Abstract 【Objective】The aim of this study was to identify ACA genes family from Brassica rapa, and to analyze the commonness and characteristics of those genes, which provided data support for further revealing the evolutionary relationship of ACA family. At the same time, the expression of BraACAs after self-pollination under abiotic stress was investigated, which laid the foundation for further exploring how BraACAs as regulate calcium to perform some biological functions. 【Method】Based on Arabidopsis genome database, the ACA gene family of Brassica rapa was identified by homologous alignment. The molecular weight, theoretical isoelectric point and other physicochemical properties were predicted by online software Expasy. The MEGA 5.0 software was used to construct phylogenetic tree, and the online software GSDS 2.0 was employed to draw gene structure map. According to the family gene location information, chromosome mapping was carried out in TBtools. Then, McscanX software was used to carry out the collinearity analysis of ACA family genes in Arabidopsis and Brassica rapa, and the online software PlantCARE was used to predict and analyze the promoter elements of BraACAs. The protein conserved domains were analyzed by online tools Pfam and MEME. The expression of BraACAs gene in different tissues, abiotic stress and self-pollination were detected by qRT-PCR. 【Result】A total of 18 ACA genes were systematically identified from Brassica rapa, which were distributed on 10 chromosomes. According to the phylogenetic tree, four groups were classified, including 3, 4, 4 and 7 members. According to promoter element analysis, there were many light, abiotic stress and hormone response elements in the promoter of ACA family genes in Brassica rapa, which indicated that ACA family genes had potential biological function of resisting stress. According to the analysis of protein domains, most of the ACA gene family proteins in Brassica rapa had four functional domains unique to the ACA family, 13 of which had N-terminal autoinhibitory domains. The results of qRT-PCR showed that BraACAs were mainly expressed in flowers and pods, and the expression of Bra002762 and Bra035649 genes were up-regulated under chill stress, suggesting that Bra002762 and Bra035649 had potential biological functions in response to chill stress; the expression of Bra031701 gene was up-regulated under salt stress, suggesting that Bra031701 had potential biological functions in response to salt stress. There were significant differences in the expression of Bra003276 and Bra024117 genes between self-pollination and cross-pollination, suggesting that Bra003276 and Bra024117 responded to the self-incompatibility of Brassica rapa. The subcellular localizations of these five genes were all located in the plasma membrane to verify their function of balancing ion concentration on the membrane. 【Conclusion】BraACAs protein structures contained four highly conserved domains. The expression of BraACAs was specific in tissues, and five ACA family gene members encoded proteins located on the cell membrane, of which Bra0002762, Bra035649, Bra031701 were associated with chilling and salt stress response, while Bra003276 and Bra024117 were associated with self-incompatibility reaction. Keywords:ACA gene family;Ca2+-ATPase;chilling stress;salt stress;self-incompatibility;Brassica rapa
PDF (7047KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 王洁, 吴晓宇, 杨柳, 段巧红, 黄家保. 大白菜ACA基因家族的全基因组鉴定与表达分析. 中国农业科学, 2021, 54(22): 4851-4868 doi:10.3864/j.issn.0578-1752.2021.22.012 WANG Jie, WU XiaoYu, YANG Liu, DUAN QiaoHong, HUANG JiaBao. Genome-Wide Identification and Expression Analysis of ACA Gene Family in Brassica rapa. Scientia Acricultura Sinica, 2021, 54(22): 4851-4868 doi:10.3864/j.issn.0578-1752.2021.22.012
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0 引言
【研究意义】大白菜(Brassica rapa L. ssp. pekinensis)为十字花科芸薹属作物,属于孢子体自交不亲和型植物,在育种生产中常喷施2%—3%食盐水来克服大白菜自交不亲和以进行亲本留种[1]。盐害和冷害也常影响大白菜的生长和产量。钙离子信号转导是植物响应各种环境因子的重要机制,逆境条件下细胞质钙离子浓度升高,引发一系列钙依赖的下游信号传递。Ca2+-ATPase(ACA)作为调节细胞内钙离子浓度的重要蛋白质之一,维持细胞内钙离子浓度的平衡[2,3]。ACA是Ca2+-ATPase最重要的亚家族成员之一,在维持细胞内钙离子浓度的平衡方面发挥着重要作用。【前人研究进展】钙离子作为第二信使,在动植物一生中都具有重要作用。PUTENEY[4]在1986年首次报道了细胞内钙离子内流的相关信息。钙离子ATP泵(又称P型ATP酶)[5]最早可以追溯到英国科学家Ringer在1885年发现钙离子对动物肌肉收缩有影响[6],10年后科学家们获得肌浆网钙泵氨基酸序列[7]。近些年拟南芥[8,9]、番茄[10,11]、水稻[12,13]、甘蓝[14]、大豆[15]和苜蓿[16]等植物陆续有钙泵的编码序列被报道。ACA蛋白属于P型ATP酶超级组基因家族的钙离子泵,典型的ACA基因家族蛋白,具有Haloacid dehalogenase- like hydrolase功能域、C端Cation transporting ATPase功能域、E1-E2 ATPase功能域和N端Cation transporter/ ATPase功能域4个高度保守的功能结构域[17]。根据其蛋白序列N端有无自抑结构域,分为内质网膜型钙离子泵和自抑制型钙离子泵[18,19,20]。目前在拟南芥、水稻、大豆和苜蓿的ACA基因家族已经作了比较全面地分析。拟南芥ACA基因家族有13个成员,其中AtACA3、AtACA5和AtACA6为内质网膜型钙离子泵;AtACA1、AtACA2、AtACA4、AtACA7、AtACA8、AtACA9、AtACA10、AtACA11、AtACA12和AtACA13属于自抑制型钙离子泵[21,22,23]。有数据显示定位于质膜上的AtACA8在拟南芥的所有器官中均有表达,同样在拟南芥所有器官表达的还有与其高度同源的AtACA10,拟南芥幼苗期外施植物激素脱落酸使AtACA8和AtACA10表达量显著升高[24]。已有研究表明与野生型相比,拟南芥突变体aca8在冷害和高温胁迫下,抗逆性增强[8]。还有研究表明,AtACA9、AtACA10和AtACA13在拟南芥有性生殖方面扮演着重要角色[24],AtACA6对提高盐离子毒害的耐受性有至关重要的作用[25]。水稻ACA基因家族由15个成员组成,其中有3个属于内质网膜型钙离子泵,12个属于自抑制型钙离子泵。水稻OsACA6在烟草中表达会积累大量的脯氨酸,从而增强烟草的抗盐和抗旱能力[26]。大豆的ACA基因家族有29个基因成员,其中有5个属于内质网膜型钙离子泵,24个属于自抑制型钙离子泵。大豆中,SUN等[15]研究证实,野生大豆Ca2+-ATPase(GsACA1)在调节植物对碳酸盐碱性和中性盐胁迫的耐受性方面有积极效果。苜蓿Ca2+-ATPase家族由16个成员组成,其中3个属于内质网膜型钙离子泵,13个属于自抑制型钙离子泵。研究发现苜蓿ACA基因家族的蛋白序列具有高度保守性,其中MtACA8和MtACA9在响应低温胁迫过程中可能发挥着重要作用[19]。大白菜中同属于Ca2+-ATP亚家族且具有钙运输功能的ECA基因家族在生长发育过程中发挥重要作用[27]。【本研究切入点】大白菜作为人们日常餐桌上的重要蔬菜,ACA蛋白可能在其育种和生产过程中的自交不亲和、盐胁迫和冷胁迫等诸多方面发挥作用,而BraACAs相关的鉴定和功能解析还未见报道。【拟解决的关键问题】本研究拟通过生物信息学手段鉴定出大白菜ACA基因家族成员,并进行特征分析和表达分析,为研究大白菜ACA家族基因的功能、基因编辑育种和开发分子标记进行辅助育种奠定基础。
1 材料与方法
试验于2020年在山东农业大学国家重点实验室和南校实验站进行。
1.1 大白菜ACA基因家族成员鉴定及基本理化性质分析
在TAIR(https://www.arabidopsis.org/)网站下载拟南芥ACA基因家族蛋白序列,同时在植物信息数据库(http://www.plantgdb.org/)中下载大白菜(版本V1.5)全基因组蛋白序列文件和gff3文件构建本地BLAST数据库,将拟南芥ACA基因家族的蛋白序列通过本地BLAST比对出大白菜候选基因(参数为E<1e-10,Identity>40%);同时利用Pfam数据库、NCBI保守结构域数据库(https://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi/)和SMART9.0(http://smart.embl-heidelberg.de/)建立大白菜全基因组蛋白结构域模型,筛选含有ACA典型结构域(Haloacid dehalogenase- like hydrolase功能域、C端Cation transporting ATPase功能域、E1-E2 ATPase功能域和N端Cation transporter/ATPase功能域)的蛋白序列[28,29]。上述结果合并,剔除重复基因和不含ACA典型结构域的基因,并人工矫正删除无完整读码框的序列。利用在线工具Expasy(http://web.expasy.org/)预测大白菜ACA基因家族的分子量、理论等电点等物理化学特性以及WoLF PSORT(http://wolfpsort.org/)和DETAIBIO(http://www.detaibio.com/tools/transmembrane.html/)预测大白菜ACA家族蛋白定位和跨膜结构[29,30],参数默认。
利用在线软件Pfam(https://pfam.xfam.org/)[28]和MEME(http://meme-suite.org)[36]对大白菜蛋白序列的保守基序与功能结构域分析,其中MEME的参数设置Maxi-mum number of motifs为10,Occurrences of a single motif为zero or one per sequence。运用DNAMAN 9对大白菜部分ACA家族基因与拟南芥AtACAs的蛋白序列进行同源比对。
采用Universal RNA Extraction Kit试剂盒(Bioteke Corporation)提取总RNA,1%琼脂糖凝胶电泳评估RNA的完整性,分光光度计(Thermo Nano Drop One)测量RNA浓度和纯度。利用HiScript® II Q RT SuperMix for qPCR Sample试剂盒(诺唯赞)进行反转录反应获得cDNA,稀释十倍后-20℃保存备用。
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