欢迎来到天天文库
浏览记录
ID:53747304
大小:1.39 MB
页数:9页
时间:2020-04-22
《棉花叶肉原生质体分离及目标基因瞬时表达体系的建立-论文.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、作物学报ACTAAGRONOMICASINICA2014,40(2):231-239http://zwxb.chinacrops.org/ISSN0496—3490;CODENTSHPA9E—mail:xbzw@chinajourna1.net.caDoI:10.3724/SP.J.1006.2014.00231棉花叶肉原生质体分离及目标基因瞬时表达体系的建立李妮娜丁林云张志远郭旺珍南京农业大学作物遗传与种质创新国家重点实验室/教育部杂交棉创制工程研究中心,江苏南京210095摘要:以棉花幼嫩子叶为材料,分析影响棉花叶肉原生质体分离及目标基因转化的主要因素,以棉花叶肉原生质体为受体,建立
2、了稳定、高效的目标基因瞬时表达与鉴定体系。技术体系包括,选择自然生长12d的棉花幼嫩子叶为材料,混合1.5%纤维素酶、O.4%离析酶、O.5molL一甘露醇、2OmmolL~KCI、20mmolL—MES、0.1molL~CaC12和1.0gL~BSA,在28*C黑暗条件下振荡酶解8h,可游离出浓度达1.O×10mL以上的纯净棉花叶肉原生质体。利用该方法将棉花锌指蛋白基因GhZFP2整合到pJIT166.GFP质粒载体,构建了G2GFP融合载体,采用40%PEG.4000介导转化,获得高转化率的棉花叶肉原生质体。对目标基因瞬时表达产物检测表明,GhZFP2蛋白清晰定位在细胞核上。关键词:
3、棉花;原生质体;PEG介导;转化;目标基因;瞬时表达IsolationofMesophyllProtoplastandEstablishmentofGeneTransientEx—pressionSysteminCottonLINi—Na,DINGLin.Yun,ZHANGZhi.Yuan,andGUOWang.ZhenStateKeyLaboratoryofCropGenetics&GermplasmEnhancement/HybridCottonResearch&DevelopmentEngineeringResearchCenter.Minis-tryofEducation,Nan
4、jingAgriculturalUniversity,Nanjing210095,ChinaAbstract:ThisPaperaimedtosetupastableandeficienttransientexpressionsystembasedoncoaonmesophyllprotoplastsproducedfromcotyrledon.WanalyzedthekeYfactorsrelatedtoisolatingefectivelycottonmesophyllprotoplastsanduseditasavehicletoexpresstransfectedgenesfor
5、functionalstudies.Theoptimizedtechniquesareasfollows:f11血ehealthy12.day.oldcotyledongrownnaturallywasselectedtoisolatecottonprotoplasts;f2)thedigestionsystemcontained1.5%eellulose,0.4%macerozyme,0.5molLmannitol,20mmolL『KCl,20mrnolL『MES,0.1molL-CaC12,and1.0gL『BSA:andf3)thedi.gestionwasconductedint
6、hedarkunder28℃for8hwithgentleshaking.Theprotoplastyieldwashigherthan1.0×10。mL-.Usingthissystemweintegratedacotonzincfingerproteingene(GhZFP2、intopJIT166.GFPplasmidvectorandconstructedtheGhZFP2:GFPfusionvector~Thetargetgenewasthentnsformedintopurifiedcotonmesophyllprotoplastmediatedwith40%PEG4000.
7、Asaresult.coRonmesophyllprotoplastwithhightransformationfrequencywasObtained.Thetransientexpres.sionassayshowedthattheGhZFP2proteinwasclearlylocatedinthecellnucleusofcoRon.Keywords:Cotton;Protoplast;PEG.mediated;Transf
此文档下载收益归作者所有