通过农杆菌介导法将哈兹木霉几丁质酶ThEn-42基因导入核桃

 通过根癌农杆菌C₅₈C₁ ATHV Rif^R介导法, 利用烟草花叶病毒35 S 双启动子和苜蓿花叶病毒引导序列控制下的含抗新霉素磷酸转移酶基因(npt Ⅱ) 和哈兹木霉几丁质酶基因(ThEn-42) 的质粒pBin19ESR 为载体, 对3 个核桃体细胞胚系进行遗传转化, 结果获得41 个抗卡那霉素的体细胞胚系。经PCR 和复式PCR 检测, 41个转化系均含有nptⅡ基因, 其中38个转化系含有ThEn-42 基因。Southern 杂交分析表明, ThEn-42基因已被整合到核桃体的基因组中。几丁质酶活性检测结果表明, 转化的体细胞胚系的几丁质酶活性比对照高几十至几千倍。遗传转化的体细胞胚系已萌发成苗。

Genetic Transformation of the Trichoderma Endochitinase Gene ThEn-42 to Som atic Embryos of English Walnut

Somatic embryos of English walnut(Juglans regia L .)were Agrobacterium-mediately transformed with the Trichoderma endochitina se gene ThEn-42 under the control of a double 35　S CaMV promoter and Alfalfa Mosaic Virus leader sequence.The selectable marker gene neomycin phospo transferaseⅡ(nptⅡ)driven by the nopaline synthase promoter was also used.A total of 41 putatively transformed somatic embryo lines were evaluated for expr ession of the nptⅡ and ThEn-42 genes by PCR and multiplex PCR.All of t he 41 somatic embryo selections expressed the nptⅡ gene，among which 38 som atic embryo selections expressed the ThEn-42 gene.Analysis of chitinase act ivity by using a fluorometric assay showed dozens to 1　000-fold higher c hitinase activity in transformed somatic embryos than that in non-transformed o nes.All of the tested chitinase-postive somatic embryo lines analyzed by South ern blotting had an intact copy of the ThEn-42 gene.Chitinase expressing so matic embryos were further germinated and are being propagated for disease resis tance assays.