观赏凤梨烯醇酶基因的克隆及分析

摘要

Enolase catalyze only one-step dehydration reaction in glycolytic pathway, and relate closely to plant stress resistance. Based on EST monoclones of enolase gene obtained from bromeliads full length cD-NA library of ornamental bromeliads,its full length cDNA sequence was obtained by Primer Walking sequencing. The gene,named for GoEnolasel (GenBank accession No. JN896863) .consisted of 1 703 bp cD-NA sequence, 1 335 bp ORF(open reading frame) which encoded a protem with 445 amino acids residues, and a putative protein which was 47. 9 kD at estimated molecular weight,5. 7 at isoelectric point and had a 'TIM-phosphate-binding superfamily' conservative domain. The secondary structure of the protein was composed of alpha helix (44. 49%), random coil (33. 71%), extend strand (13. 71%) and beta turn (8. 09%) by SPOMA analysis. Furthermore, its tertiary structure was also build based on A chain of 2PSN. Through phylogenetic tree analysis,GoEnolasel was gather to a same group with enolase protein in Oryza sativa ,Zea mays and Elaeis guineensis. The research was beneficial to breeding for stress resistance in ornamental bromeliads.%烯醇酶催化着糖酵解途径中的唯一一步脱水反应,与植物的抗逆性有着密切的联系.基于前期从擎天凤梨全长cDNA文库中获得的烯醇酶基因的EST单克隆,进行引物步移测序,获得其全长cDNA序列.序列长1703hp,编码445个氨基酸残基,命名为GoEnolasel(GenBank登录号JN896863),蛋白质理论分子质量为47.9kD,等电点为5.7,包含1个TIM磷酸结合超家族保守区,二级结构主要由α螺旋(44.49％)、随机卷曲(33.71％)、延伸链(13.71％)和β转角(8.09％)组成.系统进化树分析表明,GoEnolasel蛋白与水稻、玉米、油棕等的烯醇酶蛋白聚为一类,它们的亲缘关系最近.