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[通过粒子轰击获得携带新型cry1Ac基因的抗螟虫明恢81纯合转基因系]

[Obtaining stem borer-resistant homozygous transgenic lines of Minghui 81 harboring novel cry1Ac gene via particle bombardment].

作者信息

Zeng Qian-Chun, Wu Qian, Zhou Kai-Da, Feng De-Jiang, Wang Feng, Su Jun, Altosaar Illimar, Zhu Zhen

机构信息

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

出版信息

Yi Chuan Xue Bao. 2002 Jun;29(6):519-24.

Abstract

A modified cry1Ac gene was generated by fusing with Lys-Asp-Glu-Lue (KDEL), an endoplasmic reticulum retention signal at the 3'-ends, with signal peptide coding sequence of Soybean kunitz trypsin inhibitor (SKTI) at the 5'-ends. Vector containing the modified cry1Ac gene coding region flanked by the corn ubiquitin 1 promoter and the nopaline synthase gene (nos) terminator with Hygromycin Phosphotransferase (hpt) gene as a plant selection marker was constructed. The modified cry1Ac gene in which toxic protein targeted to endoplasmic retention was successfully transferred into Minghui 81 (Oryza sativa L. subsp. indica), an elite restoring line of commercial CMS indica hybrid rice, through particle bombardment and obtained fertile transformants. Homozygous transgenic rice lines were obtained in the third generation exploiting self-seed set reproduction and HygromycinB selection. These transgenic lines were confirmed with polymerase chain reaction (PCR) amplification, Southern blotting and ELISA detection. Pest insect-resistant bioassay indicated that some of the homozygous cry1Ac-transgenic rice plants of T2 progeny showed high-level resistance against striped stem borer (Chilo suppressalis) at field trials.

摘要

通过在3'端与内质网滞留信号赖氨酸-天冬氨酸-谷氨酸-亮氨酸(KDEL)融合,并在5'端与大豆库尼茨胰蛋白酶抑制剂(SKTI)的信号肽编码序列融合,产生了一种修饰的cry1Ac基因。构建了载体,其包含由玉米泛素1启动子和胭脂碱合成酶基因(nos)终止子侧翼的修饰cry1Ac基因编码区,以及作为植物选择标记的潮霉素磷酸转移酶(hpt)基因。通过粒子轰击将内质网滞留靶向的毒性蛋白的修饰cry1Ac基因成功转入明恢81(Oryza sativa L. subsp. indica),一种商业化三系籼型杂交水稻的优良恢复系,并获得了可育的转化体。利用自交结实繁殖和潮霉素B选择在第三代获得了纯合转基因水稻株系。通过聚合酶链反应(PCR)扩增、Southern杂交和ELISA检测对这些转基因株系进行了确认。害虫抗性生物测定表明,在田间试验中,一些T2代纯合cry1Ac转基因水稻植株对二化螟(Chilo suppressalis)表现出高水平抗性。

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