龙眼乙烯合成途径基因鉴定及响应ACC处理的分析

Identification of Ethylene Synthesis Pathway Genes in Longan and Its Response to ACC Treatment

【目的】S-腺苷甲硫氨酸合成酶(SAMS)、1-氨基环丙烷-1-羧酸合成酶(ACS)和1-氨基环丙烷-1-羧酸氧化酶(ACO)是植物合成乙烯的3个关键酶,从全基因组水平鉴定龙眼SAMS、ACS和ACO(DlSAMS、DlACS和DlACO)基因家族,并进行生物信息学、体细胞胚早期不同阶段及不同浓度1-氨基环丙烷-1-羧酸(ACC)和不同处理时间下龙眼胚性愈伤组织的表达模式分析,为进一步研究和利用DlSAMS、DlACS和DlACO基因家族奠定基础。【方法】从拟南芥数据库(TAIR)下载SAMS、ACS和ACO家族蛋白质序列作为参考序列,TBtools、NCBI Blast等工具搜索龙眼基因组数据库,鉴定DlSAMS、DlACS和DlACO基因家族。使用ExPASy、PrediSi、TMHMM Server 2.0、NetPhos 3.1 Server、Plant-PLoc、MEME、PlantCARE、STRING、TBtools等软件和在线工具预测DlSAMS、DlACS和DlACO家族成员蛋白质基本理化性质、保守基序(Motif)与互作关系,定位染色体,分析基因共线性与选择压力、基因结构及启动子顺式作用元件;Clustal W和MEGA-X软件对龙眼、拟南芥、番茄、水稻和玉米的SAMS、ACS和ACO家族成员蛋白质序列分别进行多序列比对、构建系统进化树;根据龙眼转录组数据库的FPKM值,应用TBtools软件绘制DlSAMS、DlACS和DlACO家族成员在体细胞胚早期不同阶段的表达热图;采用qRT-PCR法分析不同浓度ACC和不同处理时间下龙眼胚性愈伤组织DlSAMS、DlACS和DlACO家族成员的表达情况。【结果】龙眼第三代基因组中分别鉴定获得SAMS、ACS和ACO家族成员4个、11个和4个,在家族成员数量上与拟南芥、番茄等物种差异不大。DlSAMS家族成员最保守,所有成员的Motif相同;DlACS和DlACO家族成员也含有较多保守的Motif。3个家族共19个成员分布在11条染色体上,有6对共线性基因,与拟南芥有30对共线性基因,均受纯化选择作用。3个家族的成员均含有大量光、激素和逆境响应元件。家族成员内部的蛋白互作关系较弱,但家族成员之间互作关系非常紧密。多物种系统进化关系表明,SAMS、ACS和ACO家族均可分为3个亚家族,DlSAMS家族成员仅分布在SAMS-Ⅰ和SAMS-Ⅱ中,SAMS-Ⅲ可能为单子叶植物所特有;DlACS和DlACO家族成员在3个亚族中均有分布,且分布较为均匀;DlSAMS、DlACS和DlACO均与番茄和拟南芥的SAMS、ACS和ACO亲缘关系较近。对转录组FPKM值的分析表明,DlSAMS家族的所有成员以及DlACO4B在体细胞胚早期的3个阶段均高表达,可能在体细胞胚发生过程发挥着重要作用;DlACS1和DlACS6B在球形胚阶段高表达,可能与球形胚的形成密切相关。在胚性愈伤组织继代培养基中添加不同浓度的ACC能显著影响龙眼胚性愈伤组织的增殖量及DlSAMS、DlACS和DlACO家族成员的表达。培养20 d时,主要表现为基因的上调表达,且ACC浓度越高,上调越明显;在25—35 d则主要表现为基因的下调表达。但是在20 d时,0.01 mmol·L^(-1) ACC处理的基因主要表现为下调表达,这可能是导致其增殖量显著大于对照的原因。【结论】龙眼第三代基因组中分别有SAMS、ACS和ACO家族成员4个、11个和4个,在进化过程中的保守性均较高,且包含大量激素和逆境响应元件;3个家族成员对龙眼体细胞胚胎的发生起重要调控作用,0.01 mmol·L^(-1) ACC处理可能通过调控DlSAMS、DlACS和DlACO家族成员的表达促进龙眼胚性愈伤组织增殖。

【Objective】S-adenosylmethionine synthetase(SAMS),1-aminocyclopropane-1-carboxylic acid synthetase(ACS)and 1-aminocyclopropane-1-carboxylic acid oxidase(ACO)are three key enzymes in the biosynthesis of ethylene in plants.In this study,the gene families of SAMS,ACS and ACO in Dimocarpus longan(DlSAMS,DlACS and DlACO)were identified at the whole genome level.Bioinformatics analysis,expression pattern analysis at different early stages of somatic embryogenesis(SE),and expression pattern analysis of embryogenic callus(EC)in longan treated with 1-aminocyclopropane-1-carboxylic acid(ACC)at different time and different concentrations were carried out,so as to lay the foundation for in-depth study and utilization of DlSAMS,DlACS and DlACO family genes in longan.【Method】The protein sequences of SAMS,ACS and ACO of Arabidopsis thaliana were downloaded from TAIR as reference sequences.TBtools,NCBI blast and other tools were used to search longan genome database to identify DlSAMS,DlACS and DlACO gene families.ExPASy,PrediSi,TMHMM Server2.0,NetPhos3.1Server,Plant-PLoc,MEME,PlantCARE,STRING,TBtools and other tools were used to predict the basic physicochemical properties,chromosomal localization,gene collinearity and selection pressure,gene structures,protein conservative motifs,cis-acting elements of promoters and protein interaction relationship of DlSAMS,DlACS and DlACO gene family members.The proteins sequences of longan,Arabidopsis thaliana,tomato,rice and maize were aligned by ClustalW software,and the evolutionary trees of SAMS,ACS and ACO were constructed by MEGA-X software.According to the FPKM values of longan transcriptome database,TBtools software was used to draw the expression heat maps of DlSAMS,DlACS and DlACO members at different early stages of SE.The expression of DlSAMS,DlACS and DlACO members in EC of longan under different concentrations of ACC and treatment time were analyzed by real-time fluorescence quantitative PCR(qRT-PCR).【Result】There were 4,11 and 4 members of SAMS,ACS and ACO families in the third generation genome of longan,respectively,and the number of family members in longan had little difference with those of Arabidopsis and tomato.DlSAMS family members were the most conservative,and all members had the same motifs,DlACS and DlACO family members also had many conservative motifs.A total of 19 members of the three families were distributed on 11 chromosomes,with six pairs of collinearity genes and 30 pairs of collinearity genes with Arabidopsis thaliana,all of which were subjected to purification selection.All the members of the three gene families contained a large number of light,hormone and stress response elements.The proteins interaction within the family was weak,but the interaction between the families were very close.According to the phylogenetic relationship,SAMS,ACS and ACO gene families could be divided into three subgroups.DlSAMS family members were only distributed in SAMS-Ⅰand SAMS-Ⅱ,SAMS-Ⅲmight be unique to monocotyledons.DlACS and DlACO family members were distributed in all three subgroups,and the distribution was relatively uniform.The three gene families were closely related to SAMS,ACS and ACO in tomato and Arabidopsis thaliana.The analysis of transcriptome FPKM value showed that all members of DlSAMS family and DlACO4B were highly expressed in the early three stages of SE,which might play an important role in SE.DlACS1 and DlACS6B were highly expressed in globular embryo(GE)stage,which might be closely related to the formation of GE.The proliferation of EC and the expression of DlSAMS,DlACS and DlACO members were significantly affected by different concentrations of ACC in subculture medium.After 20 days of culture,the genes were mainly up-regulated,and the higher concentrations of ACC,the higher up regulation of genes.In 25-35 days,the expression of genes were mainly down regulated.However,at 20 d,the genes expression in EC under 0.01 mmol·L^(-1) ACC treatment was mainly down regulated,which might be the reason for its significantly higher proliferation than that of CK.【Conclusion】There were 4,11 and 4 members of SAMS,ACS and ACO family in the third generation genome of longan,which were relatively conservative in evolution,and they contained a lot of hormones and stress response elements.These three gene family members played important roles in the regulation of SE in longan.0.01 mmol·L^(-1) ACC treated might regulate and control the proliferation of EC by regulating the expression of DlSAMS,DlACS and DlACO genes.