结球甘蓝叶片卷曲过程中6个生长素相关基因的克隆与表达分析

摘要

生长素在结球甘蓝球叶向上、向内自然卷曲的过程中发挥着重要的调控作用。为挖掘重要的影响结球甘蓝叶片卷曲的生长素相关基因，通过对结球甘蓝莲座期叶片与结球期叶片的转录组进行对比分析，筛选出6个显著差异表达的生长素相关基因，分别为调控生长素动态平衡的BoILL6、BoASA1基因，控制极性运输的BoSF21、BoPIN4基因，参与信号转导的BoARF8、BoGH3.5基因。对上述6个基因进行同源克隆，分别获得全长cDNA序列，序列分析发现6个基因均含有生长素相关的结构域。分子进化树分析发现各基因与芜菁的对应基因遗传关系最近，拟南芥次之。荧光定量PCR检测结果显示， BoILL6、BoASA1与BoSF21的表达量变化最为显著，结球期叶片相对莲座期叶片的表达量差异均高达12倍以上，变化趋势与转录组数据分析结果基本一致，说明这3个基因可能与结球甘蓝叶片的卷曲发育密切相关。%Auxin plays an important role in regulating the process ofBrassica oleracea L. var.capitata L. leaf curl.In order to find out the auxin relevant genes involved in cabbage leaf curling,we analyzed transcriptome of cabbage leaf at rosette and heading stages and identified 6 significantly differentially expressed auxin-related genes,including regulation of homeostasis genes(BoILL6,BoASA1),controlling polar transport genes(BoSF21, BoPIN4)and involving in signal transduction genes(BoARF8,BoGH3.5).The full-length cDNA sequences of 6 genes were obtained,respectively using homologous clone method.The protein structure prediction revealed that 6 genes contained auxin-related domains.Phylogenetic analysis though MEGA 5.1 comparison displayed that these genes had the closest genetic relationship with correspondence genes ofBrassica rapa,andArabidopsis thaliana followed. The real-time Fluorescence quantitative PCR analysis suggested that the expression level of BoILL6,BoASA1andBoSF21 showed significantly different trend.The expression level of these 3 genes in leaf during heading stage were 12 folds more than that in the leaf at rosette stage.The trend was consistent with the results of transcriptome analysis.The up-regulated expression of these 3 genes in the leaf during heading stage maybe lead to enhance the auxin concentration and change the auxin polar contribution in the leaves,and finally lead to format of the leaf curl.