金丝楸CbuCYP71D15基因克隆及表达分析  

Cloning and expression analysis of CbuCYP71D15 gene from Catalpa bungei‘jinsi’

作　　者：徐艳红 于晓池 易飞 王军辉[2] 梁宏伟 麻文俊[2] XU Yanhong;YU Xiaochi;YI Fei;WANG Junhui;LIANG Hongwei;MA Wenjun(College of Biological and Pharmaceutical Sciences,China Three Gorges University,Yichang 443002,Hubei,China;Chinese Academy of Forestry,Beijing 100091,China;Northeast Forestry University,Harbin 150040,Heilongjiang,China)

机构地区：[1]三峡大学生物与制药学院,湖北宜昌443002 [2]中国林业科学研究院,北京100091 [3]东北林业大学,黑龙江哈尔滨150040

出　　处：《中南林业科技大学学报》2024年第8期150-158,168,共10页Journal of Central South University of Forestry & Technology

基　　金：“十四五”国家重点研发计划项目(2021YFD2200301);中国林业科学研究院基本科研业务费项目(CAFYBB2023QB001)。

摘　　要：【目的】通过对金丝楸CbuCYP71D15基因的分子特征和表达模式进行研究分析,为揭示CbuCYP71D15基因在金丝楸心材颜色形成过程中的作用提供参考依据。【方法】基于金丝楸木材样本的转录组数据,采用常规PCR扩增技术克隆获得CbuCYP71D15基因的编码区序列(CDS)全长,利用ProtParam,Protscale,PSORT Prediction等在线工具对CbuCYP71D15基因进行基础生物信息学分析。根据转录组数据,探究CbuCYP71D15基因在边材中的表达水平。同时,采用实时荧光定量PCR(qRT-PCR)的方法分析该基因在金丝楸不同组织部位中的表达模式。【结果】成功克隆得到长为1497 bp的CbuCYP71D15基因序列,生物信息学分析显示该基因编码498个氨基酸,相对分子量为56.5 kD,理论等电点为9.16,是稳定的亲水蛋白,其二级结构主要由α螺旋(46.18%)和无规则卷曲(34.34%)组成,存在跨膜区段。通过序列比对及系统进化分析发现,CbuCYP71D15基因所编码的氨基酸序列与芝麻的同源序列相似度最高,同源蛋白遗传关系最近,且CYP71Ds在进化过程中是保守的,CbuCYP71D15蛋白具有细胞色素P450超家族特征的保守结构域及血红素结合位点,属于CYP71家族。转录组数据分析显示CbuCYP71D15基因在边材中的表达水平与1,4-萘醌类化合物在边材中的表达趋势一致。qRT-PCR分析表明,CbuCYP71D15基因在金丝楸各组织部位均有表达,在边材中表达水平最高,根中表达水平最低。【结论】CbuCYP71D15基因可能在金丝楸心材呈色物质的合成代谢过程中起到羟化作用,可为进一步解析CbuCYP71D15基因在金丝楸中的生物学功能提供依据。【Objective】The molecular characteristics and expression patterns of CbuCYP71D15 gene in Catalpa bungei‘jinsi’were analyzed to provide a reference for revealing the role of CbuCYP71D15 gene in the formation of‘jinsiqiu’heartwood color.【Method】Based on the transcriptome data of‘jinsi’wood samples,the full-length coding sequence(CDS)of CbuCYP71D15 gene was cloned by conventional PCR amplification technology.The bioinformatics analysis of CbuCYP71D15 gene was carried out by ProtParam,Protscale,PSORT Prediction and other online tools.Transcriptome data were analyzed to explore the expression level of CbuCYP71D15 gene in sapwood.At the same time,the expression pattern of this gene in different tissues of‘jinsi’was analyzed by real-time fluorescence quantitative PCR(qRT-PCR).【Result】The CbuCYP71D15 gene sequence with a length of 1497 bp was successfully cloned.Bioinformatics analysis showed that the gene encodes 498 amino acids,with a relative molecular weight of 56.5 kD and a theoretical isoelectric point of 9.16.It was a stable hydrophilic protein.Its secondary structure was mainly composed of alpha helix(46.18%)and random coil(34.34%),and there was a transmembrane segment.The encoded amino acid sequence was found to have the highest similarity to the homologous sequence of S.indicum by sequence alignment and phylogenetic analysis,and the genetic relationship of homologous proteins was the closest.Moreover,CYP71Ds are conserved during evolution,and CbuCYP71D15 protein had conserved domains and heme binding sites characterized by cytochrome P450 superfamily,it belonged to CYP71 family.Transcriptome data analysis showed that the expression level of CbuCYP71D15 gene in sapwood was consistent with the trend that 1,4-naphthoquinones were significantly higher in GS1 than in GS2 and GS3.The qRT-PCR analysis showed that CbuCYP71D15 gene was expressed in all tissues of‘jinsi’,and the expression abundance was the highest in sapwood.【Conclusion】The CbuCYP71D15 gene may be involved in the synth

关 键 词：金丝楸 CYP71D15 基因克隆 表达分析 

分 类 号：S781.41[农业科学—木材科学与技术]