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

园艺学报 ›› 2001, Vol. 28 ›› Issue (1): 12-18.

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

汤浩茹^1,2;Wallbraun M.²;任正隆³;Reustle G. M.²;Krczal G. ²

(¹ 四川农业大学林学园艺学院, 雅安625014 ;²德国莱茵兰—法尔兹植物生物技术中心, 67435 Neustadt/W.;³ 四川农业大学植物遗传育种实验室, 雅安625014)

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2001-02-25 发布日期:2001-02-25

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

Tang Haoru^1,2, Wallbraun M.², Ren Zhenglong³, Reustle G. M.², and Krczal G. ²

(¹ College of Forestry and Horticulture , Sichuan Agricultural University , Ya’an , 625014;　² Zentrum Grüne Gentechnik , SLFA , 67435 Neustadt/ W. , Germany ;　³ State Key Laboratory of Plant Genetics and Breeding , Sichuan Agricultural University, Ya’an 625014)

Received:1900-01-01 Revised:1900-01-01 Online:2001-02-25 Published:2001-02-25

摘要/Abstract

摘要： 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。

关键词: 核桃, 几丁质酶, 转基因, 体细胞胚

Abstract: Somatic embryos of English walnut (Juglans regia L.) were Agrobacterium-mediately transformed with the Trichoderma endochitinase gene ThEn242 under the control of a double 35 S CaMV promoter and Alfalfa Mosaic Virus leader sequence. The selectable marker gene neomycin phospotransferase Ⅱ (npt Ⅱ) driven by the nopaline synthase promoter was also used. A total of 41 putatively transformed somatic embryo lines were evaluated for expression of the npt Ⅱand ThEn-42 genes by PCR and multiplex PCR. All of the 41 somatic embryo selections expressed the npt Ⅱgene, among which 38 somatic embryo selections expressed the ThEn-42 gene. Analysis of chitinase activity by using a fluorometric assay showed dozens to 1 000-fold higher chitinase activity in transformed somatic embryos than that in non-transformed ones. All of the tested chitinase-postive somatic embryo lines analyzed by Southern blotting had an intact copy of the ThEn-42 gene. Chitinase expressing somatic embryos were further germinated and are being propagated for disease resistance assays.

Key words: Walnut (Juglans regia), Chitinase, Genetic transformation, Somatic embryos

中图分类号:

S 664. 1

汤浩茹;Wallbraun M.;任正隆;Reustle G. M.;Krczal G. . 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[J]. 园艺学报, 2001, 28(1): 12-18.

Tang Haoru;Wallbraun M.;Ren Zhenglong;Reustle G. M.;and Krczal G. . Genetic Transformation of the Trichoderma Endochitinase Gene ThEn-42 to Somatic Embryos of English Walnut[J]. ACTA HORTICULTURAE SINICA, 2001, 28(1): 12-18.

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