基于EST库的猕猴桃脂氧合酶基因家族成员的克隆

Molecular Cloning of Lipoxygenase Gene Family Members in Kiwifruit Based on EST Database

利用猕猴桃EST库及相关生物信息学手段,从果肉组织克隆了LOX基因家族成员6个,命名为AdLox1-6。AdLox1、AdLox2、AdLox3和AdLox4为全长cDNA,编码区长度分别为2739、2595、2739和2709bp,AdLox5和AdLox6为cDNA片段,长度为1359和1557bp。猕猴桃AdLox2和AdLox5氨基酸序列同源性最高,约74%,而AdLox3和AdLox5仅为49%。生物信息学分析表明,AdLox1、AdLox3、AdLox4和AdLox6可能具有13-LOX活性,在N端含有额外约60个氨基酸残基,而AdLox2和AdLox5则属于9-LOX类型。猕猴桃AdLox1与番茄TomLoxD具有最高氨基酸序列同源性(约80%),AdLox2和AdLox5与TomLoxA高度聚类,序列同源性分别为68%和75%,AdLox4和AdLox6与TomLoxC的氨基酸序列同源性分别为62%和63%,AdLox3与番茄LOX基因序列同源性低于55%。

From Actinidia EST database, six LOX were cloned from kiwifruit flesh tissue, i.e. AdLox1-6. AdLox1, AdLox2, AdLox3 and AdLox4 contained full-length cDNAs with 2 739, 2 595, 2 739 and 2 709 bp, respectively, AdLox5 and AdLox6 were cDNA fragments of 1 359 and 1 557 bp. Among the six kiwifruit LOX genes, AdLox5 and AdLox6 had the highest amino acid sequence identity, which was 74%, while AdLox3 and AdLox5 produced the lowest amino acid sequence identity, which was 49%. Phylogenetic analysis showed that AdLox2, AdLox5 clustered as 9-LOX, AdLoxl, AdLox3, AdLox4 and AdLox6 clustered as 13-LOX. There was a signal peptide sequence of 60 amino acids in all members of the 13-LOX group, but no similar sequence in the 9-LOX genes. AdLoxl was most similar to tomato TomLoxD at 80% identity at the amino acid level, AdLox2 and AdLox5 matched to TomLoxA with 68 and 75% identity, respectively, and AdLox4 and AdLox6 showed 62% and 63% sequence identity to TomLoxC, AdLox3 had less than 55% identity to LOX genes from tomato.