摘要
【目的】搜索鉴定木薯还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NOX)基因家族成员,分析其生物学信息及在不同组织和非生物胁迫下的表达模式,为深入研究该家族基因对木薯块根发育的调控机理及提高木薯抗逆性提供理论参考。【方法】从JGI数据库下载木薯的基因组序列,由pfam数据库下载4个NOX蛋白结构域相关的HMM文件,通过HMMER程序筛选NOX基因家族成员,利用生物信息学分析软件对该家族蛋白的理化性质、氨基酸序列和结构特征进行分析。基于GEO数据库的转录组测序(RNA-Seq)数据,对木薯NOX基因家族成员在不同组织和干旱胁迫下的表达模式进行分析。【结果】从木薯基因组中共鉴定到15个NOX基因家族成员,包括9个NOX基因和6个FRO基因(NOX祖先基因)。FRO基因的外显子数(6~9个)明显少于NOX基因的外显子数(11~14个),且FRO基因的内含子序列较NOX基因短,NOX基因的第一个外显子相对保守。15个NOX基因家族成员分布在木薯的9条染色体和1个结构支架上,且均被定位在染色体末端,除6号染色体有4个基因、8号和14号染色体有2个基因外,其他染色体均只有1个基因。木薯NOX蛋白的分子量和氨基酸数明显高于FRO蛋白,而理论等电点(pI)差异不明显。FRO蛋白含6~9个保守跨膜结构域,而NOX蛋白均只含4个跨膜螺旋区,且均位于第350~750位氨基酸残基。木薯NOX家族蛋白有10个保守结构域,其中Motif1和Motif8存在于所有的NOX和FRO蛋白中,Motif6为NOX蛋白所特有,Motif3只独立存在于FRO蛋白中。NOX蛋白均具有典型的保守结构域和相似的蛋白质结构,其脱氢酶结构域明显折叠成催化域裂缝结构,且C末端氨基酸极度保守。由RNA-Seq数据的基因表达谱可知,NOX基因家族成员在不同木薯叶片、中静脉、叶柄、茎、侧芽、茎尖分生组织、根尖分生组织、易碎胚性愈伤组织、胚性愈伤组织(OES)和贮藏根中均有表达,但呈不同的表达模式,其中,Manes.14G042600和Manes.S089900在储藏根中高表达,Manes.06G128700和Manes.09G172500在须根和根尖分生组织中高表达。在木薯品种KU50和新选048的叶片中,Manes.14G042600基因在干旱胁迫下呈上调表达,推测NOX基因在特定组织及响应非生物胁迫过程中扮演重要角色。【结论】木薯NOX基因家族成员具有保守基因结构,其编码蛋白具有保守的功能域,尤其是C端氨基酸序列;NOX基因表达具有组织特异性,部分成员的表达受非生物胁迫的影响。FRO蛋白和NOX蛋白具有较明显的共进化关系。
【Objective】Gene family members of reduced nicotinamide adenine dinucleotide phosphate oxidase(NOX)in cassava were identified through bioinformatic mining of publicly available data,and gene expression profiles in different tissues and under various abiotic stresses were also analyzed,which laid theoretical references for further studies on the regulation mechanism of NOX genes on cassava tuberization and improvement of stress resistance of cassava.【Method】Cassava genome sequences were downloaded from GJI database and four NOX domain-related HMM files were downloaded from the pfam protein database,then the HMMER program was performed to characterize the NOX family members.The physical and chemical properties,amino acid sequence and structural characteristics of the family protein were analyzed by bioinformatics software.Based on transcriptome sequencing(RNA-Seq)data at GEO database,the expression profiles of cassava NOX gene family members was further analyzed in various tissues and under drought stress.【Result】A total of 15 members of the NOX gene family were identified from the cassava genome,including 9 NOX genes,6 FRO genes(or the ancestral NOX genes).The exons of NOX(11-14)gene greatly outnumbered that of the FRO family(6-9).The intron sequence of FRO gene was shorter than that of NOX gene and the first exon of NOX gene was relatively conserved.Fifteen members of the NOX gene family were distributed on 9 different chromosomes and 1 scaffold of cassava,all of them were anchored near the endpoints of chromosomes.Except for chromosome 6(4 genes)and chromosome 14(2 genes),there was only one NOX gene in each chromosome.The molecular weight and amino acid number of NOX protein in cassava were lower than FRO protein,but the theoretical isoelectric point(pI)difference was not obvious.FRO proteins harbored 6 to 9 conserved transmembrane domains,whereas all the NOX proteins had only 4 transmembrane domains.There are 10 conserved domains of NOX family proteins in cassava,with Motif1 and Motif8 in all NOX and FRO proteins,Motif6 only in NOX proteins,and Motif3 only in FRO proteins.Three-dimension homologous modeling further revealed that all NOX proteins had typical conserved domains and similar protein structures.The dehydrogenase domain folded obviously into the catalytic domain fissure structure and the C-terminal sequence appeared as an extremely conserved motif.The expression profile of the NOX genes using RNA-Seq data displayed different expression patterns across different tissues,including leaves,mid vein,petiole,stem,lateral bud,shoot apical meristem(SAM),root apical meristem(RAM),friable embryogenic callus(FEC),organized embryogenic structures(OES)and storage roots.Some of them were highly enriched in storage root,for example Manes.14G042600 and Manes.S089900,however,Manes.06G128700 and Manes.09G172500 were highly expressed in fibrous root and RAM.Notably,in the leaves of varieties cv.KU50 and Xinxuan 048,Manes.14G042600 was upregulated under drought stress,implying a potential role of NOX gene in respon-ding to abiotic stress.【Conclusion】Cassava NOX gene family members contain conserved gene structure and their encoded proteins also contain conserved functional domains,particularly in their C-terminal regions,suggesting the underlying co-evolutionary relationship between FRO protein and NOX protein.The NOX genes are tissue specific,and some of them are responsive to abiotic stress.
作者
胡广
梁大成
HU Guang;LIANG Da-cheng(Engineering Research Center of Ecology and Agricultural Use of Wetland,Ministry of Education/Hubei Key Laboratory of Waterlogging Disaster and Wetland Agriculture,Jingzhou,Hubei 434025,China;Hubei Collaborative Innovation Center for Grain Industry,Jingzhou,Hubei 434025,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2019年第10期2178-2187,共10页
Journal of Southern Agriculture
基金
国家自然科学基金项目（31671257）
