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作者简介:

车金凤(1996-),硕士研究生,主要从事植物生态学研究,(E-mail)2553426079@qq.com。

通讯作者:

李国旗,博士,研究员,主要从事植物生态学等研究工作,(E-mail)guoqilee@163.com。

中图分类号:Q943

文献标识码:A

文章编号:1000-3142(2023)10-1876-16

DOI:10.11931/guihaia.gxzw202208012

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目录contents

    摘要

    在植物响应紫外线B(ultraviolet-B,UV-B)的过程中,UV-B光受体UVR8(UV Resistance Locus 8)对植物的光形态建成和生长代谢等过程具有重要调控作用。为探究罗布麻属植物UV-B光受体信息,该研究通过罗布麻(Apocynum venetum)和大麻状罗布麻(A. cannabinum)全基因组数据进行UV-B光受体UVR8的筛选与生物信息学分析,同时利用转录组数据分析UV-B胁迫处理下的UVR8基因表达模式。结果表明:(1)罗布麻有6个UVR8基因,大麻状罗布麻有5个UVR8基因,前者分布在1、7、9和11号染色体上,后者分布在1、8和9号染色体上。(2)UVR8蛋白为亲水性稳定蛋白,定位在细胞核,不存在跨膜结构和信号肽,二级结构主要由延伸链、无规则卷曲、α-螺旋和β-转角构成。AvUVR8b和AcUVR8a蛋白三级结构与拟南芥UVR8(AtUVR8)最为类似,并且与小粒咖啡(CaUVR8)和伊德斯种咖啡(CeUVR8)的亲缘关系最近。同时发现罗布麻AvUVR8b和大麻状罗布麻AcUVR8a基因和蛋白结构与AtUVR8基因及蛋白高度相似。(3)当以一定剂量UV-B(17.52 kJ·m-2·d-1)处理两种罗布麻植株时,AvUVR8bAcUVR8a的表达量上调。据此推测在响应UV-B时,AvUVR8b基因在罗布麻中起主要作用,AcUVR8a基因在大麻状罗布麻中起主要作用。(4)顺式作用元件分析结果表明,UVR8的表达受光照、温度、水分、氧气和激素等因素的调控。该研究将为进一步研究罗布麻属UVR8的基因功能奠定基础,同时为解析罗布麻属植物适应UV-B的分子机制提供线索。

    Abstract

    In the processes of plants response to UV-B, the UV-B photoreceptor UVR8 (UV Resistance Locus 8) plays an important role in the regulation of photomorphogenesis, growth and metabolism of plants. To investigate the UV-B photoreceptors of Apocynum plant, this study is conducted to screen and bioinformatically analyze the UV-B photoreceptors UVR8 by the whole genome data of Apocynum venetum and A. cannabinum, and also to analyze the UVR8 gene expression pattern under UV-B stress treatment using transcriptome data. The results were as follows: (1) There were six UVR8 genes in A. venetum, and five UVR8 genes in A. cannabinum. The former was distributed on chromosomes 1, 7, 9 and 11, and the latter on chromosomes 1, 8 and 9. (2) UVR8 proteins were all hydrophilic stable proteins, localized in the nucleus, without transmembrane structure or signal peptides. The secondary structure consists mainly of extended strand, random coil, alpha helix and beta turn. The tertiary structures of AvUVR8b and AcUVR8a were most similar to that of Arabidopsis thaliana and were most closely related to Coffea arabica (CaUVR8) and C. eugenioides (CeUVR8). The gene and protein structures of Apocynum venetum AvUVR8b and A. cannabinum AcUVR8a were highly similar to those of AtUVR8. (3) The expression levels of AvUVR8b and AcUVR8a were up-regulated when the two Apocynum plants were treated with a certain dose of UV-B (17.52 kJ·m-2·d-1). It is speculated that in response to UV-B, the AvUVR8b in A. venetum and the AcUVR8a in A. cannabinum play a major role, respectively. (4) The analysis of cis-acting elements showed that the expression of UVR8 was regulated by light, temperature, moisture, oxygen and hormones. This study will lay a foundation for further research on the gene function of UVR8 in Apocynum, and provide clues to analyze the molecular mechanism of UV-B adaptation in the Apocynum.

  • 太阳光不仅是光合作用的能量来源,也是调控植物生长发育、昼夜节律、代谢物合成等的重要环境因子(Frohnmeyer &Staiger,2003;Jenkins,2014a,b)。紫外线(ultraviolet,UV)作为太阳光的组成部分,按其波长可分为长波紫外线(UV-A,320~400 nm),中波紫外线(UV-B,280~320 nm)和短波紫外线(UV-C,100~280 nm)(刘明雪等,2012;陈慧泽等,2021)。UV-A可穿过大气层直接到达地表,但不会对生物造成显著影响;UV-C可使大部分植物迅速死亡,但因其波长较短和穿透率差等因素,在到达地表之前就已被大气层吸收;UV-B大部分可被臭氧层吸收,作为生物有效辐射的UV-B具有双重效应。高强度UV-B是逆境胁迫因子,破坏细胞的DNA、蛋白质和脂类等生物大分子,甚至导致植物死亡;低强度UV-B是细胞信号传导的调控因子,对植物的光形态建成和代谢等生理过程具有重要作用(Frohnmeyer &Staiger,2003;Shamala et al.,2020)。

  • 早在2002年筛选对UV-B超敏感的拟南芥突变体(uvr8-1)中鉴定到了光受体UVR8(Kliebenstein et al.,2002),并于2011年证实UVR8为感受UV-B的特异性光受体(Rizzini et al.,2011)。目前,对UVR8结构和功能研究在拟南芥中进行的较多,研究结果表明,UVR8蛋白为盐桥链接的同源二聚体结构,其单体由7个片状结构首尾相连的β螺旋纵向排列围成的环形结构(Christie et al.,2012;鲍思元,2016)。高度保守的UVR8色氨酸残基(W)具有维持其蛋白结构稳定,接收和传递UV-B信号等功能(Jenkins,2014a;张宏江等,2019;Li et al.,2020),AtUVR8直接通过其W233和W258接收UV-B而无需借助其他辅助因子作为发色团(Rizzini,2011;O′Hara &Jenkins,2012; Jenkins,2014b; Yang et al.,2018)。当植株未照射UV-B时,UVR8以二聚体形式存在于细胞质;照射UV-B时,其盐桥断裂形成单体并转移到细胞核(Wu et al.,2012)与解离自CUL4-DDB1(cullin4 damaged DNA binding protein 1)E3泛素连接酶的COP1-SPA(constitutively photomor-phogenic 1-suppressor of phyA-105)形成UVR8-COP1-SPA新复合体(Rizzini,2011;Huang et al.,2013;Vanesa et al.,2019),从而减少COP1对HY5(Long Hypocotyl5)的降解(Huang,2013),同时促进HY5、HYH(HY5 Homolog)和MYB等转录因子表达,刺激黄酮类化合物合成过程中相关酶基因的转录(Hartmann et al.,2005;钱崇祯,2019;Shamala,2020;凌成婷等,2021)。当UVR8介导的下游基因过度表达时启动其负反馈机制,如激活RUP1(repressor of UV-B photomorphogenesis 1)/RUP2和STO/BBX24(Salt Tolerance/BBX24)等转录(Jenkins,2014b;Parihar et al.,2015;李国良等,2015)。UVR8与RUP1/RUP2相互作用促进其二聚体化(Cloix et al.,2012;Hideg et al.,2013),使UVR8可及时响应UV-B光信号。

  • 罗布麻(Apocynumvenetum)和大麻状罗布麻(A. cannabinum)为夹竹桃科罗布麻属多年生宿根草本或半灌木植物,具有耐旱、盐碱、贫瘠等强抗逆性(王东清等,2012);拥有“野生纤维之王”的美誉,其纺织物具有透气保暖、抗静电和紫外线防护等功能;作为药用植物,可全株入药,发挥降血压、血脂、血糖和抗衰老等作用(Li et al.,2018),其叶还可制保健茶。黄酮类化合物是罗布麻属植物的主要药用成分(张洋,2021),其合成途径复杂,受内源基因和外界环境的影响。在全球气候变化的背景下,植物面临着越来越严重的紫外线胁迫,探究植物对UV-B的响应机制与胁迫反应显得极其重要。在绿藻、苜蓿、大豆和银杏等植物中已展开对UV-B光受体UVR8结构和功能的部分研究。目前,对罗布麻属植物的研究多集中在其抗逆性、药用成分和纤维开发利用等方面,对其UVR8的研究尚未有报道。本文基于两种罗布麻的全基因组数据筛选UVR8基因,依托生物信息学分析,同时借助转录组数据探究UVR8基因表达模式,拟探讨以下问题:(1)UVR8基因结构、顺式作用元件和染色体定位;(2)UVR8保守结构域、蛋白结构和理化性质;(3)UVR8磷酸化位点和系统进化关系等;(4)UV-B胁迫下的UVR8基因表达模式。以期进一步深入解析UVR8功能及其在罗布麻属UV-B响应机制和药用成分合成等方面的研究提供线索。

  • 1 材料与方法

  • 1.1 材料和数据来源

  • 罗布麻和大麻状罗布麻的UVR8基因序列和蛋白序列,均来自本实验室前期全基因组测序工作所获得的数据(宋立肖等,2019;宋立肖,2020),并从拟南芥(Arabidopsis thaliana)数据库TAIR(https://www.arabidopsis.org/)中下载AtUVR8蛋白序列(Protein:AT5G63860.1)。

  • 1.2 方法

  • 1.2.1 两种罗布麻UVR8基因筛选

  • 本研究通过罗布麻和大麻状罗布麻全基因组的IPRSCAN、KEGG、NR和Swissport注释结果,分别筛选出注释到UV-B光受体UVR8的基因序列和蛋白序列。首先,利用BioEdit软件进行分析,以AtUVR8蛋白序列为种子序列进行本地BLAST比对,E-value<1×e-10,Identity≥30%,筛选最佳UVR8蛋白序列。然后,采用Pfam(https://pfam.xfam.org/search)和SMART(http://smart.emblheidelberg.de/)软件进行结构域验证,删除冗余序列。最后,通过以上分析分别获得罗布麻和大麻状罗布麻UVR8的基因序列和蛋白序列。

  • 1.2.2 两种罗布麻UVR8基因染色体定位

  • 基于两种罗布麻全基因组注释文件,获得UVR8基因在染色体上的位置信息,并通过软件MG2C(Chao et al.,2021)(http://mg2c.iask.in/mg2c_v2.1/)在线绘制UVR8基因染色体定位图。

  • 1.2.3 两种罗布麻UVR8基因结构和保守结构域分析

  • 两种罗布麻UVR8基因的外显子和内含子位置信息分别参考基因组注释gff3文件,采用GSDS 2.0(Hu et al.,2015)(http://gsds.gao-lab.org/index.php)工具对UVR8基因结构进行分析,利用软件MEME(Bailey et al.,2009)(http://meme-suite.org/tools/meme)对UVR8蛋白保守结构域进行预测。

  • 1.2.4 两种罗布麻UVR8蛋白基本理化性质分析

  • 利用在线软件ExPASy(Gasteiger et al.,2003)(https://web.expasy.org/cgi-bin/protparam/protpara)进行UVR8蛋白的氨基酸数、分子量、理论等电点、不稳定指数和脂肪族氨基酸指数等分析。

  • 1.2.5 两种罗布麻UVR8蛋白结构分析和亚细胞定位

  • 采用SOPMA(https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html)工具预测UVR8蛋白二级结构,以AtUVR8蛋白为模板,利用Phyre2(Kelley et al.,2015)(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index)软件分析蛋白三级结构。通过软件Cell PLoc 2.0( http://www.csbio.sjtu.edu.cn/bioinf/CellPLoc-2/),选择Euk-PLoc 2.0进行UVR8蛋白核定位分析。

  • 1.2.6 两种罗布麻UVR8蛋白跨膜结构预测、信号肽分析和磷酸化位点分析

  • 蛋白跨膜结构利用软件TMHMM 2.0(http://www.cbs.dtu.dk/services/TMHMM/)进行分析,信号肽预测通过SignalP 5.0(https://services.healthtech.dtu.dk/service.php?SignalP-5.0)进行,并采用软件NetPhots 3.1(http://www.cbs.dtu.dk/services/NetPhos/)统计蛋白磷酸化位点种类和数目。

  • 1.2.7 两种罗布麻UVR8基因顺式作用元件分析

  • 利用TBtools(Chen et al.,2020)软件获得UVR8基因上游2 000 bp序列,作为启动子序列。通过PlantCARE(http://bioinformatics.psd.ugent.be/webtools/plantcare/html/)数据库对所得序列进行预测,采用TBtools软件绘图,对主要顺式作用元件的位置和数量进行分析。

  • 1.2.8 两种罗布麻UVR8蛋白系统进化分析

  • 采用在线工具NCBI(https://www.ncbi.nlm.nih.gov/)的BLASTn进行同源性搜索,获得两种罗布麻UVR8基因的同源序列。通过MEGA 11.0软件构建系统进化树,利用Clustal W进行多重序列比对,采用邻接法(neighbour-joining,NJ)构建UVR8蛋白系统进化树,bootstrap method值设为1 000,其他参数采用系统默认值。通过工具ITQL(https://itol.embl.de)进行美化。

  • 1.2.9 两种罗布麻UVR8基因表达量分析

  • 本实验室于2021年春季在大棚内进行两种罗布麻的盆栽种植(林下土与营养土按1∶1混合),田间常规化管理。根据Gao等(2019)对UV-B辐射剂量的等级划分,结合宁夏银川地区夏季晴天的UV-B强度和两种罗布麻的强抗逆性,设自然光照(含UV-B强度8~11 W·m-2)为对照,在自然光照的基础上增加UV-B辐射处理。在生长至30~40 cm的两种罗布麻冠层上方0.5 m处安装UV-B灯管(飞利浦TL 100W/01),于每天早上10:00和下午14:00开始各处理4次,每次处理时长为10 min,每间隔10 min处理一次。可在冠层检测到增加的UV-B辐射剂量是17.52 kJ·m-2·d-1,强度为3.65 W·m-2(相当于银川地区夏季晴天UV-B强度增加33.2%~45.6%)。采用0.1 mm的醋酸纤维膜覆盖灯管以屏蔽280 nm以下的UV-C,UV-B强度由Lutron公司的UV-340A紫外线辐照计测得。期间分别在UV-B处理的第0天、第0.5天、第1天、第4天、第7天(d0、d0.5、d1、d4、d7)对植株上部成熟叶片进行取样,每个样本均为3个生物学重复,-80℃保存。本研究借助前期试验的转录组数据(暂未公开)对涉及UVR8基因的表达量进行分析,通过TBtools软件构建UVR8基因表达量热图。

  • 2 结果与分析

  • 2.1 两种罗布麻UVR8基因筛选

  • 通过罗布麻和大麻状罗布麻的全基因组测序数据,分别筛选到了10个罗布麻UVR8蛋白序列和14个大麻状罗布麻UVR8蛋白序列。以拟南芥AtUVR8蛋白序列为种子序列,将筛选结果进行序列比对及在线结构域分析。最终获得6个罗布麻UVR8(AvUVR8)基因序列和5个大麻状罗布麻UVR8(AcUVR8)基因序列,分别命名为AvUVR8aAvUVR8bAvUVR8cAvUVR8dAvUVR8eAvUVR8fAcUVR8aAcUVR8bAcUVR8cAcUVR8dAcUVR8e

  • 表1 两种罗布麻UVR8基因测序序列识别号和基因ID

  • Table1 Sequencing sequence identification number and gene ID of UVR8 genes in two species of Apocynum

  • 2.2 两种罗布麻UVR8基因染色体定位

  • 两种罗布麻的全基因组各有11条染色体,采用MG2C软件绘制UVR8基因染色体定位图,分析结果(图1)显示,UVR8基因间无串联重复现象,大麻状罗布麻AcUVR8基因不均匀地分布在3条染色体上,其中AcUVR8e分布在8号染色体,1和9号染色体则各分布有2个AcUVR8基因,分别是AcUVR8bAcUVR8cAcUVR8aAcUVR8d;罗布麻AvUVR8基因分布在4条染色体上,其中AvUVR8dAvUVR8c分别分布在7和11号染色体,1和9号染色体则各分布有2个AvUVR8基因,分别是AvUVR8fAvUVR8aAvUVR8bAvUVR8e

  • 图1 两种罗布麻UVR8基因的染色体定位

  • Fig.1 Chromosomal location of UVR8 genes in two species of Apocynum

  • 2.3 两种罗布麻UVR8基因结构和蛋白保守结构域分析

  • 通过两种罗布麻基因组注释gff3文件,采用GSDS 2.0软件对UVR8基因结构进行分析。结果表明,UVR8基因皆含上、下游非编码区、内含子和外显子,但基因长度不同,外显子数也存在差异(图2)。AvUVR8基因长度为5 115~12 672 bp,外显子数有6~16个,其中AvUVR8c外显子数最高(16个),AvUVR8a外显子数最低(6个)。AcUVR8基因长度为5 876~11 585 bp,AcUVR8外显子数有6~15个,其中AcUVR8d外显子数最高(15个),AcUVR8b外显子数最低(6个)。此外,利用MEME软件分析UVR8蛋白保守结构域(motif),motif设为7,其他参数为默认值。由图3可知,AvUVR8的motif数为4~7个,其中AvUVR8b、AvUVR8e和AvUVR8f的motif数最多(7个),AvUVR8c motif数最少(4个)。AcUVR8的motif数为6~7个,其中AcUVR8b的motif数最少(6个),其他4个AcUVR8则各有7个motif。同种罗布麻的UVR8 motif之间存在一定差异,但两种罗布麻的UVR8之间具有一定相似性,如AvUVR8a与AcUVR8b、AvUVR8b与AcUVR8a、AvUVR8e与AcUVR8d、AvUVR8f与AcUVR8c motif的数目、位置和氨基酸组成等两两相似。

  • 2.4 两种罗布麻UVR8蛋白质理化性质分析

  • 采用ExPASy工具对UVR8蛋白理化性质进行预测,结果(表2)表明,AvUVR8蛋白长度为420~534 aa,相对分子质量在45 846.10~56 827.20 Da之间,理论等电点在5.55~8.41之间。其中,AvUVR8c的氨基酸数、相对分子质量和理论等电点最高,分别为534 aa、56 827.20 Da和8.41;AvUVR8d的氨基酸数和相对分子质量最小,分别为420 aa和45 846.10 Da。当蛋白不稳定指数>40时,判定该蛋白为不稳定蛋白;当蛋白不稳定指数<40时,判定该蛋白为稳定蛋白(夏巧玉,2007)。AvUVR8蛋白的不稳定指数皆小于40,为稳定蛋白,其中AvUVR8b的不稳定指数最高(38.89),AvUVR8d的不稳定指数最低(31.12)。AvUVR8蛋白脂肪族氨基酸指数在63.50~85.17之间,亲水性平均值在-0.629~-0.134之间且小于0,为亲水性蛋白。仅AvUVR8a、AvUVR8b和AvUVR8f理论等电点<7,为酸性蛋白,其余3个为碱性蛋白。

  • 图2 两种罗布麻UVR8的基因结构

  • Fig.2 Structures of UVR8 genes in two species ofApocynum

  • 图3 两种罗布麻UVR8蛋白保守基序分布

  • Fig.3 Distribution of conserved motifs of UVR8 proteins in two species of Apocynum

  • 表2 两种罗布麻UVR8蛋白理化性质

  • Table2 Physical and chemical properties of UVR8 proteins in two species of Apocynum

  • AcUVR8蛋白长度为388~488 aa,相对分子质量在41 778.96~53 233.19 Da之间,理论等电点在5.42~8.08之间。其中AcUVR8b的氨基酸数、相对分子质量和理论等电点最高,分别为488 aa、53 233.19 Da和8.08;AcUVR8e的氨基酸数、相对分子质量和理论等电点最小,分别为388 aa、41 778.96 Da和5.42。AcUVR8蛋白的不稳定指数皆小于40,为稳定蛋白,其中AcUVR8a的不稳定指数最高(37.70),AvUVR8b的不稳定指数最低(31.56)。AcUVR8蛋白脂肪族氨基酸指数在77.74~82.42之间,亲水性平均值在-0.275~-0.047之间且小于0,为亲水性蛋白。仅AcUVR8b和 AcUVR8d理论等电点>7,为碱性蛋白,其余3个为酸性蛋白。

  • 2.5 两种罗布麻UVR8蛋白结构分析和亚细胞定位

  • 利用SOPMA软件分析UVR8蛋白二级结构,结果表明,UVR8蛋白二级结构由α-螺旋(alpha helix)、β-转角(beta turn)、延伸链(extended strand)和无规则卷曲(random coil)4部分组成(表3,图4)。其中无规则卷曲占比最高(45.49%~58.05%),其次为延伸链(22.28%~28.04%),β-转角占比最低(7.40%~9.90%),可见延伸链、无规则卷曲和α-螺旋结构为UVR8蛋白二级结构的主要组成部分。另外,本研究通过Cell PLoc 2.0在线软件,选择Euk-PLoc 2.0对UVR8蛋白进行核定位预测,结果显示UVR8蛋白皆定位在细胞核。

  • 表3 两种罗布麻UVR8蛋白二级结构组成

  • Table3 Secondary structure composition of UVR8 proteins in two species of Apocynum

  • 以AtUVR8蛋白为模板,采用软件Phyre2预测UVR8三级结构。发现仅AvUVR8b和AcUVR8a三级结构同AtUVR8相似,单体由7个完整的RCC1保守基序形成七叶β-折叠结构,而其他UVR8蛋白结构因RCC1保守基序不完整或缺少,无法形成完整的七叶β-折叠结构,表明AvUVR8b和AcUVR8a在植株响应UV-B时发挥了重要作用(图5)。

  • 图4 两种罗布麻UVR8蛋白二级结构预测

  • Fig.4 Secondary structure prediction of UVR8 proteins in two species of Apocynum

  • 2.6 两种罗布麻UVR8蛋白跨膜结构预测、信号肽分析和磷酸化位点分析

  • UVR8磷酸化位点预测结果(图6)显示,AvUVR8存在35~50个磷酸化位点,其中有13~31个丝氨酸位点,10~19个苏氨酸位点和4~8个酪氨酸位点。其中AvUVR8a磷酸化位点数最高(50个),AvUVR8d磷酸化位点数最低(35个)。AcUVR8存在31~51个磷酸化位点,其中有12~31个丝氨酸位点,10~17个苏氨酸位点和2~8个酪氨酸位点。其中AcUVR8b磷酸化位点数最高(51个),AcUVR8e磷酸化位点数最低(31个)。另外,预测结果表明两种罗布麻UVR8蛋白不存在跨膜结构和信号肽。

  • 2.7 两种罗布麻UVR8基因顺式作用元件分析

  • 本研究通过PlantCARE软件对UVR8基因编码区上游2 000 bp序列进行顺式作用元件分析,由图7可知,除了基础性元件(TATA-box和CAAT-box等)之外,UVR8基因顺式作用元件主要涉及光诱导、激素反应、逆境胁迫响应、生长发育响应等。其中,光诱导元件数量最多(117个),主要包括ATC-motif、Box 4、I-box、TCT-motif、GA-motif、GT1-motif、G-box、AT1-motif、ACE等元件;其次为激素反应元件(44个),主要有参与赤霉素反应的TATC-box、GARE-motif和P-box,响应茉莉酸甲酯的CGTCA-motif和TGACG-motif,介导水杨酸反应的TCA-element,涉及脱落酸反应的ABRE及与生长素反应相关的TGA-element,以及响应逆境胁迫元件(38个),主要有响应厌氧反应的ARE,涉及伤口反应的WUN-motif,参与干旱反应的MBS,有关低温胁迫的LTR及参与防御和压力反应的TC-rich repeats;生长发育响应元件数量最少(18个),主要涉及昼夜节律调控的circadian元件,分生组织表达元件CAT-box,胚乳表达元件GCN4-motif和调节玉米蛋白代谢的O2-site元件。以上说明两种罗布麻UVR8基因的表达不仅受光照的影响,还受到温度、水分、氧气和内源激素等因素的调控。

  • 图5 两种罗布麻UVR8蛋白三级结构预测

  • Fig.5 Tertiary structure prediction of UVR8 proteins in two species of Apocynum

  • 2.8 两种罗布麻UVR8蛋白系统进化分析

  • UV-B光受体UVR8最早发现于拟南芥(Kliebenstein,2002),并陆续在其他植物中发现。为进一步探究两种罗布麻UVR8蛋白系统进化关系,本研究通过NCBI软件的BLASTn进行同源性搜索并下载了42种植物的UVR8基因序列,涉及94条UVR8基因。借助软件MEGA 11.0的Clustal W进行多重序列比对,并采用邻接法构建UVR8蛋白系统进化树。由图8可知,聚类树分为3个亚族,两种罗布麻的UVR8蛋白分布在两个亚族上,其中AvUVR8a和AcUVR8b未与AtUVR8共处同一亚族且单独占一亚族,说明其与AtUVR8的亲缘关系存在一定差距;AvUVR8b和AcUVR8a聚集在一处且与小粒咖啡(CaUVR8)和伊德斯种咖啡(CeUVR8)的亲缘关系最近,其次与甘菊(ClUVR8)、雷公藤(TwUVR8)、澳洲坚果(MiUVR8)、三裂叶薯(ItUVR8)和牵牛(InUVR8)的亲缘关系较近;在两种罗布麻UVR8蛋白主要聚集处,其与拟南芥(AtUVR8)和枣(ZjUVR8)的亲缘关系最近,其次与可可(TcUVR8)、榴莲(DzUVR8)、木槿(HsUVR8)、陆地棉(GhUVR8)、树棉(GaUVR8)和美洲棉(GrUVR8)的亲缘关系较近。说明在两种罗布麻UVR8蛋白主要聚集的亚族上,各物种间UVR8蛋白存在明显的同源关系。

  • 图6 两种罗布麻UVR8蛋白磷酸化位点分析

  • Fig.6 Analysis of phosphorylation sites from UVR8 proteins in two species of Apocynum

  • 图7 两种罗布麻UVR8基因顺式作用元件分析

  • Fig.7 Analysis of cis-acting elements from UVR8 genes in two species of Apocynum

  • 2.9 两种罗布麻UVR8基因表达量分析

  • 利用TBtools软件构建UV-B胁迫处理下的UVR8基因表达量热图,其中d0为对照组,罗布麻苗在0和0.5 d处理后,观测到的表型变化和观测数据不明显,故对两组数据进行合并处理。由图9可知,在罗布麻中,AvUVR8bAvUVR8cAvUVR8e表达量均上调,其中AvUVR8b在d7时最高,AvUVR8e在d1时最高,随后下降,AvUVR8c呈上升趋势。AvUVR8aAvUVR8dAvUVR8f表达量均下调,其中AvUVR8a在d1时最高,d4时最低,AvUVR8d在d0时最高,d7时最低,AvUVR8f 在d1时最高,d7时最低。在大麻状罗布麻中,AcUVR8aAcUVR8b表达量均上调,其中AcUVR8a呈上升趋势,AcUVR8b在d0.5时最高。AcUVR8cAcUVR8d表达量波动较大,其中AcUVR8c开始下降,但从d0.5开始呈上升趋势,在d0.5时最低,d7时最高,AcUVR8d在d1时最高,d0.5时最低。AcUVR8e表达量下调,在d0时最高,d4时最低(图9)。表明在植物响应UV-B胁迫的过程中,AvUVR8bAvUVR8cAvUVR8e在罗布麻中起重要作用;AcUVR8aAcUVR8b在大麻状罗布麻中起重要作用。

  • 3 讨论与结论

  • 随着人类社会的发展,臭氧层变薄导致地表UV-B增强(Caldwell et al.,1989),从而影响植物的光合作用速率、代谢和生态等作用(鲍思元,2016),继而对生长发育及作物产量等造成威胁。UVR8作为UV-B的特异性光受体,研究其结构、功能及UV-B响应机制等对作物来说是必要的(Wargent &Jordan,2013)。在UV-B下,uvr8-1突变体对黄酮类化合物和花青素合成关键基因的诱导下降,查尔酮合酶mRNA和蛋白表达不再上调(Kliebenstein,2002)。当UVR8过表达时UV-B介导的光形态建成更显著,对UV-B的适应和耐受能力增强(Favory et al.,2009)。有研究发现UVR8在响应UV-B的过程中通过调控多项生命活动提高植株的适应性和抗逆性(Jenkins,2014b;Vandenbussche et al.,2014),其中低剂量UV-B辐射抑制下胚轴和根的生长(Frohnmeyer &Staiger,2003;Wellmann,1976),同时促进UV-B“防晒剂”黄酮类化合物合成等以增强适应性(Winkel-Shirley,2002;Hartmann et al.,2005;Gruber et al.,2010),UV-B损伤修复主要体现在抗氧化系统和酶修复DNA损伤(Jenkins,2014b)。这为研究罗布麻属UVR8功能及UV-B调控网络提供线索。

  • 图8 UVR8蛋白系统进化树

  • Fig.8 Phylogeneytic tree of UVR8 proteins

  • 图9 两种罗布麻UVR8基因的表达热图

  • Fig.9 Heat map of UVR8 genes expression in two species of Apocynum

  • 本研究通过罗布麻和大麻状罗布麻全基因组数据筛选UVR8蛋白序列,以AtUVR8蛋白为种子序列进一步筛选,最终获得6个罗布麻UVR8基因和5个大麻状罗布麻UVR8基因,并对其进行生物信息学分析,同时利用UV-B胁迫处理数据分析UVR8基因表达模式。研究结果显示,UVR8基因不均匀地分布在多条染色体上且不存在串联重复现象。并发现同一物种的UVR8(AvUVR8或AcUVR8)蛋白序列存在一定差异,但AvUVR8和AcUVR8之间具有相似性,如AvUVR8a与AcUVR8b,AvUVR8b与AcUVR8a等蛋白保守基序的数目、位置和种类高度相似。有研究报道,不同物种UVR8蛋白的关键氨基酸残基数目和位置高度相似,暗示UVR8蛋白在进化上相对保守(Yang et al.,2018),即光合生物的紫外线防护作用具有相似的分子功能(Rizzini,2011)。在本研究中,UVR8蛋白二级结构组成相似,但环形三级结构并不完全相同。其中,AvUVR8b和AcUVR8a的三级结构同AtUVR8最接近,单体由7个完整的RCC1保守基序形成七叶β-折叠结构,与报道的UVR8结构研究结果一致(Jenkins,2014a;鲍思元,2016;张宏江,2019)。然而,其他UVR8蛋白可能在进化过程中逐渐退化,导致RCC1保守基序不完整或缺少,无法形成完整的七叶β-折叠结构,表明AvUVR8b和AcUVR8a在响应UV-B时可能发挥主要作用。在两种罗布麻UVR8蛋白磷酸化中,以丝氨酸修饰为主,涉及苏氨酸和酪氨酸修饰。并且,UVR8蛋白为稳定性亲水蛋白,不存在信号肽和跨膜结构,与雨生红球藻、水稻等其他物种的UVR8研究结果相同(鲍思元,2016;张宏江,2019)。有研究表明,亲水蛋白含有大量亲水氨基酸,过表达时有利于提高植物耐旱、低温和高盐等抗逆性(刘盈盈,2019),说明UVR8可能参与植物的抗逆过程。

  • UVR8蛋白系统进化树主要分为3个亚族,除AvUVR8a和AcUVR8b单独处一亚族外,其余的AvUVR8和AcUVR8与拟南芥聚集于同一亚族。在AvUVR8/AcUVR8主要集聚处,其与拟南芥(AtUVR8)和枣(ZjUVR8)的亲缘关系最近,而聚集在该亚族中部的AvUVR8b和AcUVR8a与小粒咖啡(CaUVR8)和伊德斯种咖啡(CeUVR8)的亲缘关系最近。这表明在两种罗布麻UVR8主要聚集的亚族上,物种间具有明显的同源关系。顺式作用元件序列位于基因上游通过与转录因子结合的方式调控基因表达,涉及启动子、增强子、调控元件和诱导元件等(刘贺等,2022)。本研究通过UVR8基因顺式作用元件分析发现UVR8基因的表达不仅受光照影响,还受到温度、水分、氧气和内源激素等因素的调控,说明UVR8基因参与调控植物的生长发育和抗逆过程。研究发现,适当的UV-B辐射剂量对植物生长发育、品质改善、增强保鲜和抵抗逆境胁迫等存在积极调控作用(Jenkins,2009;刘一诺等,2020)。在UV-B预处理后,番茄中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的基因表达和酶活性显著升高。当UVR8功能沉默时UV-B激活的SODCAT基因表达下调,抑制UV-B缓解的氧化应激和冷害,表明UVR8参与UV-B诱导的耐寒性及抗氧化酶活性依赖于UVR8(Jiang et al.,2022)。通过对UV-B胁迫下的基因表达模式分析发现AvUVR8bAvUVR8cAvUVR8eAcUVR8aAcUVR8b基因表达量上调,其中AvUVR8cAcUVR8a表达量随处理时长的增加呈递增趋势,表明这些基因参与植物响应UV-B的过程。

  • 综上所述,AvUVR8b和AcUVR8a蛋白结构、保守基序等与AtUVR8的相关信息最接近,且在UV-B胁迫下其基因表达量上调。推测在植物响应UV-B的过程中,AvUVR8b基因在罗布麻中起主要作用,AcUVR8a基因在大麻状罗布麻中起主要作用。以AvUVR8b基因和AcUVR8a基因作为重点研究对象,将展开后续UVR8基因功能分析和UV-B胁迫反应研究,为今后深入探究罗布麻属UVR8响应UV-B的分子机制和调控网络等研究提供线索。

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