柑橘CitCEP基因家族的鉴定及对逆境和激素的响应

【目的】基于全基因组鉴定分析柑橘CEP基因(CitCEPs)家族成员,了解各成员的分类进化关系,研究各基因成员在不同组织中的表达特异性以及对激素和非生物胁迫的响应,为进一步研究CEP基因家族的生物学功能打下基础。【方法】利用BLASTp基于Phytozome数据库鉴定柑橘CEP家族成员,采用GSDS、Prot Scale Tool、EXPASY、CLUSTALX、MEGA6.0、plant CARE、Cello等软件绘制家族成员基因结构图;分析预测蛋白的相对分子质量与等电点等理化性质;构建系统进化树及亚细胞定位预测等,利用实时荧光定量PCR技术(q RT-PCR)检测柑橘CEP基因家族各成员在‘资阳香橙’(‘Citrus junos Ziyang’)不同处理下的瞬时表达情况。【结果】柑橘CEP基因家族由11个成员组成,其中CitCEP10和CitCEP11含1个内含子,其余成员均无内含子;该基因家族成员均含有SPGV/IGH保守结构域序列,其预测蛋白均为亲水性蛋白,亲水性最强的氨基酸其值为3.711,亲水性最弱的氨基酸其值为-2.778;亚细胞定位预测结果显示该基因家族成员位于细胞不同位置,其中CitCEP1、CitCEP2、CitCEP4、CitCEP5、CitCEP6、CitCEP8定位于细胞外,CitCEP3和CitCEP7在细胞外和线粒体中均存在,CitCEP9位于细胞核中,CitCEP10在细胞质膜上,而CitCEP11位于线粒体和细胞核中;聚类分析发现,该家族成员分布于不同的分支中,分别与拟南芥CEP不同成员聚在一起,表明柑橘CEP成员间具有不同生物功能。表达分析显示,CitCEP2主要在茎、叶和子叶中表达,CitCEP3主要在根和子叶中表达,而CitCEP10和CitCEP11主要在果皮中表达,其余成员在上述组织中表达极低或不表达,体现了不同成员间组织特异性的差异。在干旱条件下,CitCEP2表达下调,CitCEP3、CitCEP10和CitCEP11的相对表达量均逐渐上调;而在盐胁迫下,CitCEP2、CitCEP10的响应模式与其所对应的干旱胁迫类似,CitCEP3和CitCEP11则呈现先升后降趋势。在乙烯(ETH)、脱落酸(ABA)处理下,CitCEP2的表达量明显受到抑制,而在茉莉酸甲酯(Me JA)、水杨酸(SA)、生长素(IAA)、赤霉素(GA3)处理下均表现为先上升、后下降的趋势;在6-苄氨基腺嘌呤(6-BA)、IAA、GA3、ABA处理下CitCEP3也表现出类似趋势。CitCEP10在ETH处理下表达量显著上升,在6-BA、SA、ABA处理下表达量呈现先下降后上升趋势,而在Me JA、IAA、GA3处理中无明显规律。CitCEP11在ETH、Me JA、SA、IAA、GA3、ABA处理下表达均呈现下调趋势,与CitCEP3在ETH、SA处理下的表达模式类似。在6-BA处理下,CitCEP11表达呈现先下降后上升趋势。【结论】从柑橘全基因组上鉴定出了11个CitCEP基因成员,各成员均为亲水蛋白,并含有SPGV/IGH保守结构域,位于细胞中不同位置。在不同逆境和激素处理下,CitCEP2、CitCEP3、CitCEP10和CitCEP11呈现不同程度的响应,而其余成员响应不明显或未响应。推测CitCEP2、CitCEP3、CitCEP10与CitCEP11在柑橘生长发育和逆境响应过程中可能起着重要作用。

Identification of Citrus CitCEP Genes and Their Transcriptional Response to Stress and Hormone Treatments

【Objective】 The objectives of this study are to identify citrus CEP genes (CitCEPs) based on citrus whole genome sequences, understand the phylogenetic relationship among CitCEPs as well as their expression specificity in different tissues and response to hormone and abiotic stress, and to lay a foundation for further study on the biological function of CitCEPs.【Method】The citrus CEP genes were identified by BLASTp based on Phytozome database. Gene structure, phylogenetic tree, relative molecular mass, isoelectric point and other physical and chemical properties of CitCEP members were analyzed using GSDS, ProtScale Tool, EXPASY, CLUSTALX, MEGA6.0, plant CARE and Cello. Real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of CitCEPs under different treatments of ‘Citrus junos Ziyang’.【Result】The CitCEPs consists of 11 members, of which CitCEP10 and CitCEP11 contain 1 intron, and the other members have no intron. All CitCEPs contain conserved domain sequences (SPGV/IGH), and belong to hydrophilic protein. The magnitude of the most hydrophilic amino acid is 3.711 and that of the weakest hydrophilic amino acid is -2.778. Subcellular localization prediction showed that 11 CitCEPs were located in different positions of the cell. Among them, CitCEP1, CitCEP2, CitCEP4, CitCEP5 CitCEP6 and CitCEP8 were all located in extracellular. CitCEP3 and CitCEP7 existed in both extracellular and mitochondria. CitCEP9 and CitCEP10 were located in nucleus and plasma membrane, respectively. CitCEP11 was located in both mitochondria and nucleus. The phylogenetic analysis showed that the CitCEP members together with Arabidopsis thaliana CEPs were clustered into 3 groups, which indicated that the CitCEP members had different biological functions. Expression analysis showed that CitCEP2 was mainly expressed in stem, leaf and cotyledon, CitCEP3 was mainly expressed in root and cotyledon, while CitCEP10 and CitCEP11 were mainly expressed in pericarp, and the other members expressed very low or no expression in the above tissues, reflecting the difference in tissue specificity among different members. Under drought conditions, the expression of CitCEP2 was down-regulated, while that of CitCEP3, CitCEP10 and CitCEP11 was gradually increased. The expression of CitCEP2 and CitCEP10 under salt stress was similar to that under drought conditions, respectively, while the expression of CitCEP3 and CitCEP11 increased first and then decreased. The expression of CitCEP2 was significantly inhibited under ethylene (ETH) and abscisic acid (ABA) treatments, but the expression increased firstly and then decreased under methyl jasmonate (MeJA), salicylic acid (SA), indole acetic acid (IAA) and gibberellin (GA3) treatments. CitCEP3 showed a similar trend under the treatments of 6-benzylamino adenine (6-BA), IAA, GA3 and ABA. The expression of CitCEP10 was significantly up-regulated under ETH treatment, and decreased firstly and then increased under 6-BA, SA and ABA treatments, but there was no obvious regularity in the treatment of MeJA, IAA, GA₃. The expression of CitCEP11 showed a downward trend under the treatments of ETH, SA, MeJA, IAA, GA3 and ABA, which was similar to that of CitCEP3 under ETH and SA treatments. Under 6-BA treatment, the expression of CitCEP11 showed a trend of decreasing firstly and then increasing.【Conclusion】A total of 11 CitCEP members were identified from citrus whole genome sequences, and all of them are hydrophilic proteins which contain a conserved SPGV/IGH domain. The subcellular positions were varied among CitCEP members. Under different stress and hormonal treatments, CitCEP2, CitCEP3, CitCEP10 and CitCEP11 exhibited different degrees of response, while the response of the other member was not obvious or unresponsive. It is inferred that CitCEP2, CitCEP3, CitCEP10 and CitCEP11 may play an important role in citrus growth and development as well as stress response.