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农杆菌介导的体胚转化获得的转基因木瓜植株。

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos.

机构信息

Department of Horticulture, University of Hawaii, 96822, Honolulu, HI, USA.

出版信息

Plant Cell Rep. 1993 Mar;12(5):245-9. doi: 10.1007/BF00237128.

Abstract

Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 μg·ml(-1) kanamycin. Four putative transgenic plant lines were obtained from the cp gene(-) vector and two from the cp gene(+) vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene(+) plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.

摘要

转基因为木瓜(Carica papaya L.)植物是从胚胎培养物再生而来的,这些培养物与一种无武装的 C58 菌株的根癌农杆菌共培养,该菌株含有以下两种二元共质体载体之一:pGA482GG 或 pGA482GG/cpPRV-4。这两种二元载体的 T-DNA 区域都包含新霉素磷酸转移酶 II(NPTII)和β-葡萄糖醛酸酶(GUS)的嵌合基因。此外,木瓜环斑病毒(PRV)的植物表达外壳蛋白(cp)基因在 pGA482GG/cpPRV-4 中被 NPTII 和 GUS 基因侧翼。通过在 150μg·ml(-1)卡那霉素上进行选择,获得了具有潜在转化能力的胚胎发生木瓜组织。从 cp 基因(-)载体中获得了 4 个潜在的转基因植物系,从 cp 基因(+)载体中获得了 2 个。在所测试的所有潜在转化植物的叶片中均检测到 GUS 和 NPTII 的表达,而在 PRV cp 基因(+)植物的叶片中检测到 PRV 外壳蛋白的表达。使用聚合酶链反应扩增和 NPTII 和 PRV cp 基因的基因组印迹杂交分析了这些木瓜植物的转化状态。通过基因组印迹杂交证明了这些基因整合到木瓜基因组中。因此,与许多其他双子叶植物一样,木瓜可以用根癌农杆菌转化并再生为表型正常的表达外源基因的植物。

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