芝麻AQP家族的全基因组序列鉴定及其特征分析

Identification and Characterization of the AQP Gene Family in Sesame

【目的】从芝麻全基因组中分离鉴定水通道蛋白AQP(aquaporin)家族基因,并进行系统进化关系、连锁群定位、基因结构、跨膜结构域和亚细胞定位预测、保守性氨基酸残基以及青枯雷尔氏菌诱导表达分析,为芝麻AQP的功能研究与利用奠定基础。【方法】通过生物信息学手段,结合芝麻基因组注释信息,鉴定芝麻AQP家族成员序列信息,并用InterPro逐一进行验证。利用ClustalW2对芝麻、拟南芥和水稻的AQP以及马铃薯的XIPs进行多序列比对,用MEGA6.0构建进化树。通过MapInspect和Gene Structure Display Server 2.0进行连锁群定位和基因结构分析。采用ProtParam、WoLF PSORT和TMHMM Server v.2.0在线工具预测芝麻水通道蛋白的分子质量和等电点、亚细胞定位及跨膜结构域。通过芝麻、拟南芥和水稻AQP及马铃薯XIPs的蛋白多序列比对结果推测NPA基序、ar/R滤器及P1—P5的氨基酸残基。利用前期研究获得的转录组测序结果进行青枯雷尔氏菌诱导表达分析,并通过qRT-PCR技术对差异表达较为明显的12个芝麻AQP进行验证。【结果】系统分析鉴定了36个芝麻AQP家族基因,根据多序列比对及系统进化分析将其分为5个亚家族:13个质膜内在蛋白(PIP)、12个液胞膜内在蛋白(TIP)、8个类NOD26膜内在蛋白(NIP)、2个膜内在小分子碱性蛋白(SIP)和1个未知内在蛋白(XIP),其中有34个基因定位在12个连锁群上。同一亚家族成员在基因结构、蛋白序列、亚细胞定位预测及保守性氨基酸残基等方面都较为相似。青枯雷尔氏菌诱导表达分析显示,NIPs、SIPs和XIPs表达无明显变化,部分PIPs和TIPs能够响应青枯菌诱导。SiPIP1;2、SiPIP1;3、SiPIP2;3和SiPIP2;4受青枯菌诱导后表达上调,SiPIP1;3和SiPIP2;3为持续上调,而SiPIP1;2和SiPIP2;4的表达先下调后上调;与之相反,表达下调较为显著的有SiPIP1;4、SiPIP2;1、SiPIP2;6、SiTIP1;1、SiTIP1;3、SiTIP2;1及SiTIP2;2。上述差异表达基因的qRT-PCR验证结果与转录组测序结果一致。【结论】通过全基因组分析,在芝麻中鉴定出36个AQP家族基因,分为5个亚家族,分布于12个连锁群上,大部分基因具有1—4个内含子(除SiNIP1;2有7个内含子外)。根据ar/R滤器和P1—P5氨基酸残基组成类型,预测不同亚家族AQP可能识别的底物。青枯菌诱导表达分析表明,部分PIPs和TIPs成员的表达发生了显著变化。

【Objective】The present study aimed to identify the AQP gene family in the sesame genome and analyze its phylogenetic relationships in evolution, its linkage mapping, gene structure, transmembrane domain, conservation in amino acid residues and gene expression in response to Ralstonia solanacearum challenge.【Method】AQP gene sequences were isolated in sesame genome by standard bioinformatic techniques combined with gene annotation information and verified by Inter Pro.AQP gene sequences from sesame, Arabidopsis and rice were aligned and compared using Clustal W2, among which, XIP members of AQP were compared between sesame and potato. Phylogenetic trees were constructed with MEGA6.0. Linkage mapping and gene structure assay were performed with Map Inspect and Gene Structure Display Server 2.0, respectively. Prot Param, WoLF PSORT and TMHMM Server v2.0 were used to analyze the molecular mass and isoelectric point, the subcellular localization and the transmembrane domain respectively; multiple protein sequence alignments of AQP in sesame, Arabidopsis and rice were used to predict the NPA motif, ar/R selective filters and Froger＇s Position（P1-P5）. AQP gene expressions were analyzed based on the data of transcriptome of sesame upon Ralstonia solanacearum inoculation and further confirmed by q PCR. 【Result】A total of 36 AQP genes were identified in the sesame genome. Sequence comparison and phylogenetic tree analysis suggested that these AQP of sesame can be categorized into 5 subgroups： PIP（13 members）, TIP（12 members）, NIP（8 members）, SIP（2 members） and XIP（only 1 member）. 34 AQP genes were mapped onto 12 linkage groups. The gene structure, protein sequence, subcellular location and conservation in amino acids in the same subgroup were similar. Under Ralstonia solanacearum challenge, the expression of some members in PIP and TIP were up or down regulated, but the expression of members in NIP, SIP and XIP had no significant changes. SiPIP1;2, SiPIP1;3, SiPIP2;3, SiPIP2;4 were up-regulated; among them, SiPIP1;3 and SiPIP2;3 retain constant up-regulation, but SiPIP1;2 and SiPIP2;4 exhibited up-regulation at early stage of infection and down-regulation at later stage. SiPIP1;4, SiPIP2;1, SiPIP2;6, SiTIP1;1, SiTIP1;3, SiTIP2;1 and SiTIP2;2 were drastically down-regulated. The expression of these genes were confirmed by q PCR, which showed similar results to that of transcriptome sequencing.【Conclusion】 Through whole genome analysis, 36 members of AQP genes were found in the sesame genome. These were grouped into five subgroups, and mapped onto 12 linkage groups. Most AQP genes contain 1-4 introns（though SiNIP1;2 has 7 introns）. The substrates transported by each AQP group were predicted based on the types of ar/R selective filters and amino acid residues in Froger＇s position. Under Ralstonia solanacearum challenge,expressions of some members in PIP and TIP were significantly up or down regulated.