Construction of an antisense CYP86MF gene plasmid vector and production of a male-sterile Chinese cabbage transformant by the pollen-tube method
Construction of an antisense CYP86MF gene plasmid vector and production of a male-sterile Chinese cabbage transformant by the pollen-tube method
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DOI：
10.1080/14620316.2004.11511851
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发表时间：
2004-01
期刊：
影响因子：
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通讯作者：
Xiaolin Yu;Jiashu Cao;W. Ye;Yongqin Wang
中科院分区：
文献类型：
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作者：
Xiaolin Yu;Jiashu Cao;W. Ye;Yongqin Wang
摘要构建了含有细胞色素P86MF基因反义片段和绒毛膜特异性A9启动子的植物表达载体pBIA9-AMF。采用浸花法和花粉管转化法将载体导入小白菜。中国草(L.)Makino var.Common is Tsen et Lee，syn.白菜型油菜B.Rapa Ssp.中国草(L.)Makino var.以及开花大白菜(B.campestris SSP.中国草(L.)Makino var.Parachinens(Bailey)Tsen et Lee)。结果表明，通过T1代种子萌发试验，花粉管法可获得KanR苗，而浸花法不能获得KanR苗。其中一株KanR植株经PCR扩增和Southern blotting鉴定，证明该反义片段已整合到大白菜基因组中。Northern杂交结果表明，在A9启动子作用下，转化子中的CYP86MF基因被抑制，自花授粉后的转基因植株出现自交不育现象。用扫描电子显微镜对转基因植株和野生型植株的花器官和小孢子形态进行比较，结果表明，除了转基因植株的小孢子被一层未知物质覆盖外，没有显著差异。我们认为这一层可能是CYP86MF基因反义片段诱导雄性不育表型的原因。
Summary A plant expression vector pBIA9-AMF containing an antisense fragment of the CYP86MF gene and the tapetum-specific A9 promoter was constructed. Plasmid vectors were introduced by floral-dipping and pollen-tube transformation methods to Chinese cabbage pak-choi (Brassica campestris ssp. chinensis (L.) Makino var. communis Tsen et Lee, syn. B. rapa ssp. chinensis (L.) Makino var. communis Tsen et Lee) and flowering Chinese cabbage (B. campestris ssp. chinensis (L.) Makino var. parachinensis (Bailey) Tsen et Lee). Results showed that KanR seedlings could be obtained by the pollen-tube method through germination tests of T1 progeny seeds, but not by the floral-dipping method. One of the two KanR seedlings proved that the antisense fragment of the CYP86MF gene was integrated into the Chinese cabbage genome by PCR amplification and Southern blotting. Northern hybridization indicated that the CYP86MF gene, under the A9 promoter, was inhibited in the transformant, and self-infertility was found in the transgenic plantlets after self-pollination. Comparison of floral organs and the morphology of microspores by scanning electron microscopy of transformant and wild-type plantlets, showed no significant differences except that microspores from the transgenic plant were covered in a layer of an unknown substance. We suggest this layer might be the cause of the male-sterile phenotype induced by the antisense fragment of the CYP86MF gene.