甘蔗ACC氧化酶基因片段的克隆与序列分析

Cloning and Sequencing of ACC Oxidase Gene from Sugarcane

1 -氨基环丙烷 - 1-羧酸 (ACC)氧化酶是植物乙烯合成的一个关键酶 ,乙烯作为一种内源激素 ,对植物生长、老熟过程有多方面的调节作用。根据报道的各种植物ACC氧化酶氨基酸序列上前后两个保守区设计两个简并引物 ,以甘蔗总DNA为模板 ,通过PCR扩增到一个 940bp的基因片段。将片段序列在NCBI的BLAST软件上进行同源性搜寻 ,显示的 63个序列全部是ACC氧化酶基因 ,因而认为克隆到的片段就是甘蔗ACC氧化酶基因的一个成员。经对不同植物来源的ACC氧化酶基因家族进行比较分析 ,去除一个 10 3bp的“内含子”后 ,推导的氨基酸序列为 2 79个残基 ,占推测全长氨基酸残基总数的 86%左右。经同源性分析 ,序列与毛竹和水稻ACC氧化酶的同源率达到 86%。系统进化分析表明 ,该序列最先与水稻、其次和香蕉的ACC氧化酶聚类 ,然后再与双子叶植物的ACC氧化酶聚类 ,符合按形态特征分类的血缘关系。基因的获得对下一步了解乙烯的合成表达与甘蔗生长。

The plant hormone ethylene is not only responsible for the initiation of fruit ripening,senescence and dormancy but also for regulating many other plant developmental processes,such as seed germination,root initiation,growth,floral differentiation,sex differentiation and responding to environment stresses.One of the rate-limiting steps for ethylene biosynthesizing in plant is catalyzed by 1 aminocyclopropane 1 carboxylate (ACC) oxidase. Understanding of ethylene expressive pattern in plant is an entrance to understand the roles of ethylene on plant.In this paper,two degenerate oligonucleotide primers were designed,coding for two conservative amino acid regions in ACC oxidase protein family, the sequences of the two primers were TAGAGCTCGATGCTGGAAATGGGG and CGTCTAGAGCTTCAATCTTGGCTCCTT respectively .A PCR amplification was performed on sugarcane ( Saccharum L.Hybrid cv.ROC16) DNA template,and produced a fragment of 940 bp.By using the program of BLAST on NCBI GenBank database,the sequence presented a very high match with the ACC oxidase genes from other plants,63 searched out sequences were all ACC oxidase genes.After alignment on PCgene program,the identities of the cloned fragment with ACC oxidase genes from rice and bamboo were both reaching about 88%.So we can concluded that the cloned sequence was a member of ACC oxidase genes fragment from sugarcane.The sequence has been submitted to the GenBank database,the accession number is AF442821.According to the ACC oxidase protein family,a 'intron' of 103 bp was excluded and the sequence coded 279 amino acids,which spanned 88% of the putative whole sequence in length.Alignment and phylogenetic analysis of the amino acid sequence deduced from this fragment and the ACC oxidase sequences of other plants retrieved from GenBank were carried out by using PCgene program.The putative amino acid sequence shared a homology of 86% with the ACC oxidases of bamboo and rice,74 6% with banana,70% with tomato and potato and 68% with melon and carnation,which showed that the homology of sugarcane ACC oxidase with monocot was higher than with dicot.The results of phylogenetic analysis showed that ACC oxidase from sugarcane and ACC oxidases from rice clustered together firstly,and then came those from banana,ACC oxidases of dicot from potato,tomato,petunia,melon, Arabidopsis thaliana and carnation came subsequently.It indicated that sugarcane ACC oxidase had a closer phylogenetic affinities to the monocot ACC oxidase sequences than to the dicot ACC oxidases sequences.The clustering results of ACC oxidase molecules accorded with morphological classification system.