甘蔗ACC氧化酶全长cDNA的克隆及序列分析

ACC氧化酶是高等植物乙烯生物合成途径中的限速酶。根据报道的植物ACC氧化酶基因序列设计特异引物,从甘蔗cDNA文库中克隆到一ACC氧化酶基因片段,命名为GZ-ACO,该基因长792bp,与甘蔗基因组文库中克隆的ACO基因片段仅18个碱基之差。根据该cDNA片段序列,设计两对末端扩增的特异引物,利用RACE-PCR技术,获得GZ-ACO片段的5′端和3′端序列。用VectorNTI7.0软件对三个序列进行拼接和分析,结果得到全长的甘蔗GZ-ACO氧化酶基因。GZ-ACOcDNA核苷酸序列长1307bp,具有一个972bp完整的读码框,启动子ATG位于126bp,终止子TAA位于1097bp,推导编码323个氨基酸。系统进化分析表明,GZ-ACO基因氨基酸序列与其它植物已报道的ACC氧化酶基因具有65%~86%的同源率,且与单子叶禾本科植物首先聚类,其次与单子叶芭蕉科、兰科植物聚类,最后与双子叶植物聚类,与植物形态的系统进化结果一致。该基因已在DDBJ/EMBL/GenBank基因数据库注册,注册号为AY521566。

Cloning and sequencing of full-length cDNA encoding ACC oxidase in sugarcane

1-aminocyclopropane-1-carboxylic acid oxidase is one of the key rate-limiting enzymes for ethylene biosynthesis in higher plants. In the present study,a cDNA fragment of 792 bp,designated GZ-ACO,was obtained from sugarcane cDNA library. According to the GZ-ACO cDNA fragment sequence,full length of GZ-ACO cDNA was generated by 5′RACE and 3′RACE methods. GZ-ACO cDNA was 1 307 bp in length. Within the full-length cDNA,a clear open reading frame(ORF)was 972 nucleotides,coding 323 amino acids. The 5′end included a putative translation start codon(ATG)at position 126 bp;the 3′end included an end codon(TAA)at position 1 097 bp and had a poly(A)tail. The identity of the other polypeptides to each other varied between 65%～86%. The results of phylogenetic analysis showed that ACC oxidase from sugarcane and from Gramineae clustered together firstly,and then came those from Musaceae and Orchidaceae,dicotyledon came subsequently. The clustering results of ACC oxidase molecules accorded with morphological classification system. The sequence of GZ-ACO had been submitted to the DDBJ/EMBL/GenBank database,the accession number was AY521566.