摘要
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。
Through alignment with the sequence of NFYA5 gene from Arabidopsis thaliana （Atlg54160）, we got the homologous ESTs in Chinese cabbage [Brassica carnpestris L. ssp. pekinensis （Lour.） Makino]. Then, the full length of the gene namely BpNFYA5 was cloned by the method of Electronic Clone. The deduced protein of BpNFYA5 has the CCAAT-box binding domain, which showed 92.6% amino acid sequence identify with that ofArabidopsis. BpNFYA5 transcript is strongly induced by drought stress. The over-expression vector pBIn-NFYA5 and RNA-interference vector pFGC-NFYA5 were constructed and transformed to Arabidopsis thaliana （Col） mediated by Agrobacterium tumefaciens. The drought-tolerance was analyzed between the BpNFYA5 over-expression （OL）, RNA-interference （Ri） and the wild-type （WT） Arabidopsis through the methods of drought stress and 10% PEG6000 drought simulated treatments. The results showed that chlorophyll content of OL was higher compared with those of WT and Ri; Proline content of OL accumulated quickly and showed higher than those of WT and Ri Likewise, the rate of water loss in OL leaves was less than in WT and Ri. All above suggested that BpNFYA5 gene play an important role in drought response.
出处
《园艺学报》
CAS
CSCD
北大核心
2012年第8期1501-1510,共10页
Acta Horticulturae Sinica
基金
北京市农林科学院科技创新能力建设项目(KJCX201102003)
