以拟南芥吡哆醛激酶基因(PK)的蛋白质序列为信息探针,对大豆(Glycine max)EST数据库进行同源搜索和序列拼接,获得了全长为1102 bp的大豆吡哆醛激酶基因的cDNA序列,经RT-PCR克隆和序列分析验证,结果表明与电子克隆序列一致(GenBank登录号为DQ006813).该eDNA序列的开放阅读框(0RF)位于第119～10位,推测编码308个氨基酸.采用半定量RT-PCR方法研究了该基因的组织特异性表达情况和对强光逆境的反应,结果表明该基因在大豆根茎叶中都有表达,对强光逆境不敏感.将大豆PK基因编码的蛋白序列与马铃薯(Solanum tuberosum,AAY8186)、拟南芥(Arabidopsis thaliana,AAK94021)、水稻(Oryza sativa,ABA9978)、小麦(Triticum aestivum,AAR00318)和油菜(Brassica napus,ABE73472)的吡哆醛激酶蛋白序列进行比对,发现其一致性分别为:81%,7%,78%,79%和76%.比对结果说明在植物中该基因编码的蛋白质存在显著的相似性.根据蛋白比对结果绘制的系统发育树基本代表了它们在经典分类上的地位.
Using Arabidopsis thaliana pyridoxal kinase (PK) protein sequence as a query probe to blast Glycine max EST database,a soybean PKgene cDNA of 1 102 bp was obtained and cloned by RT2PCR and sequenced(GenBank Ac2 cession, DQ006813). The result confirmed that the in silico cloning cDNA sequence was accurate. It contains a complete open reading frame,from 119 bp to 1 045 bp, encodes 308 amino acids. Using semiquantitative RT2PCR,we found that PK gene was not regulated by illumination. It expressed in Glycine max leaves,stems and roots. The deduced amino acid sequence of Glycine max PKwas aligned against sequences from other species such as Solanum tuberosum, Arabidopsis thaliana, Oryza sativa, Triticum aestivum and Brassica napus. It showed the identity of 81 %,75 %,78 %,79 % and 76 %,respectively. The results revealed the Glycine max PKwas significant similarity in plants. Phylogenetic tree of plant PKs was mainly consistent with the classification system of these plants.
[1] Gill R W,Sanseau P.Rapid in sillco cloning of genes using expressed sequence tags(ESTs)[J].Biotech Ann Rev,2000,5:25-44.
[2] 陈润生.生物信息学及其研究进展[J].医学研究通讯.2002,31(12):2-5.
[3] 何志颖,姚玉成,胡以平.EST技术及其在基因全长cD.NA克隆上的应用策略[J].国外医学遗传学分册,2002,25(2):67-69.
[4] Wang H,Liu D,Liu C,et al.The pyridoxal kinase gene TaPdxK from wheat complements vitamin B6 synthesis-defec.tive Escherichia coli[J].Journal of Plant Physiology.2004.161:1053-1060.
[5] Tambasco-Studart M,Titiz O,Raschle T,et al.Vitamin B6 biosynthesis in higher plants[J].PNAS,2005,102(38):13687-13692.
[6] 孟宪萍,李晓晓,李雅轩,等.大豆尿黑酸叶绿基转移酶基因的克隆与进化分析[J].华北农学报,2007,22(4):14-18.
[7] 李蕊,孟宪平,胡英考,等.大豆吡哆醇生物合成蛋白基因(PDX)的电子克隆和进化分析[J].华北农学报,2007,22(1):64-68.
[8] 陈大福,牛宝龙,翁宏飚,等.蜜蜂(Apis mellifera)腺苷酸转移载体基因cDNA的电子克隆[J].中国农业科学,2004,37(6):923-927.
[9] 赵国屏.生物信息学[M].北京:科学出版社.2002.
[10] 张丽娜,牛吉山,于玲.用半定量RT-PCR方法分析小麦TaMlo.Alc基因的表达[J].西北植物学报,2005,25(7):1368-1371.
[11] 唐向荣,吴吴,贾明.水稻双链RNA结合蛋白同源基因OsRBP的克隆及其表达的分析[J].植物生理与分子生物学学报,2002,28(1):41-45.
[12] 张成岗,贺福初.生物信息学方法与实践[M].北京:科学出版社,2002.
