Comparison of promoters and selectable marker genes for use in Indica rice transformation
Comparison of promoters and selectable marker genes for use in Indica rice transformation
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DOI：
10.1023/a:1009600219477
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发表时间：
1997-01-01
影响因子：
3.1
通讯作者：
Waterhouse, PM
中科院分区：
文献类型：
--
作者：
Li, ZY;Upadhyaya, NM;Waterhouse, PM
比较了不同启动子在籼稻转化中的效果。通过粒子轰击将编码在CaMV 35 S、Emu、Actl或Ubil启动子控制下的大肠杆菌uidA（gals）基因的质粒递送到细胞悬浮培养物中。瞬时基因表达，48小时后交付，是最大的质粒利用组成型启动子，Actl和Ubil。通过用pUbil-gus质粒和含有选择性标记基因hph（其赋予潮霉素抗性）或bar（其赋予除草剂草丁膦（BASTA）抗性）的质粒轰击胚性籼稻愈伤组织来检测稳定转化组织中的基因表达，所述选择性标记基因hph或bar各自在CaMV 35 S、Emu、Actl或Ubil启动子的控制下。将轰击的愈伤组织置于适当的选择培养基上，并在射击后1天、3周和5周对GUS活性进行染色。用pUbil-hph或pEmu-hph构建体轰击的愈伤组织随着时间的推移，GUS染色区域的大小急剧增加。用pEmu-hph或pUbil-hph构建体和病毒次要外壳蛋白（cp）基因构建体共轰击愈伤组织，得到许多可育的转基因籼稻植株，含有1 - 8个拷贝的hph和cp基因。这些基因被T-1代稳定遗传。
The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gals) gene under the control of CaMV 35S, Emu, Actl or Ubil promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Actl and Ubil. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbil-gus plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Actl or the Ubil promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbil-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbil-gus and any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbil-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T-1 generation.