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我国是农业大国,土壤是立农之本,却被动承载着包括重金属在内的大部分污染物。我国重金属污染的耕地面积达2 000万hm2,每年仅镉污染的粮食达1 200万t,造成的直接经济损失超过200亿元[1]。重金属元素的难降解、易富集及食物链的可传递性给生态环境和食品安全带来严重威胁,是人类生命健康和安全的“隐形杀手”[2]。植物修复技术是近年重金属污染土壤治理的研究热点[3],其中超积累型东南景天(Sedum alfredii Hance)是为数不多的镉超积累植物之一[4],可在地上部分累积镉15 500 μg·g−1(干质量)而不呈现任何毒害作用,是应用于土壤镉污染修复的宝贵材料[5];同时,超积累型东南景天的极强耐镉特性预示着其具有丰富的抗逆基因,是研究植物耐重金属机理的理想物种。
为解析其镉耐性及超积累的机制,大量的研究聚焦于超积累型东南景天的镉吸收、转运及解毒[6-8]。研究发现,超积累型东南景天可以通过高效的根-茎转运机制将高浓度的镉离子转运到地上部分,并最终储存于叶肉的薄壁细胞、茎的髓部及皮层组织中[9]。维持植物细胞内金属离子的平衡及内稳态是提高植物耐重金属的关键[10]。研究表明,超积累型东南景天中有多个基因被发现与镉离子转运等相关,包括SaHMA3(heavy metal ATPase, HMA)[11]、SaNRAMP3(natural resistance-associated macrophage protein, NRAMP) [12]、SaNRAMP6[13]、SaMTP1(metal tolerance protein, MTP)[14]、SaZIP4[15]以及SaCAX2(cation exchangers, CAX)[16]。
ATP结合盒(ATP-binding cassette,ABC)是目前发现的最大的、功能最广泛的蛋白超家族之一,因其在跨膜运输底物时需要借助水解ATP释放的能量完成而得名[17]。ABC基因家族包括全分子和半分子2种类型,全分子蛋白结构包括2个核苷酸结合域(NBD)和2个跨膜结构域(TMD),半分子的蛋白只有1个膜结构域(MSD)和1个NBD[17]。该基因家族广泛存在于各类生物体中,在哺乳动物[18]、微生物[19]、植物[20]等生物中鉴定出ABC家族的物种已超过100种,其中,人类基因组中发现48个ABC基因[18],参与细菌耐药性、次生代谢物积累和肿瘤细胞的抗药性等,受到国内外研究者广泛关注。在植物中,ABC家族被认为参与植物体内激素[21]、脂质[22]、金属离子[23]、次生代谢物[24]以及调控植物与病原体相互作用[25]。然而,目前在超积累型东南景天中尚未开展该基因家族成员的研究。本研究以前期超积累型东南景天组学测序结果为基础,借助同源比对鉴定超积累型东南景天ABC家族基因,并开展生物信息学分析。另外,借助实时定量技术分析干旱、盐碱等不同胁迫处理下ABC基因的表达情况,为该基因功能的深入验证奠定前期基础和理论依据。
超积累型东南景天Sa12F279基因的抗逆表达响应及功能关联分析
Characterization of Expression Patterns of Sa12F279 from Sedum alfredii under Abiotic Stress and Its Functional Interaction Analysis
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摘要:
目的 对超积累型东南景天ABC转运蛋白家族成员Sa12F279开展生物信息学分析及表达分析,为探究ABC类转运蛋白在镉、干旱、盐碱等非生物胁迫中的功能提供参考。 方法 通过对超积累型东南景天转录组数据库进行比对分析,获得1个ABC家族成员的基因。通过生物信息学方法分析Sa12F279基因的进化关系、蛋白结构域构成、核心结构域同源比对及在组学数据中相关的互作蛋白分类;借助实时定量PCR技术分析该基因在盐碱、干旱及ABA激素胁迫下根中的表达变化。 结果 通过比对分析获得ABC家族成员Sa12F279基因,该基因的开放阅读框长度为4 497 bp,编码蛋白长度为1 498个氨基酸,相对分子量为167.1 KD,等电点为6.92。进化分析显示:Sa12F279基因与C亚类成员聚为一簇,且在结构域构成上符合ABC家族的保守排布。共表达网络分析显示:Sa12F279与 567个基因存在功能上的关联,对这些基因进行功能注释发现,其中39.8%的基因执行代谢相关功能,29.3%的基因与细胞内进程相关,10.2%的基因涉及生物调控,7.1%的基因参与转运活性。对镉胁迫前后转录组数据分析显示,Sa12F279基因在根、茎、叶3种组织中,取样点24 h和96 h的表达量均呈现对镉胁迫下调响应。实时定量结果显示:该基因对ABA激素、盐碱和干旱不同胁迫的应答模式存在差异,且应答响应较平缓。在ABA激素处理下,呈现先下调后上升的趋势;在盐胁迫下,呈现早期响应不显著而于胁迫后期出现上调应答;在干旱处理下,呈现先升后降又升的趋势。 结论 鉴定了超积累型东南景天ABC家族的1个Sa12F279基因,揭示了该基因在不同非生物胁迫下的表达模式。 Abstract:Objective In order to elucidate the functions of the ATP-binding cassette (ABC) protein in Sedum alfredii, a Cd hyperaccumulator, the bioinformatics analysis and expression pattern study were performed on Sa12F279 belonging to ABC protein family. Methods A local blast was carried out in the transcriptomic datasets of S. alfredii and produced a transcript categorized to ABC protein family. Further bioinformatics analysis was conducted on the phylogenetic clustering, the protein domain structures and classification of possible interaction proteins. Quantitative real-time PCR (qRT-PCR) was applied to uncover the expression patterns of Sa12F279 in the root under drought, salt, abscisic acid stresses. Result In the present work, a gene belonging to C subfamily of ABCs was identified in S. alfredii using local blast and designated as Sa12F279. The length of Sa12F279 open reading frame was 4 497 bp, coding a protein of 1 498 amino acids with pI being 6.92. The phylogenetic analysis showed that Sa12F279 was clustered with the C-subfamily of ABC protein, exhibiting uniform domain structure of TMD-NBD-TMD-NBD. Co-expression network analysis indicated that Sa12F279 served as hub genes mostly associated with metabolic process (39.8%), cellular process (29.3%), biological regulation (10.2%) and transporter activity (7.1%). The expression data from the transcriptomic datasets showed that Sa12F279 was suppressed by Cd stress in the three tissues (root, stem and leaf) at the sampling points of 24 h and 96 h. Transcriptional expression profiles responsive to drought, salt, abscisic acid (ABA) stress illustrated different response tendencies showing moderate induction. Under the treatment of ABA, the expression of Sa12F279 exhibited the tendency of decrease to elevation. Under the salt stress, Sa12F279 was up-regulated at the late stage of treatment while under the drought stress, Sa12F279 displayed the irregular tendency of elevation-decrease-elevation. Conclusion Based on the studies above, the authors characterize a gene named Sa12F279 belonging to the ABC protein family in S. alfredii and performs a comprehensive analysis including phylogenetic analysis, protein structure and co-expression network. Meanwhile, the expression profiles under different abiotic stress are also uncovered. The results could provide reference for the further studies on the ABC protein family. -
Key words:
- Sedum alfredii
- / ABC transporters
- / abiotic stress
- / expression pattern
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