龙眼DlSWC5基因克隆、亚细胞定位及表达特性分析

Cloning,subcellular localization and expression characterization of DlSWC5 gene in longan(Dimocarpus longan Lour.)

  • 摘要: 【目的】克隆龙眼ATP依赖的染色质重塑相关基因DlSWC5,并分析其亚细胞定位及表达特性,为研究该基因在龙眼生长发育及早期体胚发生过程中的调控功能提供理论依据。【方法】以龙眼品种红核子体胚发生早期胚性愈伤组织(Embryogenic callus,EC)为试验材料,结合龙眼三代基因组数据,采用RT-PCR克隆DlSWC5基因cDNA序列,对其进行生物信息学分析,基于STRING数据库预测DlSWC5蛋白互作调控网络,并通过激光扫描共聚焦试验验证蛋白亚细胞定位情况。分别利用转录组测序技术和实时荧光定量PCR检测DlSWC5基因在体胚发生早期和不同组织及不同浓度吲哚乙酸(IAA)处理下EC中的表达情况。【结果】成功克隆出DlSWC5基因的cDNA序列,与基因组中DlSWC5基因CDS序列相似性达99.80%,共编码340个氨基酸残基,该蛋白不含信号肽和跨膜结构,共含有44个磷酸化位点,分子式为C1622H2599N459O542S8,相对分子质量为37458.71 Da,理论等电点(pI)为5.74。DlSWC5蛋白结构的预测结果显示,其二级结构包含48.53%的α-螺旋、40.29%的无规则卷曲、6.47%的延伸链、4.71%的β-转角,其三级结构与漾濞槭同源蛋白高度一致。蛋白互作预测结果显示,DlSWC5蛋白与PIE1、SWC2、SWC4等10个蛋白互作。亚细胞定位显示DlSWC5蛋白定位于细胞核中。DlSWC5基因的相对表达量在早期体细胞胚胎发生中无明显差异。DlSWC5基因在体胚发生早期及种子、根、茎、叶、花蕾、花、果皮、果肉、幼果等组织中均检测到表达,与对照组(0 mg/L)相比,DlSWC5基因的相对表达量在0.5、1.0、1.5和2.0 mg/L IAA处理24 h后均显著降低(P<0.05)。【结论】成功克隆了DlSWC5基因,其编码蛋白为不稳定的亲水性蛋白,呈酸性,定位于细胞核;DlSWC5基因在龙眼体胚发生早期EC阶段被IAA诱导下调表达。

     

    Abstract: 【Objective】DlSWC5,a gene related to the ATP-dependent chromatin remodeling in longan(Dimocarpus longan Lour.),was cloned,and its subcellular localization and expression characterization were analyzed,to provide theoretical basis for investigating the regulatory function of this gene during the growth and development,and early stage of somatic embryogenesis process in longan.【Method】Embryogenic callus(EC)of the early stage of somatic embryogenesis(SE)of longan variety Honghezi was used as primary experimental materials in this study,in combination with three generations of longan genome data,cloned the cDNA sequence of longan DlSWC5 gene by RT-PCR,conducted the bioin‐formatics analysis,predicted the interaction regulation network of DlSWC5 protein based on STRING database,and verified protein subcellular localization by laser scanning confocal experiment. Transcriptome sequencing and real-time fluorescence quantitative PCR were respectively used to detect the expression of DlSWC5 gene in the early stage of somatic embryogenesis and different tissues,and in EC under different auxin(IAA)concentration treatments.【Result】The cDNA sequence of DlSWC5 gene was cloned,which existing 99.80% similarity with CDS sequence of DlSWC5 gene in longan genome,and encoded 340 amino acid residues. DlSWC5 protein,without signal peptide and transmembrane structure, contained a total of 44 phosphorylation sites,its molecular formula was C1622H2599N459O542S8,its relative molecular weight was 37458.71 Da,its isoelectric point (pI)was 5.74. The predicted result of DlSWC5 protein structure showed that its secondary structure contained 48.53% α-helix,40.29% random coil,6.47% extended strand and 4.71% β-turn,and its tertiary structure was highly consistent with the homologous protein of Acer yangbiense. The prediction of protein interaction showed that DlSWC5 protein could interact with 10 proteins such as PIE1,SWC2 and SWC4. Subcellular localization showed that DlSWC5 protein was located in the nucleus. There was no obvious difference in the relative expression level of DlSWC5 gene during the early stage of somatic embryogenesis in longan. The expression of DlSWC5 gene was detected in the early stage of somatic embrgenesis and seeds,roots,stems,leaves,buds,flowers,pericarp,pulp and young fruits. Compared with the control group(0 mg/L),the relative expression level of DlSWC5 gene was significantly decreased under 0.5,1.0,1.5 and 2.0 mg/L IAA treatments for 24 h(P<0.05).【Conclusion】DlSWC5 gene has been successfully cloned,and its encoded protein is unstable hydrophilic protein,which is acidic and localized in the nucleus. The expression of DlSWC5 gene is down-regulated by IAA induction in EC stage of the early stage of somatic embrgenesis.

     

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