-urls></urls><electronic-resource-num>10.13271/j.mpb.013.001653</electronic-resource-num><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[3]。研究培育优良综合性状的水稻品种至关重要。随着现代分子生物学技术与高通量测序手段的不断突破，科研工作者已成功分离并鉴定了众多与重要农艺性状相关的功能基因ADDINEN.CITE<EndNote><Cite><Author>Jaganathan</Author><Year>2020</Year><RecNum>4</RecNum><DisplayText><styleface="superscript">[4]</style></DisplayText><record><rec-number>4</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741523531">4</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Jaganathan,Deepa</author><author>Bohra,Abhishek</author><author>Thudi,Mahendar</author><author>Varshney,RajeevK.</author></authors></contributors><titles><title>Finemappingandgenecloninginthepost-NGSera:advancesandprospects</title><secondary-title>TheoreticalandAppliedGenetics</secondary-title></titles><periodical><full-title>TheoreticalandAppliedGenetics</full-title></periodical><pages>1791-1810</pages><volume>133</volume><number>5</number><dates><year>2020</year><pub-dates><date>May</date></pub-dates></dates><isbn>0040-5752</isbn><accession-num>WOS:000516051300001</accession-num><work-type>Review</work-type><urls><related-urls><url><GotoISI>://WOS:000516051300001</url></related-urls></urls><electronic-resource-num>10.1007/s00122-020-03560-w</electronic-resource-num></record></Cite></EndNote>[4]，同时解析了复杂的基因互作网络，这为分子设计育种奠定了理论基础。1.2GADD45蛋白概述1.2.1GADD45家族简介生长阻滞和DNA损伤诱导的45基因（growtharrestandDNAdamage-indueiblegenes,GADD45）作为关键的压力传感器，介导各种细胞反应。GADD45家族包括3个成员：GADD45α（GADD45a/DDIT1）、GADD45β（GADD45b/MyD118）及GADD45γ（GADD45g/CR6/OIG37）ADDINEN.CITE<EndNote><Cite><Author>Lal</Author><Year>2006</Year><RecNum>12</RecNum><DisplayText><styleface="superscript">[5]</style></DisplayText><record><rec-number>12</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741525282">12</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Lal,Ashish</author><author>Gorospe,Myriam</author></authors></contributors><titles><title><styleface="normal"font="default"size="100%">Egad,moreformsofgeneregulation::The</style><styleface="italic"font="default"size="100%">gadd45a</style><styleface="normal"font="default"size="100%">story</style></title><secondary-title>CellCycle</secondary-title></titles><periodical><full-title>CellCycle</full-title></periodical><pages>1422-1425</pages><volume>5</volume><number>13</number><dates><year>2006</year><pub-dates><date>Jul1</date></pub-dates></dates><isbn>1538-4101</isbn><accession-num>WOS:000239614600013</accession-num><work-type>Article</work-type><urls><related-urls><url><styleface="underline"font="default"size="100%"><GotoISI>://WOS:000239614600013</style></url></related-urls></urls><electronic-resource-num>10.4161/cc.5.13.2902</electronic-resource-num></record></Cite></EndNote>[5]ADDINEN.CITE<EndNote><Cite><Author>Patel</Author><Year>2022</Year><RecNum>13</RecNum><DisplayText><styleface="superscript">[6]</style></DisplayText><record><rec-number>13</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741525340">13</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Patel,Kishan</author><author>Murray,MaryGrace</author><author>Whelan,KellyA.</author></authors></contributors><titles><title>RolesforGADD45inDevelopmentandCancer</title><secondary-title>Advancesinexperimentalmedicineandbiology</secondary-title></titles><periodical><full-title>Advancesinexperimentalmedicineandbiology</full-title></periodical><pages>23-39</pages><volume>1360</volume><dates><year>2022</year><pub-dates><date>2022</date></pub-dates></dates><isbn>0065-2598</isbn><accession-num>MEDLINE:35505160</accession-num><work-type>;Review</work-type><urls><related-urls><url><GotoISI>://MEDLINE:35505160</url></related-urls></urls><electronic-resource-num>10.1007/978-3-030-94804-7_2</electronic-resource-num></record></Cite></EndNote>[6]。在哺乳动物中，该家族蛋白参与调控细胞应激响应、表观遗传重塑等多种关键生物学过程ADDINEN.CITE<EndNote><Cite><Author>Chandramouly</Author><Year>2022</Year><RecNum>14</RecNum><DisplayText><styleface="superscript">[7]</style></DisplayText><record><rec-number>14</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741525388">14</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Chandramouly,Gurushankar</author></authors></contributors><titles><title>Gadd45inDNADemethylationandDNARepair</title><secondary-title>Advancesinexperimentalmedicineandbiology</secondary-title></titles><periodical><full-title>Advancesinexperimentalmedicineandbiology</full-title></periodical><pages>55-67</pages><volume>1360</volume><dates><year>2022</year><pub-dates><date>2022</date></pub-dates></dates><isbn>0065-2598</isbn><accession-num>MEDLINE:35505162</accession-num><urls><related-urls><url><GotoISI>://MEDLINE:35505162</url></related-urls></urls><electronic-resource-num>10.1007/978-3-030-94804-7_4</electronic-resource-num></record></Cite></EndNote>[7]ADDINEN.CITE<EndNote><Cite><Author>Jin</Author><Year>2018</Year><RecNum>15</RecNum><DisplayText><styleface="superscript">[8]</style></DisplayText><record><rec-number>15</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741525435">15</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Jin,Zelin</author><author>Liu,Yun</author></authors></contributors><titles><title>DNAmethylationinhumandiseases</title><secondary-title>Genes&Diseases</secondary-title></titles><periodical><full-title>Genes&Diseases</full-title></periodical><pages>1-8</pages><volume>5</volume><number>1</number><dates><year>2018</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>2352-4820</isbn><accession-num>WOS:000428011500001</accession-num><work-type>Review</work-type><urls><related-urls><url><GotoISI>://WOS:000428011500001</url></related-urls></urls><electronic-resource-num>10.1016/j.gendis.2018.01.002</electronic-resource-num></record></Cite></EndNote>[8]。细胞应激响应与生存调控Gupta等人的研究表明GADD45能够参与细胞生存调节，GADD45缺失型造血细胞暴露于紫外辐射后呈现修复能力减弱、凋亡率升高等表型ADDINEN.CITE<EndNote><Cite><Author>Gupta</Author><Year>2005</Year><RecNum>16</RecNum><DisplayText><styleface="superscript">[9]</style></DisplayText><record><rec-number>16</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741525513">16</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Gupta,M.</author><author>Gupta,S.K.</author><author>Balliet,A.G.</author><author>Hollander,M.C.</author><author>Fornace,A.J.</author><author>Hoffman,B.</author><author>Liebermann,D.A.</author></authors></contributors><titles><title>HematopoieticcellsfromGadd45a-andGadd45b-deficientmicearesensitizedtogenotoxic-stress-inducedapoptosis</title><secondary-title>Oncogene</secondary-title></titles><periodical><full-title>Oncogene</full-title></periodical><pages>7170-7179</pages><volume>24</volume><number>48</number><dates><year>2005</year><pub-dates><date>Nov3</date></pub-dates></dates><isbn>0950-9232</isbn><accession-num>WOS:000232990100006</accession-num><work-type>Article</work-type><urls><related-urls><url><GotoISI>://WOS:000232990100006</url></related-urls></urls><electronic-resource-num>10.1038/sj.onc.1208847</electronic-resource-num></record></Cite></EndNote>[9]。GADD45α通过促进IκB磷酸化解除NF-κB抑制，同时激活p38激酶级联反应，从而增强细胞生存能力ADDINEN.CITE<EndNote><Cite><Author>Smith</Author><Year>2000</Year><RecNum>17</RecNum><DisplayText><styleface="superscript">[10]</style></DisplayText><record><rec-number>17</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741525846">17</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Smith,M.L.</author><author>Ford,J.M.</author><author>Hollander,M.C.</author><author>Bortnick,R.A.</author><author>Amundson,S.A.</author><author>Seo,Y.R.</author><author>Deng,C.X.</author><author>Hanawalt,P.C.</author><author>Fornace,A.J.</author></authors></contributors><titles><title><styleface="normal"font="default"size="100%">p53-mediatedDNArepairresponsestoUVradiation::Studiesofmousecellslacking</style><styleface="italic"font="default"size="100%">p53</style><styleface="normal"font="default"size="100%">,</style><styleface="italic"font="default"size="100%">p21</style><styleface="normal"font="default"size="100%">,and/or</style><styleface="italic"font="default"size="100%">gadd45</style><styleface="normal"font="default"size="100%">genes</style></title><secondary-title>MolecularandCellularBiology</secondary-title></titles><periodical><full-title>MolecularandCellularBiology</full-title></periodical><pages>3705-3714</pages><volume>20</volume><number>10</number><dates><year>2000</year><pub-dates><date>May</date></pub-dates></dates><isbn>0270-7306</isbn><accession-num>WOS:000086698100038</accession-num><work-type>Article</work-type><urls><related-urls><url><styleface="underline"font="default"size="100%"><GotoISI>://WOS:000086698100038</style></url></related-urls></urls><electronic-resource-num>10.1128/mcb.20.10.3705-3714.2000</electronic-resource-num></record></Cite></EndNote>[10]ADDINEN.CITE<EndNote><Cite><Author>Gupta</Author><Year>2006</Year><RecNum>18</RecNum><DisplayText><styleface="superscript">[11]</style></DisplayText><record><rec-number>18</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741525906">18</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Gupta,Mamta</author><author>Gupta,ShivKumar</author><author>Hoffman,Barbara</author><author>Liebermann,DanA.</author></authors></contributors><titles><title><styleface="italic"font="default"size="100%">Gadd45a</style><styleface="normal"font="default"size="100%">and</style><styleface="italic"font="default"size="100%">Gadd45b</style><styleface="normal"font="default"size="100%">protecthematopoieticcellsfromUV-inducedapoptosisviadistinctsignalingpathways,includingp38activationandJNKinhibition</style></title><secondary-title>JournalofBiologicalChemistry</secondary-title></titles><periodical><full-title>JournalofBiologicalChemistry</full-title></periodical><pages>17552-17558</pages><volume>281</volume><number>26</number><dates><year>2006</year><pub-dates><date>Jun30</date></pub-dates></dates><isbn>0021-9258</isbn><accession-num>WOS:000238490300004</accession-num><work-type>Article</work-type><urls><related-urls><url><styleface="underline"font="default"size="100%"><GotoISI>://WOS:000238490300004</style></url></related-urls></urls><electronic-resource-num>10.1074/jbc.M600950200</electronic-resource-num></record></Cite></EndNote>[11]。此外，GADD45α作为经典抑癌因子，其缺失导致基因组不稳定性加剧，表现为非整倍体累积、有丝分裂异常及DNA损伤超敏反应ADDINEN.CITE<EndNote><Cite><Author>Hollander</Author><Year>1999</Year><RecNum>19</RecNum><DisplayText><styleface="superscript">[12]</style></DisplayText><record><rec-number>19</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741526526">19</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Hollander,M.C.</author><author>Sheikh,M.S.</author><author>Bulavin,D.V.</author><author>Lundgren,K.</author><author>Augeri-Henmueller,L.</author><author>Shehee,R.</author><author>Molinaro,T.A.</author><author>Kim,K.E.</author><author>Tolosa,E.</author><author>Ashwell,J.D.</author><author>Rosenberg,M.P.</author><author>Zhan,Q.M.</author><author>Fernández-Salguero,P.M.</author><author>Morgan,W.F.</author><author>Deng,C.X.</author><author>Fornace,A.J.</author></authors></contributors><titles><title><styleface="normal"font="default"size="100%">Genomicinstabilityin</style><styleface="italic"font="default"size="100%">Gadd45a</style><styleface="normal"font="default"size="100%">-deficientmice</style></title><secondary-title>NatureGenetics</secondary-title></titles><periodical><full-title>NatureGenetics</full-title></periodical><pages>176-184</pages><volume>23</volume><number>2</number><dates><year>1999</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>1061-4036</isbn><accession-num>WOS:000082827500016</accession-num><work-type>Article</work-type><urls><related-urls><url><styleface="underline"font="default"size="100%"><GotoISI>://WOS:000082827500016</style></url></related-urls></urls><electronic-resource-num>10.1038/13802</electronic-resource-num></record></Cite></EndNote>[12]。在Ras诱导的乳腺肿瘤模型中，GADD45α敲除加速肿瘤进展，其机制与JNK/p38介导的凋亡通路抑制及衰老逃逸相关ADDINEN.CITE<EndNote><Cite><Author>Chi</Author><Year>2004</Year><RecNum>20</RecNum><DisplayText><styleface="superscript">[13]</style></DisplayText><record><rec-number>20</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741526657">20</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Chi,H.B.</author><author>Lu,B.F.</author><author>Takekawa,M.</author><author>Davis,R.J.</author><author>Flavell,R.A.</author></authors></contributors><titles><title>GADD45β/GADD45γandMEKK4compriseageneticpathwaymediatingSTAT4-independentIFNγproductioninTcells</title><secondary-title>EmboJournal</secondary-title></titles><periodical><full-title>EmboJournal</full-title></periodical><pages>1576-1586</pages><volume>23</volume><number>7</number><dates><year>2004</year><pub-dates><date>Apr7</date></pub-dates></dates><isbn>0261-4189</isbn><accession-num>WOS:000221328700017</accession-num><work-type>Article</work-type><urls><related-urls><url><GotoISI>://WOS:000221328700017</url></related-urls></urls><electronic-resource-num>10.1038/sj.emboj.7600173</electronic-resource-num></record></Cite></EndNote>[13]。值得注意的是，胰腺导管癌中GADD45α突变可致功能获得性转化，表现为促生存表型ADDINEN.CITE<EndNote><Cite><Author>Schneider</Author><Year>2006</Year><RecNum>21</RecNum><DisplayText><styleface="superscript">[14]</style></DisplayText><record><rec-number>21</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741526713">21</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Schneider,G.</author><author>Weber,A.</author><author>Zechner,U.</author><author>Oswald,F.</author><author>Friess,H.M.</author><author>Schmid,R.M.</author><author>Liptay,S.</author></authors></contributors><titles><title>GADD45αishighlyexpressedinpancreaticductaladenocarcinomacellsandrequiredfortumorcellviability</title><secondary-title>InternationalJournalofCancer</secondary-title></titles><periodical><full-title>InternationalJournalofCancer</full-title></periodical><pages>2405-2411</pages><volume>118</volume><number>10</number><dates><year>2006</year><pub-dates><date>May15</date></pub-dates></dates><isbn>0020-7136</isbn><accession-num>WOS:000237029200006</accession-num><work-type>Article</work-type><urls><related-urls><url><GotoISI>://WOS:000237029200006</url></related-urls></urls><electronic-resource-num>10.1002/ijc.21637</electronic-resource-num></record></Cite></EndNote>[14]。表观遗传调控与DNA去甲基化GADD45a可以通过去甲基化作用促进基因表达，对发育过程中的分化和转录调控起关键作用ADDINEN.CITE<EndNote><Cite><Author>Niehrs</Author><Year>2012</Year><RecNum>22</RecNum><DisplayText><styleface="superscript">[15]</style></DisplayText><record><rec-number>22</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741526760">22</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Niehrs,Christof</author><author>Schaefer,Andrea</author></authors></contributors><titles><title>ActiveDNAdemethylationbyGadd45andDNArepair</title><secondary-title>TrendsinCellBiology</secondary-title></titles><periodical><full-title>TrendsinCellBiology</full-title></periodical><pages>220-227</pages><volume>22</volume><number>4</number><dates><year>2012</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>0962-8924</isbn><accession-num>WOS:000303075400006</accession-num><work-type>Review</work-type><urls><related-urls><url><GotoISI>://WOS:000303075400006</url></related-urls></urls><electronic-resource-num>10.1016/j.tcb.2012.01.002</electronic-resource-num></record></Cite></EndNote>[15]ADDINEN.CITE<EndNote><Cite><Author>Chandramouly</Author><Year>2022</Year><RecNum>23</RecNum><DisplayText><styleface="superscript">[7]</style></DisplayText><record><rec-number>23</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741526824">23</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Chandramouly,Gurushankar</author></authors></contributors><titles><title>Gadd45inDNADemethylationandDNARepair</title><secondary-title>Advancesinexperimentalmedicineandbiology</secondary-title></titles><periodical><full-title>Advancesinexperimentalmedicineandbiology</full-title></periodical><pages>55-67</pages><volume>1360</volume><dates><year>2022</year><pub-dates><date>2022</date></pub-dates></dates><isbn>0065-2598</isbn><accession-num>MEDLINE:35505162</accession-num><urls><related-urls><url><GotoISI>://MEDLINE:35505162</url></related-urls></urls><electronic-resource-num>10.1007/978-3-030-94804-7_4</electronic-resource-num></record></Cite></EndNote>[7]。GADD45α通过招募TET1（Ten-ElevenTranslocation1）至R环结构，协同TDG（ThymineDNAGlycosylase）启动CpG岛局部主动去甲基化ADDINEN.CITE<EndNote><Cite><Author>Kienhoefer</Author><Year>2015</Year><RecNum>25</RecNum><DisplayText><styleface="superscript">[16]</style></DisplayText><record><rec-number>25</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741526968">25</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Kienhoefer,Sabine</author><author>Musheev,MichaelU.</author><author>Stapf,Ulrike</author><author>Helm,Mark</author><author>Schomacher,Lars</author><author>Niehrs,Christof</author><author>Schaefer,Andrea</author></authors></contributors><titles><title>GADD45aphysicallyandfunctionallyinteractswithTET1</title><secondary-title>Differentiation</secondary-title></titles><periodical><full-title>Differentiation</full-title></periodical><pages>59-68</pages><volume>90</volume><number>1-3</number><dates><year>2015</year><pub-dates><date>Jul-Oct</date></pub-dates></dates><isbn>0301-4681</isbn><accession-num>WOS:000365802600006</accession-num><work-type>Article</work-type><urls><related-urls><url><GotoISI>://WOS:000365802600006</url></related-urls></urls><electronic-resource-num>10.1016/j.diff.2015.10.003</electronic-resource-num></record></Cite></EndNote>[16]ADDINEN.CITE<EndNote><Cite><Author>Arab</Author><Year>2019</Year><RecNum>26</RecNum><DisplayText><styleface="superscript">[17]</style></DisplayText><record><rec-number>26</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741527025">26</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Arab,Khelifa</author><author>Karaulanov,Emil</author><author>Musheev,Michael</author><author>Trnka,Philipp</author><author>Schaefer,Andrea</author><author>Grummt,Ingrid</author><author>Niehrs,Christof</author></authors></contributors><titles><title>GADD45AbindsR-loopsandrecruitsTET1toCpGislandpromoters</title><secondary-title>NatureGenetics</secondary-title></titles><periodical><full-title>NatureGenetics</full-title></periodical><pages>217-+</pages><volume>51</volume><number>2</number><dates><year>2019</year><pub-dates><date>Feb</date></pub-dates></dates><isbn>1061-4036</isbn><accession-num>WOS:000457314300007</accession-num><work-type>Article</work-type><urls><related-urls><url><GotoISI>://WOS:000457314300007</url></related-urls></urls><electronic-resource-num>10.1038/s41588-018-0306-6</electronic-resource-num></record></Cite></EndNote>[17]。该过程遵循Tet-TDG通路：5-甲基胞嘧啶（5mC）经氧化脱氨生成5-羟甲基尿嘧啶（5hmU），随后TDG启动碱基切除修复（BER）实现胞嘧啶重置ADDINEN.CITE<EndNote><Cite><Author>Morgan</Author><Year>2004</Year><RecNum>27</RecNum><DisplayText><styleface="superscript">[18]</style></DisplayText><record><rec-number>27</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741527077">27</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Morgan,H.D.</author><author>Dean,W.</author><author>Coker,H.A.</author><author>Reik,W.</author><author>Petersen-Mahrt,S.K.</author></authors></contributors><titles><title>Activation-inducedcytidinedeaminasedeaminates5-methylcytosineinDNAandisexpressedinpluripotenttissues-Implicationsforepigeneticreprogramming</title><secondary-title>JournalofBiologicalChemistry</secondary-title></titles><periodical><full-title>JournalofBiologicalChemistry</full-title></periodical><pages>52353-52360</pages><volume>279</volume><number>50</number><dates><year>2004</year><pub-dates><date>Dec10</date></pub-dates></dates><isbn>0021-9258</isbn><accession-num>WOS:000225493400078</accession-num><work-type>Article</work-type><urls><related-urls><url><GotoISI>://WOS:000225493400078</url></related-urls></urls><electronic-resource-num>10.1074/jbc.M407695200</electronic-resource-num></record></Cite></EndNote>[18]ADDINEN.CITE<EndNote><Cite><Author>Guo</Author><Year>2011</Year><RecNum>28</RecNum><DisplayText><styleface="superscript">[19]</style></DisplayText><record><rec-number>28</rec-number><foreign-keys><keyapp="EN"db-id="vzxesd0wbe22eoew52f5sas25zzttttde29p"timestamp="1741527131">28</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Guo,JunjieU.</author><author>Su,Yijing</author><author>Zhong,Chun</author><author>Ming,Guo-li</author><author>Song,Hongjun</author></authors></contr