.word可编辑.园艺学报2013,40(12:2419–2428http://www.ahs.ac.cnActaHorticulturaeSinicaE-mail:yuanyixuebao@126.com拟南芥At-pri-miR828基因的克隆及其对番茄的遗传转化贾小云1,于治芹1,*,梁建萍1,唐贵良1,2,3,**,金雷皓1,张莉4,贺立恒4,李润植4,**(1山西农业大学生命科学学院,山西太谷030801;2密歇根理工大学生物科学院,美国霍顿49931;3河南农业大学,郑州450002;4山西农业大学农学院,山西太谷030801摘要:MicroRNA828(miRNA828是一种新近发现的生物学功能还未全面研究的miRNA。为从不同角度阐明miRNA828的生物学功能,从拟南芥中克隆到At-pri-miR828基因并构建了该基因过量表达的植物表达载体pC2300-pOT2-At-pri-miR828,通过农杆菌介导的叶盘法将pC2300-pOT2-At-pri-miR828‘导入异源植物番茄品种AilsaCraig’中。PCR鉴定结果显示,外源基因At-pri-miR828已成功整合到转基因番茄基因组中,共获得9个转基因株系,67株转基因植株。定量PCR检测结果显示,与野生型番茄植株相比,转基因植株中miR828的表达量显著增加,而生物信息学所预测的miR828靶基因Sly-myb-like1的表达水平则相应降低。花青素含量测定结果显示,miR828过量表达的转基因番茄植株花青素含量明显低于野生型植株,表明miR828参与了番茄花青素的生物合成调控。.专业.专注..word可编辑.关键词:番茄;miR828;Sly-myb-like1;花青素;实时定量PCR中图分类号:S641.2文献标志码:A文章编号:0513-353X(201312-2419-10CloningofArabidopsisAt-pri-miR828GeneandItsGeneticTransformationintoTomatoJIAXiao-yun1,YUZhi-qin1,*,LIANGJian-ping1,TANGGui-liang1,2,3,**,JINLei-hao1,ZHANGLi4,HELi-heng4,andLIRun-zhi4,**(1CollegeofLifeSciences,ShanxiAgriculturalUniversity,Taigu,Shanxi030801,China;2DepartmentofBiologicalSciences,MichiganTechnologicalUniversity,Houghton,MI49931,USA;3HenanAgriculturalUniversity,Zhengzhou450002,China;4CollegeofAgriculture,ShanxiAgriculturalUniversity,Taigu,Shanxi030801,ChinaAbstract:MicroRNA828(miR828isanewlyidentifiedsmallRNAwhosebiologicalfunctionsarestillnotveryclear.ToexplorethebiologicalfunctionsofmiR828,particularlyintomato,theAt-pri-miR828genewasisolatedfromArabidopsisthaliana,andclonedintothecorrespondingsiteofthepCAMBIA2300toconstructtheplantexpressionvectorpC2300-pOT2-At-pri-miR828.ThisAt-pri-miR828收稿日期:2013–06–29;修回日期:2013–11–20基金项目:国家自然科学基金项目(31101555;山西省青年基金项目(2011021032-2;山西省留学基金项目(2011051;山西省高等学校优秀青年学术带头人项目(2012;山西人才引进与开发专项(614191;山西农业大学科技创新基金项目(2010021*并列第一作者**通信作者Authorforcorrespondence(E-mail:rli2001@hotmail.com;gtang1@mtu.edu.专业.专注..word可编辑.致谢:感谢构建pCAMBIA2300-PacI载体的康文俊同学。2420园艺学报40卷overexpressingvectorwasthenintroducedintotomato(SolanumlycopersicumMill.‘AilsaGraig’usingAgrobacterium-mediatedtransformation.Totalof9transgeniclinesand67transgenicplantscarryingtheAt-pri-miR828geneunderthecontrolofthe35Spromoterweregenerated.PCRamplificationshowedthatAt-pri-miR828wasintegratedintothegenomeDNAoftransgenictomatoes.Real-timePCRanalysisdemonstratedthatmiR828mRNAlevelinthetransgenictomatoeswasincreasedgreatly.Accordingly,expressionofSly-myb-like1,apotentialtargetgeneofmiR828predictedbyinformatics,wassuppressed.FurtheranthocyanincontentanalysisrevealedthatanthocyanincontentwasgreatlyreducedinmiR828overexpressedtransgenicplants,demonstratingmiR828mightfunctionintheanthocyaninbiosynthesisoftomato.Keywords:tomato;miR828;Sly-myb-like1;anthocyanin;real-timePCRMicroRNA(miRNA,也称微小RNA,是一类内源性的单链非编码小分子RNA,长度约为21~24个核苷酸(Bartel,2004。miRNA介导了一种新的基因表达调控模式,在植物体...