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菜薹因为没有好的雄性不育材料或自交不亲和系,至今尚无一代杂种用于生产,根据拟南芥及白菜型油菜的花药不开裂基因DAD1的保守序列设计引物,扩增菜薹的DAD1基因片段(DAD1F),构建反义DAD1F植物表达载体,用农杆菌介导法转化菜薹,对转基因植株进行分子检测,鉴定其雄性不育性并进行育性恢复试验。克隆得到的菜薹的DAD1基因片段大小为678bp,命名为BrcpDAD1F,其序列与拟南芥和白菜型油菜的DAD1高度同源,同源率分别为88%和99%;共得到了12株转基因植株,有6株在转录水平上得到表达,表现为雄性不育,花器官畸形,花粉活力低,萌发率不到10%,且开花后不能结角果或结空角果,或者得到极少种子但种子不萌发;用对照的花粉给转基因植株授粉可使其正常结实。以500μmol.L-1茉莉酸甲酯处理可使其雄性不育得到恢复,花粉可以在柱头和培养基上萌发,具有受精能力。T1代可育株与不育株的比例都呈1∶3分离,T2代不同株系的育性分离比例不同,有些株系继续呈1∶3的分离,有些株系全是可育株或全是不育株,说明反义抑制呈单基因稳定遗传。
Because there is no good male sterile material or self-incompatibility lines, there are no generation of hybrids for production. Based on the conserved sequence of anther dehiscence gene DAD1 of Arabidopsis and Brassica campestris, The DAD1 gene fragment of sedge (DAD1F) was constructed to construct antisense DAD1F plant expression vector. The Agrobacterium tumefaciens-mediated transformation of Campanulaceae was used to detect the molecular marker of the transgenic plants. The male sterility of the transgenic plants was identified and the fertility restoration test was carried out. The cloned DAD1 gene fragment was 678bp in length and named BrcpDAD1F. Its sequence was highly homologous to DAD1 in Arabidopsis and Brassica campestris, and its homology was 88% and 99% respectively. A total of 12 transgenic Six plants were expressed at the transcriptional level, showing male sterility, floral organ deformity, low pollen viability, germination rate less than 10%, and no anther or anther fruit after flowering, or very few Seeds, however, did not germinate; pollination of transgenic plants with control pollen allowed for normal seed setting. Male sterility was restored by treating with 500μmol.L-1 methyl jasmonate, and pollen germinated on stigma and medium with fertilizing ability. The ratio of fertile plants in T1 generation was separated by 1: 3. The percentage of fertility segregation in different strains of T2 generation was different. Some plants continued to show a separation of 1: 3, and some of them were all fertile plants or All sterile plants, indicating antisense inhibition was single-gene stable inheritance.