Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S‐adenosyl‐L‐methionine synthetase activity
Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S‐adenosyl‐L‐methionine synthetase activity
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用来自拟南芥的 sam1 转化罂粟细胞悬浮培养物，产生具有不同 S-腺苷-L-蛋氨酸合成酶活性的细胞系
DOI：
10.1111/j.1399-3054.1997.tb05407.x
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
A. David
中科院分区：
文献类型：
--
作者：
Muriel Belny;D. Hérouart;B. Thomasset;H. David;A. Jacquin;A. David
我们开发了一种利用根癌农杆菌介导法进行罂粟(Papaver Somniferum)转基因的方法。用含有双元载体pTHW136或双元载体pO35SSAM的根癌农杆菌菌株GV3101(PMP90)与睡莲下胚轴细胞悬浮培养。前者含有uidA报告基因和nptII选择基因，分别编码β-葡萄糖醛酸酶和新霉素磷酸转移酶II。后者包括编码S-腺苷-L-蛋氨酸合成酶的sam基因和npt11基因。在添加帕罗霉素的培养液中筛选转化细胞系。杂交前用和不用PCR扩增的Southern杂交分析证实外源基因的整合。测定了5个转化细胞系提取液中SAM合成酶的活性。其中一个细胞表现出明显的过度活性，而另外两个细胞株的活性低于对照细胞系，这让我们质疑可能存在的本地和外来SAM基因的部分共抑制。据我们所知，这是根癌农杆菌介导的罂粟悬浮细胞转化的首次报道。
We have developed a procedure for opium poppy (Papaver somniferum) transformation using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl-derived cell suspension cultures of P. somniferum were cocultivated with A. tumefaciens strain GV3101 (pMP90) harbouring either the binary vector pTHW136 or the binary vector pO35SSAM. The former contained the uidA reporter gene and the nptII selectable gene, encoding the enzymes β-glucuronidase and neomycin phosphotransferase II, respectively. The latter contained the saml gene encoding the enzyme S-adenosyl-L-methionine (SAM) synthetase and the nptll gene. Putatively transformed cell lines were selected on media supplemented with paromomycin. Integration of the foreign genes was confirmed by Southern blot analysis with and without PCR amplification prior to hybridization. SAM synthetase activity was measured in extracts of 5 transformed cell lines. One of them expressed a significant overactivity while two others had a lower activity than the control cell line, leading us to question the possible partial cosuppression of both the resident and the foreign sam genes. To our knowledge, this is the first report of Agrobacterium tumefaciens-mediated transformation of Papaver somniferum cell suspension cultures.