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中国农学通报 ›› 2010, Vol. 26 ›› Issue (15): 32-37.

所属专题: 生物技术 园艺

• 生物技术科学 • 上一篇    下一篇

花粉介导法将萝卜抗菌肽基因导入野罂粟的影响因素研究

魏玉杰 张金文 何庆祥   

  • 收稿日期:2010-03-02 修回日期:2010-04-08 出版日期:2010-08-05 发布日期:2010-08-05

  • Received:2010-03-02 Revised:2010-04-08 Online:2010-08-05 Published:2010-08-05

摘要:

以野罂粟开放花蕾为受体,载有萝卜抗菌肽AFP(antifungal protein)基因的质粒pBIAFP为供体,在不同浓度的蔗塘溶液中,加入新鲜花粉与含有目的基因的质粒DNA,应用SKH250LH型超声波震荡器进行震荡处理得到花粉处理液,在不同的时间用带有Coolsnap的OLYPUS光学显微镜下观察野罂粟花粉萌发情况;在不同授粉时间后,辅以人工授粉的方法将花粉处理液滴注野罂粟柱头上,套袋后获得种子,测定其结实率。实验证明:利用花粉介导法进行萝卜抗菌肽基因转化,以蔗糖浓度11%,处理后10-15h后的野罂粟花粉萌发最好;在蔗糖浓度8%的溶液中,以5h的结籽量较好。后代经卡那霉素筛选和PCR检测,初步证明外源AFP基因整合到野罂粟基因组中,并获得转基因T1代。为特殊中药材育种提供一个新方法。

关键词: 辛硫磷, 辛硫磷, 油菜, 土壤, 残留, 消解动态

Abstract:

Abstract: Selecting opening wild poppy bud as receptors , plasmid pBIAFP containing the radish peptide AFP (antifungal protein) gene as donor, in different concentrations of cane sugar solution, adding fresh pollen and plasmid DNA containing the target gene , shocking with SKH250LH-type ultrasonic oscillator for treatment pollen processing fluid. After all these process, we observed its pollen germination under OLYPUS light microscope with Coolsnap at different times. After different pollination time, dropping the pollen on wild poppy stigma supplemented with artificial method, bagging for seed and measuring seed-setting rate. The results show that, after transformation of radish peptide with pollen-mediated method , 11% sucrose concentration and 10-15h treatment is best for wild poppy pollen germination , when the sucrose solution concentration is 8%, 5h can get better seed-setting rate. After future kanamycin selection and PCR detection , we verified that exogenous AFP genes integrate into the genome of wild poppy initially and obtained transgenic T1 generation .It provide a new approach for special Chinese herbal medicine breeding .