白菜雄性不育相关基因BcMF4的反义RNA验证

根据普通白菜(Brassica campestris ssp.chinensis var.communis)雄性不育相关基因BcMF4的cDNA序列设计特异引物,从普通白菜花蕾cDNA中扩增出607 bp的片断,然后将该片段连接至双元载体pBI12l中,得到反义RNA植物表达载体并导入农杆菌LBA4404菌株中;通过组织培养途径转化菜心(B.campestris ssp.chinensis var.parachinensis),得到了12株转基因植株,其中5株的43.7%的花粉为缩小空瘪畸形,29.6%的花粉缺乏生活力,而且其花粉离体萌发率降低至24.7%.结果表明,反义RNA技术沉默BcMF4基因导致了菜心转基因植株的部分花粉发育不良,BcMF4基因在普通白菜和菜心等花粉发育中起着重要作用.

Functional validation of BcMF4 gene related to male sterility of Chinese cabbage by antisense RNA

Based on the cDNA sequence of BcMF4 related to male sterility of Chinese cabbage-pak-choi(Brassica campestris ssp.chinensis var.communis,syn.B.rapa ssp.chinensis var.communis),a pair of specific primers were designed and a 607 bp fragment was amplified from the cDNA of flower buds of Chinese cabbage-pak-choi.The fragment was introduced to binary vector pBI121,and the generated antisene RNA vector was then mobilized into Agrobacterium tumefaciens strain LBA4404.The A.tumefaciens harboring the BcMF4 fragment was transformed to flowering Chinese cabbage(B.campestris ssp.chinensis var.parachinensis) via tissue culture,and 12 independent transgenic plants were obtained.Approximately 43.7% of the pollen grains from 5 transgenic plants exhibited abnormal shapes,and only 24.7% of pollen grains from these plants germinated normally.These results indicated that the functional interrupted by antisense RNA resulted in partial pollen grains abortion in flowering Chinese cabbage,suggesting that the product of BcMF4 gene plays an important role during pollen development of Chinese cabbage crops such as Chinese cabbage-pak-choi and flowering Chinese cabbage.