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以拟南芥(Arabidopsis thaliana)NFYA5基因(GenBank登录号:At1g54160)的序列为基准,通过比对获得芸薹属大白菜[Brassi cacampestrisL.ssp.pekinensis(Lour.)Makino]同源EST序列;采用电子克隆的方法从大白菜中克隆到相关基因全长序列,命名为BpNFYA5。该基因包含3个内含子,其推导蛋白与拟南芥NFYA5CCAAT-box结合域相似性达92.6%。对大白菜进行干旱胁迫,发现BpNFYA5显著上调表达。构建了植物过表达载体pBIn-NFYA5和RNA干扰载体pFGC-NFYA5,通过根癌农杆菌(Agrobacterium tumefaciens)介导浸花法转化拟南芥。经除草剂、潮霉素筛选及PCR鉴定获得转基因植株。采用干旱和10%PEG6000模拟亏水胁迫,对转基因材料的T2代进行了耐旱性鉴定。结果显示:过表达植株(OL)较野生型植株(WT)和干扰表达植株(Ri)叶绿素含量高;渗透胁迫下OL植株脯氨酸含量迅速积累,积累量显著高于WT和Ri植株;控水干旱2周OL株系较WT及Ri材料具有更好的干旱耐受性;OL株系离体叶片的失水速率较小,叶片有效水分利用率较WT和Ri高。结果表明所克隆的基因BpNFYA5在亏水胁迫调控中具有抗旱作用。
Based on the sequence of Arabidopsis thaliana NFYA5 gene (GenBank accession number: At1g54160), the homologous EST sequence of Brassica campestris L.ssp.pekinensis (Lour.) Makino was obtained by comparison; Electronic cloning method from the Chinese cabbage cloned to the full length of the relevant gene sequence, named BpNFYA5. The gene contains three introns, which deduced that the deduced protein has 92.6% similarity to Arabidopsis NFYA5CCAAT-box binding domain. Drought stress on Chinese cabbage, found that BpNFYA5 significantly up-regulated expression. Plant over-expression vector pBIn-NFYA5 and RNA interference vector pFGC-NFYA5 were constructed and transformed into Arabidopsis by Agrobacterium tumefaciens-mediated maceration. After the herbicide, hygromycin selection and PCR identification of transgenic plants. T2 generation of transgenic plants was identified for drought tolerance using drought and 10% PEG6000 simulated deficit stress. The results showed that the content of chlorophyll in overexpression plants (OLs) was higher than that in wild type plants (WTs) and interference expression plants (Ri). The proline content of OL plants increased rapidly under osmotic stress and the accumulation was significantly higher than that of WT and Ri plants Compared with WT and Ri, the water-desiccated OL strain had better drought tolerance than the WT and Ri materials. The rate of water loss in detached leaves of OL plants was smaller and the available water use efficiency of leaves was higher than that of WT and Ri. The results showed that the cloned gene BpNFYA5 has a drought-resistant role in the regulation of deficient water stress.