甘蓝BobHLH121基因克隆、亚细胞定位及表达分析

Cloning,subcellular localization and expression analysis of BobHLH121 gene from Brassica oleracea L.

  • 摘要: 【目的】克隆甘蓝bHLH转录因子(BobHLH121)基因,分析其在不同组织及低温胁迫下的表达模式,为深入研究bHLH转录因子在甘蓝低温响应中的作用机理提供理论参考。【方法】克隆BobHLH121基因编码区(CDS)序列,利用生物信息学软件进行预测分析,并构建pCAMBIA1300-FaFKF1-GFP融合表达载体,通过农杆菌介导转染烟草表皮细胞,观察荧光信号以确定蛋白亚细胞定位情况。采用实时荧光定量PCR(qRT-PCR)检测BobHLH121基因在低温胁迫下的相对表达量。【结果】BobHLH121基因CDS长度为1367bp,定位于C06染色体上,其编码311个氨基酸,蛋白分子量为34.89 kD,理论等电点(pI)为5.42,不稳定系数为52.29,疏水性平均系数为-0.733,说明该蛋白为不稳定的亲水性蛋白,且呈酸性,定位于细胞核。BobHLH121蛋白的二级结构主要由α-螺旋(占30.87%)、延伸链(占9.32%)、β-转角(占2.89%)和无规则卷曲(占56.91%)组成,具有保守的HLH结构域。BobHLH121蛋白与拟南芥(NP_191768.2)、琴叶拟 南 芥(EFH52923.1)、亚 麻 荠(XP_01909381.1)、荠 菜(XP_023638997.1)、油 菜(CDY65446.1)、白 菜(XP_009104362.1)和萝卜(XP_018442860.1)等多种植物bHLH蛋白的氨基酸相似性分别为55.6%、54.8%、52.0%、50.3%、76.4%、73.9%和61.1%。系统发育进化树分析结果显示,BobHLH121与拟南芥(NP_191768.2)和琴叶拟南芥(EFH52923.1)bHLH蛋白在同一小分支上。BobHLH121基因启动子区域存在植物生长发育、非生物胁迫、激素应答和光响应大量的顺式作用元件。BobHLH121基因在叶片的相对表达量显著高于其他组织(P<0.05,下同),在其他组织中的相对表达量均较低。低温胁迫6~12h时BobHLH121基因的相对表达量较低温处理0h(对照)显著降低,在低温处理24h时急剧升高,显著高于其他处理时间,低温处理48 h时又显著降低,表明该基因可能在低温胁迫下延迟表达。【结论】BobHLH121基因含有bHLH家族典型的HLH保守结构域序列,具有明显的组织表达特异性,可能参与甘蓝叶片的生长发育调控,且响应低温胁迫,可能在甘蓝耐寒性中发挥调控作用。

     

    Abstract: 【Objective】The whole length of Brassica oleracea L. bHLH transcription(BobHLH121 gene)was cloned by homologous cloning method,and its expression pattern under organ/tissue and low temperature stress was analyzed, which provided theoretical reference for studying the mechanism of bHLH transcription factor in low temperature response. 【Method】The sequence of the coding region(CDS)of BobHLH121 gene was cloned,the prediction analysis was performed using bioinformatics software,and the pCAMBIA1300-BobHLH121-GFP fusion expression vector was constructed through the transfection of tobacco epidermal cells,and the fluorescent signal was observed to determine the subcellular localization of the protein. The relative expression of the BobHLH121 gene under low temperature stress was measured using real-time fluorescence quantitative PCR(qRT-PCR).【Result】The CDS length of BobHLH121 gene was 1367 bp, localized on chromosome C06,encoding 311 amino acids,protein molecular weight of 34.89 kD,theoretical isoelectric point(pI)was 5.42,the instability coefficient was 52.29,and the average hydrophobicity was -0.733,indicating that the protein was an unstable hydrophilic protein,which was acidic and localized in the nucleus. The secondary structure of BobHLH121 protein mainly consisted of α-helix(30.87%),extended chain(9.32%),β-turn(2.89%)and random coil (56.91%),with a conserved HLH domain. BobHLH121 protein was closely related to bHLH proteins in Arabidopsis thaliana(NP_191768.2),A. lyrata(EFH52923.1),Camelina sativa (XP_01909381.1),Capsella brusa-pastoris(XP_ 023638997.1),B. napus(CDY65446.1),Chinese cabbage(XP_009104362.1)and radish(XP_018442860.1),and had amino acid similarities of 55.6%,54.8%,52.0%,50.3%,76.4%,73.9% and 61.1%,respectively. Phylogenetic tree analysis showed that BobHLH121 was on the same small clade as the A. thaliana(NP_191768.2)and A. lyrata(EFH52923.1) bHLH protein. A large number of cis-acting elements for plant growth and development,abiotic stress,hormone response,and light response existed in the promoter region of the BobHLH121 gene. The relative expression of the BobHLH121 gene was significantly higher in leaves than in other tissues(P<0.05,the same below)and lower in all other tissues. The relative expression of BobHLH121 gene was significantly decreased under low temperature treatment for 6-12 h under low temperature treatment at 0 h(control),and increased sharply at 24 h under low temperature treatment,which was significantly higher than that of other treatments,and decreased significantly at 48 h under low temperature treatment. It indicated that the gene might be delayed in expression under low temperature stress.【Conclusion】BobHLH121 has a conserved HLH domain typical of the bHLH family,has obvious tissue expression specificity,may participate in the regulation of growth and development of cabbage leaves,and respond to low temperature stress,may play a regulatory role in cabbage cold tolerance.

     

/

返回文章
返回