番茄脂肪氧化酶D基因的克隆分析和植物表达载体的构建

Cloning and Analysis of Lipoxygenase D from Tomato and Plant Expression Cassettes Construction

利用RT-PCR方法从番茄中克隆了TomloxD的cDNA。采用生物信息学,ChloroP,SMART方法对TomloxD分析与研究,结果表明,TomloxD基因编码是一个由908个氨基酸残基组成的蛋白,蛋白分子量为102.31 kD。在蛋白的N-端有77个氨基酸组成的叶绿体信号肽。TomloxD是一种植物脂肪氧化酶,具有两个保守结构域:在靠近N-端区域有PLAT/LH2结构域,在C-端有pfam:lipoxygenase结构域。TomloxD蛋白与大豆LOX3蛋白三维结构高度重叠。TomloxD与来自马铃薯、烟草、油橄榄、拟南芥和玉米的脂肪氧化酶的一致性分别为96%、90%、82%、72%和67%。系统进化关系分析表明TomloxD与来源于双子叶植物的脂肪氧化酶蛋白的亲缘关系更近,而与单子叶植物的相对较远。构建植物高效表达载体TomloxD-pBIN19,并导入根癌农杆菌LBA4404中,得到植物转化工程菌,这为下一步用于番茄等作物的转基因操作,进一步研究TomloxD基因的功能,获得高抗逆性的转基因作物奠定了基础。

The eDNA sequence of Torn^xD was cloned from tomato by RT-PCR,whieh was studied with the method of bioinformatics, Chlo- roP and SMART, the result showed that TomloxD encoded a protein of 908 amino acid residues with a calculated molecular mass of 102.31 kDa, and had a chloroplast transit peptide （cTP）, which was a stretch of 77 amino acids in the N-terminal region of protein sequences. TomloxD belonged to plant lipoxygenase and had two conserved domain： a PLAT/LH2 domain in the N-terminal region and a pfam： lipoxyge- nase domain in the （;-terminal region. The three dimensional structure alignments of TomloxD protein and soybean LOX3 protein suggested they were high overlap. Alignment of the amino acid sequence of TomloxD shared extensive homology with other members of higher plants, of which the identity computed as follows： maximum 96 % with Solanum tuberosum LOX-H3, 90 % with Nicotiana attenuate Lox3, 82 % with Olea europaea lox2, 76 % with Arabidopsis thaliana LOX3, and minimum 67 % with Zea mays lox 9. The phylogenetie relationship a- nalysis indicated that TomloxD was more closely related to lipoxygenases from dicots than that from monocots. After constructing the recombi- nant vectors TomloxD-pBIN19, TomloxD-pBIN19 vector was introduced into A. tumefaciens strain LBA4d04, and ready to be used in trans- formation of tomato and researching the function of TomloxD gene and developing transgenic crop plants with improved tolerance to biotic and abiotie stresses.