杧果矮化基因GA2ox的克隆、亚细胞定位及表达分析

Cloning,subcellular localization and expression analysis on dwarfing gene GA2ox in Mangifera indica

【目的】为研究杧果树体发育过程中GA合成调控以及GA信号转导的作用提供参考,并为矮生型杧果品种及矮化砧木的选育提供参考。【方法】以广西地方具有矮化特性杧果品种‘桂热杧82号’无病虫侵害的嫩叶为材料,对其GA2ox基因克隆,并进行亚细胞定位及表达分析。通过改良Trizol法提取叶片总RNA,利用简并引物克隆GA2ox基因片段,采用RACE法得到基因的全长cDNA序列,命名为MiGA2ox。通过生物信息学方法分析其结构特征,利用GFP进行亚细胞定位,采用实时荧光定量RT-PCR技术研究MiGA2ox基因在‘桂热杧82号’与乔化品种‘金煌杧’中的表达特性。【结果】MiGA2ox全长cDNA序列为2 051 bp,含有3′非翻译区约900 bp,5′非翻译区约400 bp。GA2ox序列均含有完整的开放阅读框,终止密码子为TGA,编码341个氨基酸,与橙子(XM_006467404.3)、柑橘(XM_006449629.2)、木薯(XM_021738345.1)、巴西橡胶树(XM_021820571.1)、陆地棉(XM_016896568.1)、中棉(XM_012597405.1)的一致性分别为79%、79%、78%、78%、76%、75%。MiGA2ox编码蛋白的相对分子质量为38 729.4 Da,等电点为6.56,为稳定蛋白,无信号肽,无明显疏水区,无跨膜结构域。根据共聚焦激光扫描显微镜观察结果可知,MiGA2ox编码蛋白定位于细胞核中。在随机选择2个时间节点,矮化品种‘桂热杧82号’MiGA2ox的表达量均高于乔化品种‘金煌杧’。【结论】‘桂热杧82号’MiGA2ox基因的表达与其矮化性状有关。

【Objective】To provide some references for studies on GA synthesis regulation and GA signal transduction during development of Mangifera indica tree,and to provided some references for selection of dwarf M.indica varieties and dwarf rootstocks.【Method】M.indica L.cv.Guire No.82 in Guangxi has dwarf characteristics,and its tender leaves without disease and insect pests were used as test materials.GA2ox gene was cloned and performed subcellular localization and expression analysis.Total RNA in leaves was extracted by the modified Trizol method.GA2ox gene fragment was cloned by using degenerate primers.The full-length cDNA sequence of the gene was obtained by RACE amplification and was named as MiGA2ox.Gene structure charcateristics were analyzed by using some bioinformatics methods.Subcellular localization was performed by using GFP.Expression characterisitics of MiGA2ox in dwarf‘Guire No.82’and arborization‘Jinhuangmang’were researched by using quantitative real-time PCR(qRT-PCR)technology.【Result】The full-length sequences of MiGA2ox gene is 2051 bp,containing about 900 bp of 3′untranslated region and about 400 bp of 5′untranslated region.GA2ox sequence contains a complete open reading frame,and the termination codon is TGA,encoding 341 amino acids.Uniformity of MiGA2ox gene with GA2ox sequences in orange(XM_006467404.3),citrus(XM_006449629.2),cassava(XM_021738345.1),Brazilian rubber tree(XM_021820571.1),upland cotton(XM_016896568.1)and Zhongmian(XM_012597405.1)are 79%,79%,78%,78%,76%and 75%,respectively.The MiGA2ox encoded protein has a relatively molecular mass of 38729.4 Da and an isoelectric point of 6.56.It is of stable protein,has no signal peptide,no obvious hydrophobic region and no transmembrane domain.The results of confocal laser scanning microscopy shows that the MiGA2ox encoding protein is localized in nucleus.Expression of MiGA2ox in dwarfing variety‘Guire No.82’is higher than that in arborization variety‘Jinhuangmang’.【Conclusion】Expression of MiGA2ox gene in‘Guire No.82’is related to its dwarf characteristics.