作物杂志, 2018, 34(6): 58-67 doi: 10.16035/j.issn.1001-7283.2018.06.010

遗传育种·种质资源·生物技术

小麦GASA基因家族生物信息学分析

吕亮杰, 陈希勇, 张业伦, 刘茜, 王莉梅, 马乐, 李辉

河北省农林科学院粮油作物研究所/河北省作物遗传育种实验室,050035,河北石家庄

Bioinformatics Identification of GASA Gene Family Expression Profiles in Wheat

Lü Liangjie, Chen Xiyong, Zhang Yelun, Liu Qian, Wang Limei, Ma Le, Li Hui

Hebei Academy of Agriculture and Forestry Sciences, Institute of Cereal and Oil Crops/Crop Genetics and Breeding Laboratory of Hebei, Shijiazhuang 050035, Hebei, China

通讯作者: 李辉,研究员,主要研究方向为小麦分子技术与遗传育种

收稿日期: 2018-06-18   修回日期: 2018-09-6   网络出版日期: 2018-12-15

基金资助: 河北省现代农业科技创新工程项目.  F18R01;494-0402-YBN-RDC4
河北省农林科学院财政专项.  F17R0013;2018060303
河北省农林科学院粮油作物研究所开放课题.  LYS2016004

Received: 2018-06-18   Revised: 2018-09-6   Online: 2018-12-15

作者简介 About authors

吕亮杰,助理研究员,主要从事小麦遗传育种研究 。

摘要

赤霉素调节的GASA(Gibberellic Acid-Stimulated in Arabidopsis)基因家族是植物特有的转录因子家族,在调控植物生长发育过程中起重要作用。关于小麦GASA基因家族全基因组分析的研究尚未见报道。为进一步探讨小麦GASA基因的功能,通过对小麦最新基因组数据进行分析,获得了35个TaGASA基因,命名为TaGASAs,根据染色体编号排列为TaGASA1~TaGASA35。结合公布的小麦品种Chinese Spring的基因组数据,采用生物信息学方法对其基因结构、染色体分布、蛋白结构、系统进化及表达谱进行了分析。结果表明,35个小麦TaGASA基因分布于除3A、4A、3B、3D染色体外的其余17条染色体上,基因编码长度为78~264个氨基酸的蛋白质,基因外显子数量从2个到7个不等;串联重复片段分析结果表明,片段复制和串联重复是小麦TaGASA家族基因扩张的主要模式;小麦TaGASA蛋白进化树和7种作物GASA基因的系统进化树表明,GASA基因分为4个类别,同一类之间的结构较为相似;小麦35个TaGASA基因家族成员含有10个motif,推测小麦TaGASA基因家族应都含有motif1、motif2和motif3。在13个组织器官中都检测到了35个TaGASA基因的转录本,不同组织器官中TaGASA基因的表达存在明显差异。

关键词: 小麦 ; GASA ; 生物信息学 ; 进化树 ; 表达谱

Abstract

The Gibberellic Acid-Stimulated in Arabidopsis (GASA) gene family is a specific transcription factor in the plant that plays an important role in the regulation of plant growth and development. However,genome-wide analysis of the GASA gene family has not been reported in wheat. To further explore the function of the wheat GASA gene, 35 TaGASA genes, named TaGASAs, were obtained by analyzing the latest genomic data of wheat and were ranked according to the chromosome number as TaGASA1-TaGASA35. Combined with the published genome data of cultivar Chinese Spring, genes structure, chromosome distribution, the conserved domain of proteins, phylogenetic trees and gene expression profiles of the wheat cultivars were analyzed using bioinformatics methods. The results showed that 35 wheat TaGASA genes were distributed on the remaining 17 chromosomes except for 3A, 4A, 3B and 3D chromosomes. The genes encoded 78-264 amino acids in length and the number of gene exons was from 2 to 7. The results of tandem repeat analysis showed that fragment replication and tandem repeats were the main patterns of gene expansion in the wheat TaGASA family. The phylogenetic tree of wheat TaGASA proteins and the seven crops GASA proteins showed that GASA genes were divided into four categories, and the structure of the same class was similar. The 35 TaGASA genes family in wheat contain 10 motifs, and it is speculated that the wheat TaGASA gene family should contain motif1, motif2 and motif3. 35 TaGASA genes were all detected in 13 tissues and organs, and the expression of TaGASA genes in different tissues were significantly different.

Keywords: Wheat ; GASA ; Bioinformatics ; Phylogenetic tree ; Expression profiling

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吕亮杰, 陈希勇, 张业伦, 刘茜, 王莉梅, 马乐, 李辉. 小麦GASA基因家族生物信息学分析[J]. 作物杂志, 2018, 34(6): 58-67 doi:10.16035/j.issn.1001-7283.2018.06.010

Lü Liangjie, Chen Xiyong, Zhang Yelun, Liu Qian, Wang Limei, Ma Le, Li Hui. Bioinformatics Identification of GASA Gene Family Expression Profiles in Wheat[J]. Crops, 2018, 34(6): 58-67 doi:10.16035/j.issn.1001-7283.2018.06.010

小麦(Triticum aestivum L.)作为世界上最重要的粮食作物之一,是产量仅次于玉米和水稻的第三大粮食作物。GASA(Gibberellic Acid-Stimulated in Arabidopsis)基因家族被推测是一类与植物生长发育有关的基因,对拟南芥、水稻GASA基因家族的功能已有较深研究,而对小麦GASA基因功能研究的报道较少。GASA基因家族是一类受赤霉素(GA)调节的富含半胱氨酸的基因[1],大多定位于细胞壁,在植物生长发育和激素信号转导过程中发挥重要作用。GASA蛋白结构域一般都含有N-端信号肽,具有胞外分泌蛋白的特性,由18~29个氨基酸残基组成;信号肽后面紧接亲水区域,主要是由极性氨基酸残基组成;C-端则是由大约60个左右的氨基酸组成,包含12个完全保守的半胱氨酸残基(GASA结构域),该结构域被认为是GASA蛋白维持空间结构和发挥功能的关键区域[2]

GASA基因家族在植物中分布广泛,编码基因数量多,多数成员受GA调控[3,4,5,6]。GAST1(GA-stimulated transcript 1)是最早从番茄突变体gib1中被分离鉴定的GASA家族成员[7],之后陆续在番茄(Lycopersicum esculentum)[8]、矮牵牛(Petunia hybrida)[9]、马铃薯(Solanum tuberosum)[10]、水稻(Oryza sativa)[4,11-12]、非洲菊(Gerbera hybrida)[13]、草莓(Fragaria ananassa)[14]、棉花(Gossypium spp)[15]以及拟南芥(Arabidopsis thaliana)[16]中分离鉴定出GASA基因和蛋白。对已分离获得的GASA基因进行的分析表明,该基因家族编码的蛋白可能在植物生长发育过程中发挥重要的调控作用,包括参与种子萌发[17]、侧根形成[18]、茎伸长[19]、开花时间[1,19]、果实发育[20,21]、生物胁迫[22]与非生物胁迫[23]应答以及激素信号转导[24]等多个过程。

随着各类植物GASA基因的深入研究,大量GASA基因已被克隆,部分基因的功能也得到鉴定。张盛春等[16]对拟南芥GASA基因家族中的15个成员进行表达分析表明,GASA基因家族成员都在生殖器官尤其是花中有较强的表达,多数成员在茎尖分生组织、根尖生长点和花序轴分枝处表达,所以推测拟南芥GASA基因家族编码的蛋白可能调控细胞分裂。Roxrud等[1]的研究也证实GASA4基因在拟南芥中直接控制种子形成,并通过影响花序的分枝来间接调控种子的产量。在水稻中,GASA基因家族的OsGASR1和OsGASR2基因也被证实与花序的分化有关[11]。Huang等[25]选用分布于世界各地的950份水稻材料进行全基因组关联分析,鉴定出1个可能与水稻粒长相关的基因GASR7,该基因编码假定的Snakin/GASA蛋白。在小麦A基因组供体种乌拉尔图小麦(Triticum urartu)中,GASR7的同源基因TuGASR7与粒长和粒重表型显著相关;同时也在普通小麦中发现TaGASR7基因与产量性状相关,表明GASR7可以用于小麦产量的遗传改良[26,27]

关于GASA基因的研究不仅限于植物地上部分,根部发育方面的研究也取得了一定成果。Aubert等[2]研究发现拟南芥中GASA4基因在主根和侧根中都有表达。Zimmermann等[18]将野生型以及突变体lrt l和rum 1进行的根组织基因特异性分析,发现GASA基因家族成员ZmGSL(Zea mays Gibberellic Acid Stimulated-Like)可能受到赤霉素的调节并且与侧根的发育有关。

小麦是我国最重要的粮食作物之一,小麦的产量和品质严重影响了我国小麦的发展。小麦基因组是由A、B、D 3个亚基因组整合在一起形成的异源六倍体,其基因组大小约为17Gb,重复序列达85%。高质量的小麦基因组序列己释放(http://www.wheat genome.org/)[28],小麦A基因组供体乌拉尔图小麦和D基因组供体粗山羊草(Aegliops tauschii)的基因组测序工作也已由中国完成[29,30,31],这为筛选小麦生长发育基因及研究其进化过程奠定了基础。目前,小麦中关于GASA基因家族分析的报道较少,还没有对该基因家族的生物信息学和表达进行分析,因此有必要利用最新公布的小麦基因组数据对GASA基因家族进行系统分析。本研究利用生物信息学方法在最新的小麦基因组数据上鉴定小麦GASA基因,并对其基因结构、染色体分布、串联重复片段、分子进化、蛋白结构及表达谱进行分析,以期为进一步探讨小麦GASA基因的功能奠定基础,为利用分子生物学技术改良小麦性状提供理论依据。

1 材料和方法

1.1 小麦TaGASA基因家族的鉴定

从EnsemblPlants数据库(http://plants.ensembl.org/index.html/)下载小麦的基因组序列、基因注释和蛋白序列文件,利用NCBI(National Center for Biotechnology Information)https://www.ncbi.nlm.nih.gov/blast已报道的GASA蛋白序列并与Pfam数据库(http://pfam.xfam.org/)进行比对(e-value<1e-5),获得BRX基因家族的Pfam ID及其序列。搜索小麦TaGASA基因家族的同源蛋白,删除重复序列,利用在线软件Pfam(http://www.sanger.ac.uk/Software/Pfam/search.shtml)进行保守结构域分析验证,剔除冗余蛋白。将GASA基因家族成员按照染色体顺序编号命名并映射到不同染色体上,将散在的非染色体序列合并为U染色体。同时Blast各物种的蛋白序列与相应基因家族Pfam进行比对,得到每个物种GASA基因家族序列和蛋白序列。借助ProtParam(https://web.expasy.org/protparam/)对GASA基因家族蛋白进行分子量、等电点和氨基酸信息预测。

1.2 小麦TaGASA基因家族结构分析

根据EnsemblPlants数据库中的DNA数据库检索小麦TaGASA基因的内含子、外显子、染色体位置等信息,利用GSDS 2.0(Gene Structure Display Server)(http://gsds.cbi.pku.edu.cn/)在线绘制GASA基因的内含子和外显子的组成及基因家族进化树。利用Inparanoid分析小麦的同源蛋白(orthologous groups,OG),使用circos基于基因注释信息对OG关系作图,删除OG聚类过程中没有同源关系的基因使得同源基因在图片中显示。

1.3 小麦TaGASA基因家族的分子进化树构建

从EnsemblPlants数据库下载大麦(Hordeum vulgare)、拟南芥(Arabidopsis thaliana)、二穗短柄草(Brachypodium distachyum)、水稻(Oryza sativa)、玉米(Zea mays)、高粱(Sorghum bicolor)基因组和蛋白序列数据;利用MUSCLE[32]对小麦、大麦、二穗短柄草、水稻、玉米、高粱、拟南芥GASA蛋白序列进行多重比对,将结果输入MEGA 7.0[33],采用邻接法(Neighbor-Joining,NJ)分别构建小麦GASA基因家族进化树及小麦、大麦、二穗短柄草、水稻、玉米、高粱、拟南芥的系统进化树,其中,校验参数(bootstrap)设置为1000,其余均为默认值。借助FigTree绘制小麦GASA基因家族进化树及小麦、大麦、二穗短柄草、水稻、玉米、高粱、拟南芥的系统进化树。

1.4 小麦TaGASA基因家族motif结构和三级结构预测分析

基于The MEME suite的在线工具MEME(http://meme-suite.org/tools/meme)对小麦TaGASA基因家族的Motif序列进行分析;利用在线软件ExPaSy提供的SWISS-MODEL(https://swissmodel.expasy.org/interactive)对小麦TaGASA基因家族的蛋白质空间模型进行三维结构同源建模。

1.5 小麦TaGASA基因的表达谱分析

利用已公布的小麦RNA-seq数据,检索小麦TaGASA基因的表达谱(http://www.plexdb.org/modules/tools/plexdb_blast.php)。数据库中提供了小麦品种Chinese Spring的13个组织器官的表达数据,包括胚芽鞘、胚根、胚乳、根、花冠、叶、幼穗、花苞、雌蕊、花药、3~5 DAP(days after pollination)颖果、22 DAP胚、22 DAP胚乳,获取数据库中13个组织器官的FPKM(fragments per kilobase of transcript permillionmapped reads)值作为TaGASA基因的组织表达谱数据,用Heatmapper构建基因表达热图(http://www.heatmapper.ca/)。热图中可以表示出基因表达的强度。

2 结果与分析

2.1 小麦TaGASA基因家族的鉴定及蛋白特性分析

经EMBL-EBI确认,GASA基因家族的Pfam号为PF02704,利用HMM程序搜索得到小麦的TaGASA基因家族,结合已报道的水稻、拟南芥和葡萄GASA基因序列在Ensembl Plants数据库中进行BLASTP比对,得到47个小麦候选GASA基因;利用Pfam(http://www.ranger.ac.uk/Soft-ware/Pfam/search.shtml)对获得的小麦候选TaGASA基因编码蛋白质序列进行保守结构域分析,剔除不含有典型GASA结构域的冗余蛋白后,最终获得了35个家族成员,按照染色体顺序分别命名为TaGASA1~TaGASA35(表1)。对35个TaGASA基因综合分析发现,这35个小麦TaGASA基因分布于除3A、4A、3B、3D染色体外的其余17条染色体上。1B、2B上含有的基因数目最多,为4个;其次为1A、5A、1D和5D,基因数目均为3个;2A、6A、7A、4B、6B、4D和6D上含有的TaGASA基因数目最少,均仅有1个。序列分析显示,35个TaGASA基因编码长度为78~264个氨基酸的蛋白质,其中TaGASA16编码的氨基酸数目最多(264个),而TaGASA1最少(78个);蛋白质的相对分子质量为8 614.10~28 627.28kDa,TaGASA16的相对分子质量最大(28 627.28kDa),而TaGASA1最小(8 614.10kDa);等电点范围为8.27~9.41,TaGASA8预测的等电点最低(8.27),而TaGASA29最高(9.41),见表1

表1   35个小麦TaGASA基因的基本信息

Table 1  Basic information of 35 wheat TaGASA genes

基因名
Gene name
基因号
Gene ID
染色体
Chromosome
基因位置
Gene position
编码区长度(bp)
Coding sequence length
蛋白质预测Protein prediction
氨基酸
Amino acid
分子量(kDa)
Molecular mass
等电点
Isoelectric point
TaGASA1TRIAE_CS42_1AL_TGACv1_000841_AA00201401AL11 587-12 505234788 614.108.86
TaGASA2TRIAE_CS42_1AL_TGACv1_001082_AA00246201AL45 226-52 080276929 682.228.63
TaGASA3TRIAE_CS42_1AL_TGACv1_001087_AA00247401AL80 457-81 33530910311 179.178.81
TaGASA4TRIAE_CS42_2AS_TGACv1_113018_AA03497902AS10 196-10 8652769210 008.828.63
TaGASA5TRIAE_CS42_5AL_TGACv1_374351_AA11977505AL109 217-110 18533611212 493.618.72
TaGASA6TRIAE_CS42_5AL_TGACv1_375002_AA12137505AL39 823-40 585282949 785.418.90
TaGASA7TRIAE_CS42_5AL_TGACv1_377138_AA12444405AL8 818-10 7782949810 405.239.01
TaGASA8TRIAE_CS42_6AL_TGACv1_470886_AA14976006AL203 827-205 47534811611 953.768.27
TaGASA9TRIAE_CS42_7AS_TGACv1_569647_AA18211207AS74 216-74 91030310110 250.038.97
TaGASA10TRIAE_CS42_1BL_TGACv1_030884_AA01029801BL104 154-105 07530910311 352.468.80
TaGASA11TRIAE_CS42_1BL_TGACv1_031264_AA01105801BL17 666-18 665276929 775.398.62
TaGASA12TRIAE_CS42_1BL_TGACv1_031280_AA01108201BL12 426-13 1002889610 283.139.34
TaGASA13TRIAE_CS42_1BL_TGACv1_031520_AA01155101BL89 188-90 015276929 748.328.46
TaGASA14TRIAE_CS42_2BS_TGACv1_146134_AA04562802BS148 955-149 94531510510 795.848.47
TaGASA15TRIAE_CS42_2BS_TGACv1_146482_AA04661702BS94 799-95 305276929 946.668.44
TaGASA16TRIAE_CS42_2BS_TGACv1_146482_AA04662102BS106 417-115 67579226428 627.288.64
TaGASA17TRIAE_CS42_2BL_TGACv1_130684_AA04160602BL40 977-41 82532110710 985.858.90
TaGASA18TRIAE_CS42_4BS_TGACv1_328307_AA10860104BS137 696-138 7202949810 390.359.30
TaGASA19TRIAE_CS42_5BL_TGACv1_404222_AA12911705BL136 136-149 51050116717 405.479.34
基因名
Gene name
基因号
Gene ID
染色体
Chromosome
基因位置
Gene position
编码区长度(bp)
Coding sequence length
蛋白质预测Protein prediction
氨基酸
Amino acid
分子量(kDa)
Molecular mass
等电点
Isoelectric point
TaGASA20TRIAE_CS42_5BL_TGACv1_405876_AA13368805BL8 923-10 10433611212 488.548.44
TaGASA21TRIAE_CS42_6BL_TGACv1_499717_AA15899006BL81 146-82 97932110710 970.838.77
TaGASA22TRIAE_CS42_7BS_TGACv1_593258_AA19499207BS57 596-58 5082979910 235.109.06
TaGASA23TRIAE_CS42_7BL_TGACv1_578066_AA18887407BL32 717-33 64536612213 359.779.30
TaGASA24TRIAE_CS42_1DL_TGACv1_061126_AA01862701DL98 006-98 923276929 792.378.46
TaGASA25TRIAE_CS42_1DL_TGACv1_061126_AA01862901DL149 160-150 029276929 720.318.62
TaGASA26TRIAE_CS42_1DL_TGACv1_061906_AA02054901DL58 046-58 96030910311 217.248.86
TaGASA27TRIAE_CS42_2DS_TGACv1_177275_AA05718602DS135 526-136 135276929 919.818.99
TaGASA28TRIAE_CS42_2DS_TGACv1_179436_AA06071302DS19 436-20 15431210410 670.678.47
TaGASA29TRIAE_CS42_4DS_TGACv1_362006_AA11756204DS35 367-36 2952949810 387.399.41
TaGASA30TRIAE_CS42_5DL_TGACv1_433605_AA14173205DL17 722-18 84533311112 308.258.44
TaGASA31TRIAE_CS42_5DL_TGACv1_433682_AA14192905DL8 699-10 0052949810 386.188.90
TaGASA32TRIAE_CS42_5DL_TGACv1_435968_AA14567505DL11 236-15 21074424826 889.909.14
TaGASA33TRIAE_CS42_6DL_TGACv1_527263_AA17015306DL12 943-14 52035411812 265.158.47
TaGASA34TRIAE_CS42_7DS_TGACv1_622044_AA20315507DS2 526-3 744282949 921.889.24
TaGASA35TRIAE_CS42_7DL_TGACv1_602652_AA19639307DL31 497-33 92538712914 160.539.18

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2.2 小麦TaGASA基因家族同源进化分析

片段复制和串联重复是家族基因扩张的主要模式,为了分析小麦TaGASA家族基因与祖先材料的同源进化关系,采用生物信息学方法将TaGASA基因定位到不同染色体,并对串联重复片段进行分析,对具有同源关系的基因进行相关的连线说明(图1)。从同源关系来看,TaGASA5、TaGASA16、TaGASA17、TaGASA19等12个基因与其他染色体上的基因没有同源对应关系,而TaGASA1、TaGASA2、TaGASA3、TaGASA4等23个基因与其他染色体上的基因具有同源关系。1D染色体上的TaGASA26基因与1A染色体上的TaGASA1、TaGASA2、TaGASA3基因和1B染色体上的TaGASA10、TaGASA11、TaGASA12基因串联重复,说明这7个基因具有一定的同源关系,其中6个基因都与TaGASA26相关,推测该6个基因由TaGASA26同源重复而来。5A染色体的TaGASA6与5B的TaGASA20和5D的TaGASA30基因串联重复,而2A染色体的TaGASA4和4B染色体的TaGASA18均仅有一个串联重复,分别对应染色体2B的TaGASA15和4D的TaGASA29,说明这两个GASA基因在D基因组和A基因组没有拷贝。

图1

图1   小麦TaGASA基因同源进化分析

Fig.1   Homologous evolution analysis of TaGASA genes in wheat


2.3 小麦TaGASA基因家族蛋白进化树及基因结构分析

利用本研究中小麦GASA全长蛋白序列构建小麦系统进化树,结果(图2)显示,在35个编码小麦TaGASA蛋白的基因中检测到了10个旁系同源基因对,即TaGASA20和TaGASA5、TaGASA9和TaGASA22、TaGASA1和TaGASA3、TaGASA29和TaGASA18、TaGASA28和TaGASA14、TaGASA31和TaGASA12、TaGASA6和TaGASA19、TaGASA24和TaGASA11、TaGASA8和TaGASA33、TaGASA15和TaGASA16。基因结构分析表明,小麦35个TaGASA基因外显子数量变化从2个(TaGASA29、TaGASA18等)到7个(TaGASA16),小麦TaGASA基因家族蛋白进化树显示,同一类之间的结构较为相似。总体来看,TaGASA基因家族结构较为简单,多数还有2~3个外显子,这些基因可能产生或分化的时间较晚,推测其功能相对专一。具有相似外显子和内含子的结构,在蛋白进化树上也具有很高的同源性,表明亲缘关系近的基因在进化过程中其外显子、内含子具有一定的相似性。

图2

图2   小麦TaGASA基因家族的蛋白系统进化树和基因结构

Fig.2   Phylogenetic tree and gene structures of wheat TaGASA gene family


小麦(35个)、大麦(14个)、二穗短柄草(8个)、玉米(15个)、水稻(10个)、高粱(13个)及拟南芥(15个)GASA基因的系统进化树分析表明,来自7种作物的110个GASA基因分为3个类别,而本研究中得到的35个小麦TaGASA基因也可以归于这3个类别(图3)。

图3

图3   小麦与其他物种GASA基因的系统进化树

Fig.3   The phylogenetic analysis of GASA genes in wheat and other species


2.4 小麦TaGASA基因家族的蛋白结构

模体(motif)是蛋白质分子结构中介于二级结构与三级结构之间的结构层次,又称超二级结构,是蛋白质分子具有特定功能或作为独立结构域一部分的二级结构聚合体。基因家族所有的或者大多数成员共有的motif极可能是该家族执行重要功能或组成结构不可缺少的部分,如一些具有序列特异性的蛋白的结合位点(转录因子)或者是涉及到重要生物过程的RNA起始、终止、剪切等。识别基因家族共同的motif就能了解该基因家族的特征,从而可以利用这些特征来发掘基因家族新成员。本研究中小麦35个TaGASA基因家族成员含有10个motif,其中TaGASA32含有最多的motif结构(9个),TaGASA19、TaGASA26、TaGASA10、TaGASA3分别含有8、6、6、6个motif,其余30个TaGASA基因都是含有4个或者5个motif。35个TaGASA都还有motif1、motif2、motif3模型;只有TaGASA32含有motif9,且含有5个;仅TaGASA1含有motif8(图4)。分析结果说明,TaGASA基因家族应都含有motif1、motif2、motif3,TaGASA1和TaGASA32是TaGASA基因家族具有特异性功能的基因。这个预测有助于发现TaGASA基因家族的新成员。

图4

图4   小麦TaGASA基因家族的motif分析

Fig.4   Motif analysis of the wheat TaGASA genes family


本研究通过对小麦35个TaGASA基因家族成员的氨基酸序列进行三维结构同源建模,通过在线软件Swiss-Model分析显示35个成员的氨基酸序列三级结构相似性较高。所以,从小麦35个TaGASA基因家族成员氨基酸序列中选取4条最具代表性的序列(TaGASA11、TaGASA19、TaGASA30、TaGASA32)进行同源三级结构建模(图5)。这4条序列都含有3个α-螺旋,但其三级结构不完全相同,这可能与α-螺旋、β-折叠的长度以及无规则卷曲的长度不同有关,这些相似或差异之处可能是导致它们功能上相似或不同的原因。

图5

图5   小麦TaGASA基因家族的蛋白三级结构

Fig.5   Protein tertiary structure of the wheat TaGASA gene family


2.5 小麦TaGASA基因的表达谱分析

小麦品种Chinese Spring 13个组织器官的RNA-seq数据分析结果(图6)显示,35个TaGASA基因在13个组织器官中都检测到了转录本。如图6所示,TaGASA34、TaGASA5、TaGASA9、TaGASA22、TaGASA30、TaGASA10、TaGASA20、TaGASA26、TaGASA1、TaGASA3在胚芽鞘、胚乳、花冠、叶、幼穗、雌蕊、3~5 DAP颖果7个组织器官中均具有较高的表达量,这和刘秋华等[12]研究的水稻GASA基因结果基本一致;TaGASA15、TaGASA4、TaGASA23、TaGASA27、TaGASA21、TaGASA8、TaGASA33、TaGASA35在胚芽鞘、胚乳、花冠、幼穗4个组织器官中也表现出了较高的表达量,这与赵腾等[34]研究结果基本一致;TaGASA18、TaGASA29、TaGASA6、TaGASA19、TaGASA12、TaGASA32仅在雌蕊、3~5 DAP颖果和22 DAP胚中表达量较高;TaGASA24、TaGASA25、TaGASA2、TaGASA13仅在花药和22 DAP胚中表达量较高;TaGASA7、TaGASA31仅在3~5 DAP颖果和22 DAP胚乳中表达量较高。

图6

图6   小麦TaGASA基因在不同组织器官中的表达谱分析

1:胚芽鞘;2:胚根;3:胚乳;4:根;5:花冠;6:叶;7:幼穗;8:花苞;9:雌蕊;10:花药;11:3~5 DAP颖果;12:22 DAP胚;13:22 DAP胚乳

Fig.6   Expression profile of TaGASA genes in thirteen tissues of wheat

1: Coleoptile; 2: Seed root; 3: Embryo; 4: Root; 5: Crown; 6: Leaf; 7: Immature inflorescence; 8: Floral bracts; 9: Pistil; 10: Anthers; 11: 3-5 DAP caryopsis; 12: 22 DAP embryo; 13: 22 DAP endosperm


3 讨论

普通小麦是典型的异源六倍体,包含A、B、D 3个基因组。通过基因家族研究发现,在漫长的进化中D基因组的抗病相关基因、品质相关基因以及抗非生物应激反应的基因均不同程度地发生了扩增。异源六倍体基因组存在丰富动态变化,在驯化和多倍化过程中,有些基因家族成员丢失,而涉及生长、代谢和能量采集的基因家族则表现为扩张,这可能与作物长期产量选择有关。在禾谷类作物中,子粒的粒长、粒宽和粒厚等表型性状往往对作物产量影响较大。如在水稻中发现了一些GASA家族影响粒长、粒宽和粒厚的基因,在小麦中也发现了影响粒长的GASA基因家族基因(TaGASR7)。为进一步探讨小麦TaGASA基因的功能,本研究利用生物信息学方法根据小麦最新数据库,首次对六倍体小麦进行了GASA基因家族的研究,并在获得的35个TaGASA基因的基础上,对其基因结构、染色体定位、系统进化、蛋白保守结构域及表达谱进行了分析。

本研究在小麦全基因组水平上共检测到35个编码TaGASA蛋白的基因,所有成员均具有1个保守的GASA结构域,包含了60个左右的氨基酸,整个家族蛋白可能均属于分泌型胞外蛋白。从系统进化角度看,GASA基因在大多数植物中都是由多成员组成,它们属于横向同源基因,处于物种形成后的同一进化分支中。小麦中GASA基因家族成员多于已报道的拟南芥(15个)、水稻(9个)、大豆(2个)、野杨(18个)等植物GASA基因家族成员数目[16,34-35],可能是片段复制和串联重复导致小麦GASA基因扩张。总之,小麦TaGASA基因家族的进化过程可能受局部片段重复多倍化、不等价交换及转座子等多种因素影响,导致其进化错综复杂。小麦TaGASA蛋白进化树和7种作物GASA基因的系统进化树表明,GASA基因分为4个类别,同一类之间的结构较为相似,不同亚族间氨基酸组成和蛋白结构的区别可能导致功能上的差异。本研究获得的35个TaGASA基因家族成员中的TaGASA9、TaGASA22、TaGASA34与已研究的小麦TaGASR7基因序列基本一致,分别位于7AS、7BS、7DS上;TaGASA6、TaGASA19、TaGASA32与已研究的水稻OsGASR1基因序列基本一致,分别位于5AL、5BL、5DL上;TaGASA17与已研究的水稻OsGASR2基因序列基本一致,位于2BL上。小麦35个TaGASA基因家族成员含有10个motif,推测小麦TaGASA基因家族应都含有motif1、motif2、motif3。TaGASA在小麦的13个组织器官中都检测到了转录本,且表达模式存在明显的差异,可能存在功能分化,表明这类基因在植物生长发育过程中具有不同的功能。但是关于小麦TaGASA基因如何调控赤霉素的基因表达、参与小麦生长发育还需要进一步的探讨。

综上所述,本文通过生物信息学的方法对普通小麦TaGASA基因家族进行分析,预测基因的结构与功能,并进行了相关的同源进化分析,这为GASA基因在小麦生长发育过程中基因的挖掘和利用提供了理论与试验依据。

The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。

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DOI:10.1046/j.1365-313X.2003.01950.x      URL     PMID:14690507      [本文引用: 1]

The Petunia hybrida GA-induced proteins (GIPs) belong to a large group of proteins identified in numerous plant species. These proteins share a similar C-terminal region containing 12 cysteine residues in conserved positions. To date, the function of these proteins remains unclear. We previously found that GIP1 expression coincides with cell elongation in stems and flowers and is induced by gibberellic acid (GA 3 ). Transient expression of a GIP1:green fluorescent protein (GFP) fusion in tobacco bright yellow 2 (BY2) cells and immunoblot analyses suggest microsomal compartmentalization with possible endoplasmic reticulum (ER) localization. However, the polyclonal anti-GIP1 antibodies also reacted with proteins extracted from the cell wall. Three novel GIP homologs, GIP2 , GIP4 , and GIP5 , were isolated. While GIP4, similar to GIP1, is putatively localized to the ER membrane, the cleavable hydrophobic N-terminal sequences of GIP2 and GIP5 suggest cell wall localization. GIP1 and GIP2 are expressed during cell elongation, whereas GIP4 and GIP5 are expressed during cell division; nevertheless, they all were induced by GA 3 . We generated transgenic petunia in which we repressed the putative cell wall protein GIP2. The transgenic plants exhibited late flowering and reduced stem elongation. These phenotypic alterations were found under low, but not moderate-high temperatures, suggesting functional redundancy under normal growth conditions. The expression pattern and cellular localization of GIP2, its regulation by GA, and the phenotype of the transgenic plants suggest a role in GA-mediated cell elongation and transition to flowering.

Berrocallobo M, Segura A, Moreno M , et al.

Snakin-2,an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection

Plant Physiology, 2002,128(3):951-961.

DOI:10.1104/pp.010685      URL     [本文引用: 1]

Furukawa T, Sakaguchi N, Shimada H .

Two OsGASR genes,rice GAST homologue genes that are abundant in proliferating tissues,show different expression patterns in developing panicles

Genes & Genetic Systems, 2006,81(3):171-180.

DOI:10.1266/ggs.81.171      URL     PMID:16905871      [本文引用: 2]

Two different types of genes for rice GA-stimulated transcript (GAST) homologue genes, Oryza sativa GA-stimulated transcript-related gene 1 (OsGASR1) and gene 2 (OsGASR2), were found. Both OsGASR proteins contain a cysteine-rich domain highly conserved among GAST family proteins in their C-terminal regions. Gibberellin A3 (GA3) stimulated expression of both OsGASRs in the wild-type Nipponbare and GA3 synthesis-deficient mutant. Expression of both OsGASRs apparently increased when cell proliferation entered the logarithmic phase, and rapidly reduced when cell proliferation was temporarily halted. RT-PCR analysis indicated different expression patterns of these genes in developing panicles. OsGASR1 was limitedly but strongly expressed in florets while OsGASR2 was expressed in both florets and branches. In situ hybridization showed that they were strongly expressed in the root apical meristem (RAM) and shoot apical meristem (SAM), but little signals were detected in mature leaves. Transient expression of OsGASR-GFP fusion proteins in onion epidermal cells revealed that both OsGASR proteins localized to the apoplasm or cell wall. These results suggest that OsGASR1 and OsGASR2 were involved in cell division and might play diverse roles in differentation of panicles.

刘秋华, 罗曼, 彭建宗 , .

水稻OsGASR4基因及其启动子的克隆与表达分析

华南师范大学学报, 2015(1):81-86.

DOI:10.6054/j.jscnun.2014.11.006      URL     [本文引用: 2]

对前期克隆的水稻GAST基因家族新成员OsGASR4开展研究,利用DNAMAN软件分析水稻OsGASR4进化树;检测GA处理野生型和d-18水稻突变体后OsGASR4表达变化;利用RT-PCR方法分析该基因的时空表达特性;克隆了该基因上游长约2082 bp调控序列,并利用网络工具预测启动子顺式作用元件,构建OsGASR4启动子GUS融合表达载体,通过农杆菌介导的水稻遗传转化。结果表明:OsGASR4蛋白具有典型的GASA保守结构域,启动子里含有多个与激素响应元件、光诱导相关元件以及逆境胁迫响应元件;GA3处理降低了OsGASR4转录水平;RT-PCR检测和GUS染色的结果表明,OsGASR4在水稻茎和幼穗的表达量相对较高。表明OsGASR4可能作为GA信号途径的抑制因子参与水稻茎和穗的早期发育。

Kotilainen M, Helariutta Y, Mehto M , et al.

GEG participates in the regulation of cell and organ shape during corolla and carpel development in Gerbera hybrida

Plant Cell, 1999,11(6):1093-1104.

DOI:10.1105/tpc.11.6.1093      URL     [本文引用: 1]

De la Fuente J I AI, Castillejo C, Sanchez-Sevilla JF , et al.

The strawberry gene FaGAST affects plant growth through inhibition of cell elongation

Journal of Experimental Botany, 2006,57(19):2401-2411.

DOI:10.1093/jxb/erj213      URL     PMID:16804055      [本文引用: 1]

The strawberry (Fragaria ananassa) FaGAST gene encodes a small protein with 12 cysteine residues conserved in the C-terminal region similar to a group of proteins identified in other species with diverse assigned functions such as cell division, elongation, or elongation arrest. This gene is expressed in the fruit receptacle, with two peaks during ripening at the white and the red-ripe stages, both coincident with an arrest in the growth pattern. Expression is also high in the roots but confined to the cells at the end of the elongation zone. Exogenous application of gibberellin increased the transcript level of the FaGAST gene in strawberry fruits. Ectopic expression of FaGAST in transgenic Fragaria vesca under the control of the CaMV-35S promoter caused both delayed growth of the plant and fruits with reduced size. The same growth defect was observed in Arabidopsis thaliana plants overexpressing FaGAST. In addition, the transgenic plants exhibited late flowering and low sensitivity to exogenous gibberellin. Taken together, the expression pattern, the regulation by gibberellin, and the transgenic phenotypes point to a role for FaGAST in arresting cell elongation during strawberry fruit ripening.

Liu Z H, Zhu L, Shi H Y , et al.

Cotton GASL genes encoding putative gibberellin-regulated proteins are involved in response to GA signaling in fiber development

Molecular Biology Reports, 2013,40(7):4561-4570.

DOI:10.1007/s11033-013-2543-1      URL     PMID:23645033      [本文引用: 1]

GAST (GA-stimulated transcript)-like genes have been reported as targets of GA regulation in some plant species. In this study, we isolated seven GAST-like cDNAs from cotton (Gossypium hirsutum) cDNA libraries (designated as GhGASL1-GhGASL7). Meanwhile, the genomic DNA clones corresponding to the seven GhGASL genes were isolated by using PCR amplification technique. Analysis of gene structure revealed that four genes (GhGASL1/3/5/6) contain two exons and one intron, while the rest have four exons and three introns. All of the deduced GhGASL proteins contain a putative signal peptide in the N-terminus and a conservative cysteine-rich C-terminal domain. Quantitative RT-PCR analysis indicated that the seven GhGASL genes are differentially expressed in cotton tissues. Among them, GhGASL1/4/7 were predominantly expressed in cotyledons, while the transcripts of GhGASL2/5 were preferentially accumulated at hypocotyls. GhGASL3 mRNA was largely accumulated in fibers, while GhGASL6 transcripts were mainly detected in ovules. Furthermore, GhGASL2/3/5 displayed a relatively high expression levels during early fiber elongation stages, and were regulated by GA. These data suggested that GhGASL genes may be involved in fiber elongation and in response to GA signaling during fiber development.

张盛春, 王小菁 .

拟南芥DELLA下游的GASA基因表达研究

科学通报, 2008,53(22):2760.

DOI:10.1360/csb2008-53-22-2760      URL     Magsci     [本文引用: 3]

<p>赤霉素(gibberellins, GAs)信号转导途径相关组分的分离与功能研究对于最终阐明GA作用机制十分重要, 目前赤霉素信号途径关键因子DELLA的下游组分研究较少. 拟南芥中受GA调控的<em>GASA</em> (<em>GA</em>-<em>Stimulated in Arabidopsis</em>)家族共有15个基因, 预测所有GASA蛋白的N端均有一可剪切的信号肽, C端含有12个半胱氨酸的GASA保守结构域. RT-PCR结果证实, 在<em>DELLA</em>突变体<em>gai-t6</em>和<em>rga-24</em>以及二者双突变体中, <em>GASA4</em>和<em>GASA6</em>的表达上调而<em>GASA1</em>和<em>GASA9</em>下调, 与<em>GASA4</em>和<em>GASA6</em>表达受外源赤霉酸(GA<sub>3</sub>)促进而<em>GASA1</em>和<em>GASA9</em>表达受GA<sub>3</sub>抑制的结果相一致. 除此之外, 其他一些<em>GASA</em>基因表达也分别受GA<em>3</em>和脱落酸(ABA)的独立或共同调控. 大部分<em>GASA</em>基因在根、茎、叶、花和幼嫩荚果中均有表达. 利用启动子驱动GUS报告基因的方法研究了<em>GASA6</em>, <em>GASA7</em>, <em>GASA8</em>, <em>GASA9</em>, <em>GASA10</em>, <em>GASA11</em>和<em>GASA12</em>等7个基因的器官表达特异性, 发现在生长分化旺盛的组织器官及离层区均有这些基因的表达, 可能与细胞的分裂和生长有关. 本研究为<em>GASA</em>基因家族在GA和ABA 信号途径中的功能研究提供重要依据.</p>

Rubinovich L, Weiss D .

The Arabidopsis cysteine-rich protein GASA4 promotes GA responses and exhibits redox activity in bacteria and in planta

Plant Journal, 2010,64(6):1018-1027.

DOI:10.1111/j.1365-313X.2010.04390.x      URL     PMID:21143681      [本文引用: 1]

Although the gibberellin (GA) signaling pathway has been elucidated, very little is known about the steps linking first transcriptional activation to physiological responses. Among the few identified GA-induced genes are the plant-specific GAST1-like genes, which encode small proteins with a conserved cysteine-rich domain. The role of these proteins in plant development and GA responses is not yet clear. The Arabidopsis GAST1-like gene family consists of 14 members, GASA1 14. Here we show that over-expression of the GA-induced GASA4 gene in Arabidopsis promoted GA responses such as flowering and seed germination. Suppression of several GASA genes using synthetic microRNA (miRGASA) also promoted seed germination. This was probably caused by suppression of GASA5, which acts as a repressor of GA responses. Previously, we proposed that GAST1-like proteins are involved in redox reactions via their cysteine-rich domain. The results of this study support this hypothesis, as over-expression of GASA4 suppressed ROS accumulation and the transgenic seeds were partially resistant to the NO donor sodium nitroprusside (SNP). Moreover, Escherichia coli expressing intact GASA4 or a truncated version containing only the cysteine-rich domain were resistant to SNP. Mutated GASA4, in which conserved cysteines were replaced by alanines, lost its redox activity and the ability to promote GA responses, suggesting that the two functions are linked. We propose that GA induces some GAST1-like genes and suppresses others to regulate its own responses. We also suggest that the encoded proteins regulate the redox status of specific components to promote or suppress these responses.

Zimmermann R, Sakai H, Hochholdinger F .

The gibberellic acid stimulated-like gene family in maize and its role in lateral root development

Plant Physiology, 2010,152(1):356-365.

DOI:10.1104/pp.109.149054      URL     PMID:19926801      [本文引用: 2]

In an approach to study lateral root development in monocots, genome-wide searches for homologs of the Gibberellic Acid Stimulated Transcript-like (GAST-like) gene family in rice (Oryza sativa) and maize (Zea mays) were carried out. Six novel GAST-like genes in rice and 10 members of the gene family in maize, which were designated ZmGSL (for Z. mays Gibberellic Acid Stimulated-Like), were identified. The ZmGSL family encodes small proteins of 75 to 128 amino acids, which are characterized by a conserved 59 to 64 amino acid C-terminal domain. Within this domain, 17 amino acids, including 12 cysteines, are perfectly conserved. The transcript of the ZmGSL1 gene is differentially spliced into the alternative variants ZmGSL1a and ZmGSL1b, the latter of which is translated into a premature protein that lacks the C-terminal domain. The presence of an additional N-terminal cleavable signal sequence in eight of the 10 ZmGSL proteins suggests that they are secreted into the extracellular matrix. In-depth root-specific gene expression analyses carried out in the wild type and the lateral root mutants lrt1 and rum1 suggest a role for ZmGSL genes in early lateral root development, which is likely regulated by gibberellic acid. Expression patterns of ZmGSL1a and ZmGSL1b propose antagonistic functions of these splice variants during early lateral root formation.

Zhang S C, Yang C W, Peng J Z , et al.

GASA5,a regulator of flowering time and stem growth in Arabidopsis thaliana

Plant Molecular Biology, 2009,69(6):745-759.

DOI:10.1007/s11103-009-9452-7      URL     PMID:202020522020202020202020202020      [本文引用: 2]

Flowering is a critical event in the life cycle of plants and is regulated by a combination of endogenous controls and environmental cues. In the present work, we provide clear genetic evidence that GASA5 , a GASA family gene in Arabidopsis ( Arabidopsis thaliana ), is involved in controlling flowering time and stem growth. GASA5 expression was present in all tissues of Arabidopsis plants, as detected by RT-PCR, and robust GUS staining was observed in the shoot apex of 8-day-old seedlings and inflorescence meristems during reproductive development. Phenotypic analysis showed that a GASA5 null mutant ( gasa5-1 ) flowered earlier than wild type with a faster stem growth rate under both long-day (LD) and short-day (SD) photoperiods. In contrast, transgenic plants overexpressing GASA5 demonstrated delayed flowering, with a slower stem growth rate compared to wild-type plants. However, neither the GASA5 null mutants nor the GASA5 overexpressing plants revealed obvious differences in flowering time upon treatment with gibberellic acid (GA 3 ), indicating that GASA5 is involved in gibberellin (GA)-promoted flowering. GAI ( GA INSENSITIVE ), one of the five DELLAs in Arabidopsis, was more highly expressed in GASA5 -overexpressing plants, but it was lower in gasa5-1. Further transcript profiling analysis suggested that GASA5 delayed flowering by enhancing FLOWERING LOCUS C ( FLC ) expression and repressing the expression of key flowering-time genes, FLOWERING LOCUS T ( FT ) and LEAFY ( LFY ). Our results suggest that GASA5 is a negative regulator of GA-induced flowering and stem growth.

Moyano-Cañete E, Bellido M L, García-Caparrós N , et al.

FaGAST2,a strawberry ripening-related gene,acts together with FaGAST1 to determine cell size of the fruit receptacle

Plant & Cell Physiology, 2013,54(2):218-236.

DOI:10.1093/pcp/pcs167      URL     PMID:23231876      [本文引用: 1]

Numerous GAST-like genes have been reported in higher plants, but only one GAST-like gene (FaGAST1) has been described in strawberry so far. Herein, we have identified a novel strawberry FaGAST gene (FaGAST2) whose expression showed an increase throughout fruit receptacle development and ripening, coinciding with those stages where a decrease in fruit expansion processes (G3-W and R-OR stages) occurs. FaGAST2 only shares 31% and 15.7% amino acid and nucleotide sequence homology, respectively, with the previously reported FaGAST1 gene, but both genes contain a signal peptide and a highly conserved GASA domain (cysteine-rich domain) in the C-terminal region. FaGAST2 expression is mainly confined to the fruit receptacle and is not regulated by auxins, GA(3) or ABA, but is regulated by ethephon, an intracellular generator of ethylene. In addition, the expression of the FaGAST2 gene also increased under oxidative stress conditions (H2O2 or Colletotrichum acutatum infection), suggesting a direct role for FaGAST2 protein in reactive oxygen species scavenging during fruit growth and ripening and during fungal infection. On the other hand, the overexpression of the FaGAST2 gene in different transgenic lines analyzed caused a delay in the growth of strawberry plants and a reduction in the size of the transgenic fruits. The histological studies performed in these fruits showed that their parenchymal cells were smaller than those of the controls, supporting a relationship between FaGAST2 gene expression, strawberry fruit cell elongation and fruit size. However, transitory silencing of FaGAST2 gene expression through RNA interference approaches revealed an increase in FaGAST1 expression, but no changes in fruit cell size were observed. These results support the hypothesis that both genes must act synergistically to determine fruit cell size during fruit development and ripening.

Blancoportales R, Lópezraéz J A, Bellido M L , et al.

A strawberry fruit-specific and ripening-related gene codes for a HyPRP protein involved in polyphenol anchoring

Plant Molecular Biology, 2004,55(6):763-780.

DOI:10.1007/s11103-004-1966-4      URL     PMID:15604715      [本文引用: 1]

A strawberry ( Fragaria x ananassa cv. Chandler) fruit cDNA ( Fahyprp - cDNA ) and its corresponding gene ( Fahyprp ) showing sequence homology to higher plant hyprp genes have been isolated. The cDNA contains an open reading frame encoding a 1602kDa protein with 156 amino acids. The peptide has an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxy-terminal region homologous to other HyPRP proteins. Northern blot and QRT-PCR analysis have shown that the strawberry transcript is specifically expressed in fruit, not being detected in other plant tissues. “ In situ ” hybridization and immunolocalization studies have indicated that the Fahyprp gene is strongly expressed in achene sclerenchyma cells, in the vascular and receptacle cells of immature green fruit and in the vascular cells of mature red fruits. The achenes removal from unripe green fruits induced the expression of this Fahyprp gene. This induction was reverted by treatment of deachened fruit with the auxin NAA, supporting the idea that Fahyprp gene expression is regulated by auxins. Furthermore, the HyPRP protein has been localized in parenchymatic cells of immature fruits associated to structures containing condensed tannins. The results are discussed supporting a putative role of this protein in the anchoring of polymeric polyphenols in the strawberry fruit during growth and ripening.

Mao Z C, Zheng J Y, Wang Y S , et al.

The new CaSn gene belonging to the snakin family induces resistance against root-knot nematode infection in pepper

Phytoparasitica, 2011,39(2):151-164.

DOI:10.1007/s12600-011-0149-5      URL     [本文引用: 1]

The new CaSn gene belonging to the snakin family in pepper ( Capsicum annuum ) encodes a novel antimicrobial peptide and responds to root-knot nematode ( Meloidogyne spp.) infection. CaSn was isolated and cloned using suppression subtractive hybridization (SSH), and the gene was characterized and expressed in Escherichia coli . The CaSnakin protein encoded by CaSn is an antimicrobial peptide consisting of a signal peptide of 23 amino acid residues, an acidic peptide of 14 amino acid residues (pI = 4.18), and a mature protein of 66 amino acid residues that corresponds to a molecular mass of 7.03 kDa. The peptide sequence has 12 conserved cysteines forming six disulfide bridges. CaSnakin is highly homologous to the peptide snakin-2 (StSN2) of potato ( Solanum tuberosum ); CaSnakin also shows 88.5% identity to StSN2. Phylogenetic tree analysis indicated that the CaSn gene belongs to subfamily II of the snakin family. Real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) results showed that the CaSn gene was induced and expressed evidently by root-knot nematode infection; CaSn is also expressed in buds, stems, roots and leaves. The CaSnakin protein expressed in E. coli showed strong antimicrobial activity against free-living nematodes ( Caenorhabditis elegans ) and root-knot nematodes in vitro . In addition, the virus-induced gene silencing (VIGS) results revealed that the CaSn gene participates in the defense of plants against nematodes. In conclusion, the CaSn gene can be activated by nematode infections, and it plays an important role in host defense. As far as we know, this is the first investigation reporting the role of a snakin gene in the defense of plants against nematodes. In addition, the CaSn gene is the first gene of the snakin family isolated from pepper.

Zhang S C, Wang X J .

Over expression of GASA5 increases the sensitivity of Arabidopsis to heat stress

Journal of Plant Physiology, 2011,168(17):2093-2101.

DOI:10.1016/j.jplph.2011.06.010      URL     [本文引用: 1]

Rubinovich L, Ruthstein S, Weiss D .

The Arabidopsis cysteine-rich GASA5 is a redox-active metalloprotein that suppresses gibberellin responses

Molecular Plant, 2014,7(1):244-247.

DOI:10.1093/mp/sst141      URL     PMID:24157610      [本文引用: 1]

Huang X H, Zhao Y, Wei X H , et al.

Genome-wide association study of flowering time and grain yield traits in a worldwide collection of rice germplasm

Nature Genetics, 2011,44(1):32-39.

DOI:10.1038/ng.1018      URL     PMID:22138690      [本文引用: 1]

A high-density haplotype map recently enabled a genome-wide association study (GWAS) in a population of indica subspecies of Chinese rice landraces. Here we extend this methodology to a larger and more diverse sample of 950 worldwide rice varieties, including the Oryza sativa indica and Oryza sativa japonica subspecies, to perform an additional GWAS. We identified a total of 32 new loci associated with flowering time and with ten grain-related traits, indicating that the larger sample increased the power to detect trait-associated variants using GWAS. To characterize various alleles and complex genetic variation, we developed an analytical framework for haplotype-based de novo assembly of the low-coverage sequencing data in rice. We identified candidate genes for 18 associated loci through detailed annotation. This study shows that the integrated approach of sequence-based GWAS and functional genome annotation has the potential to match complex traits to their causal polymorphisms in rice.

Ling H Q, Zhao S C, Liu D C , et al.

The draft genome of Triticum urartu

Nature, 2013,496:487-490.

[本文引用: 1]

Zhang D D, Wang B N, Zhao J M , et al.

Divergence in homoeolog expression of the grain length-associated gene GASR7 during wheat allohexaploidization

The Crop Journal, 2015,3(1):1-9.

DOI:10.1016/j.cj.2014.08.005      URL     [本文引用: 1]

Hexaploid wheat has triplicated homoeologs for most of the genes that are located in subgenomes A, B, and D. GASR7, a member of the Snakin/GASA gene family, has been associated with grain length development in wheat. However, little is known about divergence of its homoeolog expression in wheat polyploids. We studied the expression patterns of the GASR7 homoeologs in immature seeds in a synthetic hexaploid wheat line whose kernels are slender like those of its maternal parent (Triticum turgidum, AABB, PI 94655) in contrast to the round seed shape of its paternal progenitor (Aegilops tauschii, DD, AS2404). We found that the B homoeolog of GASR7 was the main contributor to the total expression level of this gene in both the maternal tetraploid progenitor and the hexaploid progeny, whereas the expression levels of the A and D homoeologs were much lower. To understand possible mechanisms regulating different GASR7 homoeologs, we firstly analyzed the promoter sequences of three homoeologous genes and found that all of them contained gibberellic acid (GA) response elements, with the TaGASR7B promoter (pTaGASR7B) uniquely characterized by an additional predicted transcriptional enhancer. This was confirmed by the GA treatment of spikes where all three homoeologs were induced, with a much stronger response for TaGASR7B. McrBC enzyme assays showed that the methylation status at pTaGASR7D was increased during allohexaploidization, consistent with the repressed expression of TaGASR7D. For pTaGASR7A, the distribution of repetitive sequence-derived 24-nucleotide (nt) small interfering RNAs (siRNAs) were found which suggests possible epigenetic regulation because 24-nt siRNAs are known to mediate RNA-dependent DNA methylation. Our results thus indicate that both genetic and epigenetic mechanisms may be involved in the divergence of GASR7 homoeolog expression in polyploid wheat.

Avni R, Nave M, Barad O , et al.

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication

Science, 2017,357(6346):93.

DOI:10.1126/science.aan0032      URL     PMID:28684525      [本文引用: 1]

Wheat (Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer (T. turgidum ssp. dicoccoides). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 (TtBtr1) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties.

Jia J Z, Zhao S C, Kong X Y , et al.

Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation

Nature, 2013,496(7443):91-95.

DOI:10.1038/nature12028      URL     PMID:23535592      [本文引用: 1]

About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD)(1,2). Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker's flour(2). Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.

Ling H Q, Zhao S C, Liu D C , et al.

Draft genome of the wheat A-genome progenitor Triticum urartu

Science Foundation in China, 2013,496(2):87-90.

DOI:10.1038/nature11997      URL     PMID:20      [本文引用: 1]

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m)A(m)), is central to wheat evolution, domestication and genetic improvement(1). The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu(2), which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome(3)) and Ae. tauschii (the donor of the D genome(4)), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

Choulet F, Alberti A, Theil S , et al.

Structural and functional partitioning of bread wheat chromosome 3B

Science, 2014,345(6194):1249721.

DOI:10.1126/science.1249721      URL     PMID:25035497      [本文引用: 1]

We produced a reference sequence of the 1-gigabase chromosome 3B of hexaploid bread wheat. By sequencing 8452 bacterial artificial chromosomes in pools, we assembled a sequence of 774 megabases carrying 5326 protein-coding genes, 1938 pseudogenes, and 85% of transposable elements. The distribution of structural and functional features along the chromosome revealed partitioning correlated with meiotic recombination. Comparative analyses indicated high wheat-specific inter- and intrachromosomal gene duplication activities that are potential sources of variability for adaption. In addition to providing a better understanding of the organization, function, and evolution of a large and polyploid genome, the availability of a high-quality sequence anchored to genetic maps will accelerate the identification of genes underlying important agronomic traits.

Edgar R C .

MUSCLE:multiple sequence alignment with high accuracy and high throughput

Nucleic Acids Research, 2004,32(5):1792-1797.

DOI:10.1093/nar/gkh340      URL     [本文引用: 1]

Kumar S, Stecher G, Tamura K .

MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets

Molecular Biology & Evolution, 2016,33(7):1870-1874.

[本文引用: 1]

赵腾, 夏新莉, 尹伟伦 .

黑杨GASA基因的克隆和功能分析

广东农业科学, 2012,39(8):138-140.

DOI:10.3969/j.issn.1004-874X.2012.08.043      URL     [本文引用: 2]

为研究黑杨的速生分子调控机 制,采用PCR技术,从欧美杂种黑杨R270(Populus deltoides×Populus nigra)中克隆得到GASA基因,并构建其植物表达载体,通过农杆菌花序侵染法转入拟南芥,获得转基因植株,进行黑杨GASA基因的功能分析。结果表 明:克隆得到的黑杨GASA基因cDNA片段总长为330 bp,可编码109个氨基酸残基;与野生型拟南芥相比,超量表达PdGASA4能促进拟南芥的提早抽薹、叶片的伸长及茎的增高和加粗。

李昆仑, 柏锡, 卢姗 , .

碱胁迫应答GsGASA1及GsGASA2基因表达特性研究

东北农业大学学报, 2012,43(1):143-148.

DOI:10.3969/j.issn.1005-9369.2012.01.025      URL     [本文引用: 1]

野生大豆具有很强的抗逆性和适应能力,是利用基因工程手段进行作物抗逆分子育种的重要基因来源供体材料。为了获得具有自主知识产权、在植物渗透胁反应中起关键作用的功能基因,利用前期构建的野生大豆碱胁迫基因芯片表达谱,从中选取两个碱胁迫处理下显著上调表达,经分析预测属于GASA基因家族的基因(probesets分别为Gma.15958.1.S1_at;GmaAffx.90343.1.S1_at),分别命名为GsGASA1、GsGASA2。对上述两个基因进行了芯片结果的sqRT-PCR验证,并分析了其在野生大豆中经盐、干旱、冷胁迫处理下的表达特性。结果表明,GsGASA1、GsGASA2基因对这三种胁迫处理均表现出应答反应,虽然在不同胁迫条件下表达高峰出现的时间和表达强度上存在差异,但均呈现出在短期内显著上调表达的表达模式,推测岱甜SAI、GsGASA2基因在非生物胁迫中将起到一定作用。另外,GsGASA1、白GA跗2基因的表达受GA诱导7LPAC、ABA的抑制。研究将为下一步GsGASA1、GsGASA2全长基因的克隆及其在非生物胁迫中的功能研究奠定基础,也为研究GA和ABA信号通路的相互作用提供线索。

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