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利用同源克隆方法从强耐旱栽培大豆(Glycine max)品种齐黄22中克隆得到2个编码大豆△1-吡咯啉-5-羧酸合成酶(P5CS)的基因,分别命名为Gm P5CS1和Gm P5CS2。氨基酸序列分析发现,Gm P5CS1包含1个长度为2 163 bp的ORF,编码720个氨基酸,等电点p I为6.70,分子量大小为78.38 k Da;Gm P5CS2包含1个长度为2 271 bp的ORF,编码756个氨基酸,等电点p I为6.10,分子量大小为82.18 k Da。与NCBI公布的部分物种蛋白质序列比对发现,Gm P5CS1与紫花苜蓿(Medicago sativa)的亲缘关系最高,Gm P5CS2与豇豆(Vigna unguiculata)的亲缘关系最高。组织表达分析表明,Gm P5CS1与Gm P5CS2基因在大豆的各个组织中均有表达,其中叶和根中的表达量相对较高,茎中表达量次之,花和籽粒中的表达量相对较低。利用Gateway技术得到植物过表达载体p Earley Gate103-Gm P5CS,再转入根癌农杆菌EHA105中,为Gm P5CS基因的转化及功能分析奠定了基础。
Two homologous cloning methods were used to clone two genes coding for soybean △ 1-pyrroline-5-carboxylic acid synthase (P5CS) from strong drought-tolerant cultivated Glycine max Qihuang 22 and named as Gm P5CS1 and Gm P5CS2. Amino acid sequence analysis revealed that Gm P5CS1 contains a 2 163 bp ORF encoding 720 amino acids with an isoelectric point p I of 6.70 and a molecular weight of 78.38 kDa. Gm P5CS2 contains an ORF of 2 271 bp , Encoding 756 amino acids, the isoelectric point p I was 6.10 and the molecular weight was 82.18 kDa. Compared with NCBI published protein sequences of some species, Gm P5CS1 showed the highest genetic relationship with Medicago sativa, and Gm P5CS2 had the highest genetic relationship with Vigna unguiculata. Tissue expression analysis showed that Gm P5CS1 and Gm P5CS2 genes were expressed in all tissues of soybean, and the expression of Gm P5CS1 and Gm P5CS2 was relatively high in leaves and roots, followed by expression in stems and lower in flowers and grains . Using Gateway technology, the plant over-expression vector pEleyley Gate103-Gm P5CS was transformed into Agrobacterium tumefaciens EHA105, which laid the foundation for the transformation and function analysis of Gm P5CS gene.