百合无性繁殖器官病毒检测及LlMADS2调控珠芽发生的初步解析

Detection of lily asexual reproductive organ virus and preliminary analysis of LlMADS2 regulating bulbilformation

【目的】探究百合无性繁殖器官的带毒率,讨论以珠芽作为百合新型繁殖器官的意义。克隆卷丹(Lilium lancifolim)MADS-box转录因子MADS2,分析其在卷丹珠芽形成过程中及珠芽发生的不同位置、不同组织中的表达水平,为分析LlMADS2基因在卷丹珠芽发生中的作用机制提供理论参考。【方法】利用PCR的方法对卷丹(Lilium lancifolium)、重瓣卷丹(‘Flora pleno’)、泸定百合(Lilium sargentiae)、淡黄花(Lilium sulphureum)百合中的黄瓜花叶病毒(Cucumber mosaic virus)、百合无症病毒(Lily symptomless virus)和百合斑驳病毒(Lily mottle virus)进行检测。从卷丹珠芽形成过程的转录组中筛选得到参与珠芽发生的关键基因LlMADS2,从卷丹中扩增MADS2基因编码区(CDS)序列,利用生物信息学软件对其进行预测分析,通过qRT-PCR的方法分析其在卷丹形成过程中不同时期、不同位置及不同组织的表达特性。【结果】LlMADS2的CDS全长为741 bp,编码246个氨基酸,相对分子质量为28 170.95 D,理论等电点为7.05,脂肪系数为78.90,不稳定系数为61.14,蛋白亲水性平均值为-0.698,不存在跨膜运输结构,属于MADS-box蛋白家族;qRT-PCR结果显示,LlMADS2在珠芽发生不同时期及不同位置的表达量呈现显著差异,时空表达的结果表明,其在卷丹的各个部位均有表达,但在花柱部位表达量最高。【结论】在4种能自然产生珠芽的百合中,珠芽中百合病毒的种类少,作为无性繁殖器官确实具有低毒优势。LlMADS2基因含有MADS-box家族典型的MADS保守结构域序列,在卷丹珠芽形成过程具有组织表达特异性,在不同时期和位置也表现出明显的表达差异,可能参与卷丹珠芽发生的调控,且发挥促进作用。

【Objective】The virus content in the vegetative organ of lily was investigated and the significance of using bulbil as a new reproductive organ of lily was discussed.The MADS2 transcription factor of the MADS-box of Lilium lancifolium was cloned and analyzed for expression levels in different tissues, different positions and different stages during the process of bulbils formation, which would provide a theoretical reference for exploring the mechanism of LlMADS2 gene in the formation of bulbil of Lilium lancifolium. 【Method】PCR was used to detect the cucumber mosaic virus, lily symptomless virus and lily mottle virus in lily varieties like Lilium lancifolium, Flora pleno, Lilium sargentiae and Lilium sulphureum. The key gene LlMADS2 involved in bulbil formation was screened from the transcriptome of Lilium lancifolium bulbil formation, and the MADS2 coding region(CDS) sequence was amplified from Lilium lancifolium. Bioinformatics software was used for prediction and analysis. The expression characteristics of LlMADS2 in different stages, different positions and different tissues during the formation of Lilium lancifolium were analyzed by qRT-PCR.【Result】The full length of LlMADS2 CDS is 741 bp, encoding 246 amino acids, with a molecular weight of 28 170.95 Da, a theoretical isoelectric point of 7.05, a fat coefficient of 78.90, an instability coefficient of 61.14, and an average protein hydrophilicity of-0.698. There was no transmembrane transport structure, indicating it belongings to the MADS-box protein family. qRT-PCR results showed that the expression levels of LlMADS2 at different stages of bulbil formation and at different positions showed significant differences. The spatio-temporal expression results showed that LlMADS 2 was expressed in all parts of Lilium lancifolium, but its expression level was the highest in the style.【Conclusion】Among the four lilies that can naturally produce bulbils, the species of lily virus in bulbils is less, so it does have the advantage of low toxicity as an asexual reproductive organ. The LlMADS2 gene contains the typical MADS conservative domain sequence of the MADS-box family, and it has tissue expression specificity in the process of bulblet formation. It also shows significant expression differences at different stages and locations, which indicates that it may participate in the regulation of bulbil formation and play a promoting role.