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中国农学通报 ›› 2017, Vol. 33 ›› Issue (20): 29-34.doi: 10.11924/j.issn.1000-6850.casb16070132

所属专题: 生物技术 小麦

• 生物技术科学 • 上一篇    下一篇

细胞穿透肽介导的小黑麦小孢子遗传转化研究

刘大普,杨凤萍,张晓东   

  1. 北京市农林科学院北京农业生物技术研究中心,北京市农林科学院北京农业生物技术研究中心,北京市农林科学院北京农业生物技术研究中心
  • 收稿日期:2016-07-27 修回日期:2017-06-30 接受日期:2016-10-25 出版日期:2017-07-18 发布日期:2017-07-18
  • 通讯作者: 杨凤萍
  • 基金资助:
    北京市优秀人才培养资助项目“麦类作物抗逆转录因子基因的挖掘和利用”(PYZZ130826001696);北京市农林科学院科技创新能力建设 专项“小麦小孢子转化系统的建立”(KJCX201102003);北京市留学人员科技活动择优资助项目资助“利用大麦条纹花叶病毒研究小黑麦茎秆NAC转 录因子基因”。

Cell Penetrating Peptides Mediated Transformation of Triticale Microspore

  • Received:2016-07-27 Revised:2017-06-30 Accepted:2016-10-25 Online:2017-07-18 Published:2017-07-18

摘要: 本研究选用小黑麦小孢子作为供试材料,进行细胞穿透肽介导的植物单倍体细胞遗传转化可行性研究。提取单核靠边期的小黑麦小孢子进行悬浮培养,将细胞穿透肽与报告基因GFP 的表达框复合物对小孢子进行转化。转化植株进行了PCR分子检测,并在荧光体视显微镜下观察外源GFP 基因的表达情况。共获得21 株候选转基因植株,其中染色体自然加倍植株为10 株。T1代加倍植株的PCR检测结果显示,加倍的转基因株系中有3 个株系的PCR检测结果呈阳性。通过荧光体视显微镜可在转基因植株的幼胚部位观测到GFP蛋白的荧光。以上结果表明外源GFP基因已经整合到小黑麦基因组上并且得到表达并稳定遗传。因此,由细胞穿透肽介导的植物单细胞转化是一种有效、可行的植物单细胞转基因方法。

关键词: 银槭, 银槭, 盐胁迫, 生理指标

Abstract: In this study, the microspores from Triticale c.v. AC Ultima were used as explant to study the feasibility of cell penetrating peptides (CPPs) mediated transformation to a plant single cell. The microspores at mid-to-late-uninucleate stage were isolated and cultured in liquid suspension midium. The mexture of cell penetrating peptides (CPPs) and the plasmid DNA only with GFP gene’s expression frame were introduced into microspores. The transformants were screened by PCR and the expression of foreign GFP gene was identfied with fluorescence stereoscope. 21 transformed plants were regenerated, and 10 transformants were homozygous doubled haploid (DH) plants among them. The analysis of PCR confirmed 3 DH lines with positive results in T1 progeny, and GFP fluorescence could also be observed in the immature embryo of these lines. All the results indicated that exogenous GFP gene was well integrated into the genome of Triticale and inherited to offspring. This paper provides an effctive and feasible approch of CPPs mediated transformation, which will be applied in genetic transformation of plant single cell.

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