西瓜交替氧化酶AOX2基因的克隆与分析

Cloning and analysis of the alternative oxidase-2(AOX2) gene in watermelon

为了研究西瓜交替氧化酶基因家族在西瓜植株中可能发挥的功能,以西瓜(Citrullus lanatus)耐冷种质IVSM9为材料,根据植物不同物种交替氧化酶基因核苷酸保守区序列设计兼并引物,得到西瓜交替氧化酶(alterna-tive oxidase)AOX基因的中间片段。在已知序列的基础上,分别设计5’和3’末端扩增的特异引物,采用cDNA末端快速扩增(RACE)的方法得到西瓜交替氧化酶基因的5’端和3’端序列,用DNAMAN5.22软件对3个序列进行拼接和分析,获得全长序列。得到的基因片段同其他物种AOX基因家族中的alternative oxidase-2(AOX2)基因具有高度的同源性,因此命名ClAOX2。根据拼接得到的cDNA全长序列设计5’和3’两端引物,得到ClAOX2基因DNA和cDNA的全长序列。该基因cDNA全长为1 263 bp,开放阅读框架为1 050 bp,编码349个氨基酸。DNA全长1 983bp,存在3个内含子。目前该基因已在GenBank注册,登录号为ADD84880。荧光定量分析表明,ClAOX2基因在西瓜根、茎、叶、花、果等各个组织中均有表达,且在果实中的表达量最高。因此推测该基因可能在西瓜果实的发育中发挥作用。

In order to study the possible functions of the alternative oxidase gene family in watermelon plants,high-quality RNA was extracted from cold-tolerant watermelon（Citrullus lanatus） germplasm IVSM No.9 and then cDNA was obtained through reverse transcription kit.A cDNA fragment of alternative oxidase was amplified using degenerate oligonucleotide primers designed on the basis of the conserved domain of alternative oxidase genes in other plants.According to the cDNA fragment sequence,the 5＇ and 3＇ end fragment sequences were obtained using RACE technology respectively.The full length cDNA of ClAOX2 was obtained by DNAMAN5.22 from the above three sequences.The cDNA fragment exhibited high homology to the alternative oxidase-2（AOX2） of other species and could be referred to as ClAOX2.Then the full lengths of cDNA and DNA were obtained by PCR according to the known cDNA sequence.The full-length cDNA of ClAOX2 was 1 263 bp in size with a 1050 bp open reading frame（ORF）.The full-length DNA was 1 983 bp which has consisted of four exons interrupted by three introns.The accession number of ClAOX2 in GenBank database is ADD84880.Real-time PCR showed that the ClAOX2 gene expressed in root,stem,leaf,flower,and fruit of watermelon seedling and significantly higher in the fruit.These results demonstrated that ClAOX2 may play a role during fruit development.