类固醇5α-还原酶基因(steroid 5α-reductase 2,DET2)是油菜素内酯合成的关键基因,在抵抗非生物胁迫伤害中起着重要的作用.本研究克隆了杨树(Populus L) PtDET2基因,cDNA编码区全长774 bp,编码257个氨基酸;进化树分析表明,PtDET2基因编码的氨基酸序列与碧桃(Prunus persica)相似性为78％.构建植物表达载体pSH737-35S-PtDET2,利用农杆菌(Agrobacterium tumefaciens)介导的叶盘法遗传转化烟草.选取3个转基因株系TP10、TP12和TP15,用不同浓度甘露醇和NaCl模拟干旱胁迫和盐胁迫,对其种子发芽率和幼苗期进行耐旱性和耐盐性分析.结果发现,在300 mmol/L甘露醇处理15d,种子发芽率比野生型高40％以上,通过观察苗期根的生长发现,野生型植株根系生长明显受到抑制;在200 mmol/L NaCl处理种子15d,转基因植株种子发芽率比野生型高50％以上,野生型植株根系生长也受到明显的抑制.分别用20％ PEG 6 000和200 mmol/L NaCl处理转基因植株和野生型植株(0、1、3、5和7d),结果显示,在干旱和盐胁迫下,转基因植株的超氧化物歧化酶(Superoxide dis-mutase,SOD)、过氧化氢酶(Catalase,CAT)和过氧化物酶(Peroxidase,POD)的活性均显著高于野生型,丙二醛(Malondialdehyde,MDA)含量显著低于野生型植株.因此,PtDET2基因表达提高了烟草植株的耐旱与耐盐能力,研究结果为进一步探讨该基因功能提供依据,为创制转基因耐旱和耐盐提供基础资料.%The steroid 5αt-reductase gene is a key gene in the synthesis of brassinolide and plays an important role in abiotic stress tolerance.Currently,the steroid 5α-reductase 2 gene (PtDET2) were cloned from poplar (Populus tomentosa Carr.).Its coding region of the cDNA was 774 bp and encoded 257 amino acids.The phylogenetic tree analysis showed that the sequence of the cDNA encoding the poplar PtDET2 gene was 78％ similar to that of Prunus persica.The plant expression vector pSH737-35S-PtDET2 was constructed and transformed into tobacco using Agrobacterium tumefaciens-mediated leaf disc.Three different transgenic lines,i.e.TP10,TP12 and TP15 as well as the wild type,were subjected to the drought tolerance and salt tolerance of seed germination rate and seedling stage with different concentrations of mannitol and NaCl simulated drought stress and salt stress,The results showed that the seed germination rate was more than 40％ higher than that of wild type when treated with 300 mmol / L mannitol for 15 days.The growth of wild type plant roots was obviously inhibited by observing the growth of seedling roots.The seeds were treated with 200 mmol / L NaCl 15 d,the seed germination rate of transgenic plants was 50％ higher than that of wild type,and the growth of wild type plant roots was also inhibited obviously.The transgenic plants and wild plants 0 d,1 d,3 d,5 d and 7 d were treated with 20％ PEG 6000 and 200 mmol / L NaCl respectively.The results showed that under drought and salt stress,the superoxide dismutase,The activities of enzymes and peroxidase were significantly higher than those of wild type,and the content of malondialdehyde was significantly lower than that of wild type plants.Studies have shown that PtDET2 gene expression may improve the drought tolerance and salt tolerance of tobacco plants.This study provides the basis for further study of the gene function,and provides the basic data for the establishment of transgenic drought tolerance and salt tolerance.(it should be considerably improved).
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