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1、作物学报ACTAAGRONOMICASINICA2014,40(7):1190−1196http://zwxb.chinacrops.org/ISSN0496-3490;CODENTSHPA9E-mail:xbzw@chinajournal.net.cnDOI:10.3724/SP.J.1006.2014.01190土壤宏基因组中抗草甘膦新基因的克隆与转化水稻的研究1,2121,*1,2,*王云鹏马景勇马瑞马建刘文国12吉林农业大学农学院,吉林长春130118;吉林省农业科学院,吉林长春130033摘要:EP
2、SPS(5-烯醇丙酮莽草酸-3-磷酸合酶,EC2.5.1.19)是植物芳香族氨基酸和植物次生代谢产物生物合成中莽草酸途径的关键酶;同时也是广谱性除草剂草甘膦的作用目标。本实验通过对草甘膦污染土壤宏基因组文库的建立及筛选,成功克隆了一个新的草甘膦抗性的EPSPS基因(命名为soilEPSPS)。序列分析表明,soilEPSPS基因全长1404bp,其编码的467个氨基酸中未涉及已公布专利中保护的氨基酸序列。原核功能验证表明,该基因对草甘膦的耐受能力优于EPSPSCP4基因。将该基因与水稻RubiscoSSU引导
3、肽相融合构建由actin启动子驱动的植物表达载体,用农杆菌介导法实现了水稻的遗传转化。抗性再生植株的PCR和Southern杂交结果表明所获得的26株再生植株均为转基–1因阳性植株,其中共有3个单拷贝转化事件。草甘膦抗性鉴定证明纯合体T2代植株能够耐受高达500mmolL的草甘膦。本研究为转基因抗除草剂水稻新品种的培育奠定了基础。关键词:宏基因组;水稻;农杆菌;遗传转化CloningofNewHerbicideResistantGeneinSoilMetagenomicsandGenera-tionofTra
4、nsgenicRicePlants1,2121,*1,2,*WANGYun-Peng,MAJing-Yong,MARui,MAJian,andLIUWen-Guo12FacultyofAgronomy,JilinAgriculturalUniversity,Changchun130118,China;JilinAcademyofAgriculturalSciences,Changchun130033,ChinaAbstract:TheEPSPS(5-enolpyruvylshikimate-3-phospha
5、tesynthase,EC2.5.1.19)isconsideredasoneofthecrucialenzymesintheshikimatepathwayforthebiosynthesisofessentialaromaticaminoacidsandsecondarymetabolitesinplant.Itisalsoprovedasaspecifictargetofbroadspectrumherbicideglyphosate.Inthisresearch,anewglyphosateresis
6、tantEPSPSgenenamedsoilEPSPSwasisolatedbyscreeningthemetagenomiclibrary.ThesequenceanalysissuggestedthatsoilEPSPSconsistsof1404bpandencodesapolypeptideof467aminoacidsthatarenotinvolvedintheaminoacidsequenceprotectedbythepublishedpatent-sions.Theresultofproka
7、ryoticexpressionconfirmedthatsoilEPSPShadabetterabilityofglyphosateresistancethanEPSPSCP4gene.FusingthegeneandtheRubiscoSSUsignalpeptideconstructedtheplantexpressingvectorwiththeactinpromoterdriven,andtransformationofricemediatedbyAgrobacteriumtumefaciens.P
8、CRandSouthernanalysesshowedthatatotalof26trans-genicplantswereobtainedandthreeofthemweresinglecopyinsertionevents.Resistantexperimentshowedthatthehomozy-–1gousT2plantsintheseeventscouldtoleratethespray
