葡萄VviSEP2基因的克隆及表达分析

Cloning and expression of Vitis vinifera VviSEP2 gene

【目的】克隆葡萄VviSEP2基因的完整开放阅读框(ORF)序列,明确其与胚珠败育型无核葡萄胚珠败育的关系。【方法】通过RT-PCR技术在‘无核白’葡萄中克隆葡萄VviSEP2基因的完整ORF序列,并对该序列及其编码产物进行生物信息学分析,采用半定量RT-PCR和实时荧光定量PCR技术分析VviSEP2的表达模式。【结果】克隆得到一个无核葡萄胚珠发育相关基因,该基因cDNA序列长度为1 132bp,ORF为741bp,编码246个氨基酸。氨基酸多序列比对和进化树分析确认,该基因是E类MADS-box基因家族成员,命名为VviSEP2。表达分析结果表明,VviSEP2只在花蕾、花和胚珠中有表达,而在根、茎、叶中无表达,并且该基因在有核葡萄‘黑比诺’花与花蕾中的相对表达水平明显高于无核葡萄‘无核白’,同时VviSEP2基因在‘黑比诺’胚珠发育各时期的相对表达水平均高于‘无核白’,为后者的3~5倍。【结论】VviSEP2基因与无核葡萄的胚败育可能存在一定关系。

【Objective】This study aimed to clone the whole open reading frame（ORF）of VviSEP2 fromVitis vinifera and verify the relationship between VviSEP2 and agronomic traits of stenospermocarpic（seedless）grapevines.【Method】The full-length ORF sequence of VviSEP2 was isolated fromV.viniferacv.‘Thompson Seedless＇ and analyzed with bioinformatics methods.Semi-quantitative real-time PCR（sqRT-PCR）and quantitative real-time PCR（qRT-PCR）were also used to detect the expression pattern of VviSEP2.【Result】A gene related to ovule development of stenospermocarpic grapevine was obtained.The cDNA sequence length of this gene was 1 132 bp and the ORF was 741 bp,encoding 246 amino acids.It was confirmed that the gene was a member of the Class E MADS-box family by amino acid sequence alignment and phylogenetic tree analysis,and it was designated as VviSEP2.The VviSEP2 expressed in flower bud,flower and ovule,but not in root,stem and leaf.The expression levels of this gene in flower and flower bud of‘Pinot Noir＇were significantly higher than that of‘Thompson Seedless＇.The relative expression levels of the VviSEP2 gene in‘Pinot Noir＇in each development period were 3-5 times of those in ‘Thompson Seedless＇.【Conclusion】The VviSEP2 gene was associated with stenospermo-carpy.