黄瓜脂氧合酶基因CsLOX2的克隆及表达分析

【目的】以华北型黄瓜种质26号为材料克隆黄瓜脂氧合酶基因CsLOX2的cDNA全长并分析其序列特征，在此基础上研究该基因在果实发育进程中的表达模式、酶活性变化及相应的C6醛类物质含量，为研究脂氧合酶基因在黄瓜果实醛类香气物质形成中的作用机制奠定基础。【方法】以华北型黄瓜种质26号为材料，通过RT-PCR和RACE技术克隆黄瓜的脂氧合酶基因CsLOX2的cDNA全长并分析其生物信息学特征，对CsLOX2在果实发育进程中的表达模式进行Real-time PCR检测，通过气质联用（GC-MS）技术测定相应的C6醛类香气物质的含量并利用分光光度法测定LOX酶活性。【结果】华北型黄瓜种质26号的CsLOX2基因cDNA全长2 878 bp，ORF区为2 640 bp，编码879个氨基酸，推导的蛋白质分子量为99.39 kD，等电点为6.28。通过氨基酸序列比对发现，该基因编码的氨基酸序列具有脂氧合酶家族的3个重要特征区域及植物LOX中皆具有的6个高度保守的组氨酸，该序列与其它物种的脂氧合酶氨基酸序列具有较高的同源性。根据其N端序列特征推测CsLOX2属于I类LOX，具有13-LOX活性。Real time-PCR分析结果表明，该基因在花后3 d表达量最高，此后下降，至花后15 d最低；脂氧合酶的活性自开花当天逐渐上升，至花后12 d达到最高，此后下降；C6醛类香气含量在果实发育始期较高，随果实发育进程逐渐下降，至花后12 d最低。【结论】利用RACE技术克隆了华北型黄瓜种质26号脂氧合酶基因CsLOX2的cDNA全长，序列分析表明该基因具有植物LOXs的特征结构域。CsLOX2基因表达高峰先于脂氧合酶活性高峰的出现，C6醛类香气含量在果实发育始期较高，随果实发育进程逐渐下降。该研究结果将为进一步探讨脂氧合酶基因在黄瓜果实醛类香气物质形成中的作用机制奠定基础。

Cloning and Expression Analysis of Lipoxygenase Gene CsLOX2 in Cucumis sativus (Cucumber)

【Objective】To investigate the mechanism of cucumber lipoxygenase gene participating in the formation of fruit aldehyde aroma, a full length cDNA named CsLOX2 was cloned from the Northern China germplasm No. 26 and the sequence characteristics of CsLOX2 were analyzed. Furthermore, the expression mode, the relative C6 aldehyde content and the lipoxygenase activity of cucumber fruit during fruit development were studied. 【Method】The full length cDNA of CsLOX2 gene was cloned using the methods of RT-PCR and 3′RACE, and the structure and the coded protein were analyzed. The expression mode of CsLOX2 gene using Real-time PCR and LOX activity were measured during the fruit developing. The head space-solid phase microextraction (HS-SPME) combining with gas chromatography-mass spectrometry (GC-MS) method were used to measure the relative C6 aldehyde content of cucumber fruits. 【Result】The full length cDNA of cucumber lipoxygenase gene named CsLOX2 was 2 878 bp. The open reading frame encompassed 2 640 bp and encoded 879 polypeptides. The calculated protein molecular mass was 99.39 kD and isoelectric point was 6.28. The amino acids sequence of CsLOX2 shared three conserved regions and six highly conserved histidines with other plant LOXs. It showed that the sequence of CsLOX2 had high identity compared with other lipoxygenases by NCBI BLAST. The RT-PCR analysis showed that CsLOX2 gene expressed differently during the fruit development, and highest on the 3 d after anthesis, and then down-regulated gradually and lowest on the 15 d after anthesis. The total LOX activity rose from the 3 d to 12 d after anthesis, and it was up to the highest activity on the 12 d after anthesis, and then went down. The expression peak of CsLOX2 gene preceded that of the LOX activity during the fruit development. The relative C6 aldehyde content was higher at fruit early development stage and decreased gradually during the fruit development and which was the lowest on 12 d after anthesis.【Conclusion】A lipoxygenase gene CsLOX2 was cloned from the Northern China germplasm No. 26 by RT-PCR and RACE. The deduced amino acid sequence of CsLOX2 gene contained the conserved motifs of LOXs family. The expression peak of CsLOX2 gene preceded that of the LOX activity during the fruit development. This study will provide a basis for probing the mechanism of CsLOX2 gene participating in the biosynthesis of aldehydes of cucumber fruits.