拟南芥35S∶NAS2载体构建和转基因株系的筛选

Construction of Arabidopsis thaliana 35S∶NAS2 vector and screening of transgenic lines

为了研究基因NAS2和BZIP44在拟南芥缺铁胁迫响应中的遗传关系,文章以野生型(wild type,WT)拟南芥为材料构建35S∶NAS2/WT转基因植株,以拟南芥突变体bzip44为材料构建35S∶NAS2/bzip44转基因植株;提取野生型拟南芥的RNA,以其反转成的cDNA作为模板克隆NAS2的CDS区域,将双酶切后的目的片段与同样双酶切的pART27质粒进行连接,得到的连接产物转入大肠杆菌DH5α感受态中,通过菌落聚合酶链式反应(polymerase chain reaction,PCR)鉴定和测序得到阳性单克隆,然后将重组质粒转入农杆菌感受态GV3101中,获得阳性单克隆菌落;用浸花法分别侵染野生型拟南芥和纯合的拟南芥bzip44突变体植株,通过筛选和鉴定得到纯合的35S∶NAS2/WT和35S∶NAS2/bzip44转基因植株。研究结果为基因NAS2和BZIP44在拟南芥缺铁胁迫响应中的遗传关系提供参考。

In order to study the genetic relationship between the genes NAS2 and BZIP44 in the response of Arabidopsis thaliana to iron deficiency stress,35S∶NAS2/WT transgenic plants were constructed using wild type(WT)Arabidopsis thaliana as materials,and 35S∶NAS2/bzip44 transgenic plants were constructed using Arabidopsis thaliana mutant bzip44 as materials.The RNA of WT Arabidopsis thaliana was extracted,and the CDS region of NAS2 was cloned with the inverted cDNA as a template.The target fragment after double enzyme digestion was ligated with the pART27 plasmid of the same double enzyme digestion,and the resulting ligation product was transferred into E.coli DH5αcompetent cells.A positive single clone was obtained by polymerase chain reaction(PCR)and sequencing,and then the recombinant plasmid was transferred into Agrobacterium GV3101 competent cells to obtain a positive single clone.Then,the WT and homozygous Arabidopsis thaliana bzip44 mutant plants were infected with the floral dip method,respectively.The homozygous 35S∶NAS2/WT and 35S∶NAS2/bzip44 transgenic plants were obtained through screening and identification.The results of this study provide reference for the genetic relationship of genes NAS2 and BZIP44 in the response to iron deficiency stress in Arabidopsis thaliana.