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Volume 34 Issue 4
Aug.  2021
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Cloning, Expression and Protein Interaction of ThSHR3 Genes in Taxodium hybrid ‘Zhongshanshan406’

  • Corresponding author: HUA Jian-feng, jfhua@cnbg.net
  • Received Date: 2020-08-19
    Accepted Date: 2021-02-24
  • Objective To obtain the SHORT-ROOT 3 gene (ThSHR3) from Taxodiumhybrid 'Zhongshanshan 406' by cloning and to identify and analyze its characteristics and related functions based on proteome and transcriptome data of adventitious roots of Taxodium hybrid 'Zhongshanshan406'. Method The full length of ThSHR3 gene was cloned by RACE technology and the bioinformatics characteristics were analyzed by bioinformatics software. Semi-quantitative PCR and real-time quantitative PCR were used to detect the expression characteristics of ThSHR3. The subcellular localization of ThSHR3 protein was confirmed by transient expression of protoplasts, and the protein interaction of ThSHR3 was verified by bimolecular fluorescence complementation (BiFC) technique. Result The authors assembled a full-length of ThSHR3 gene, which consists of 2 019 bp nucleotide sequence, containing a 1 446 bp open reading frame (ORF) encoding 482 amino acid proteins. ThSHR3 protein has conserved GRAS domains in the C-terminal, such as LHRI, VHIID, LHRII, PFYRE and SAW. Phylogenetic analysis showed that ThSHR3 belongs to the SHR subfamily. ThSHR3 gene showed a gradually increased expression pattern in the development of adventitious root. Transient expression analysis of protoplasts showed that ThSHR3 protein was located in the nucleus. Further BiFC experiment revealed that ThSHR3 protein and ThSCR protein which also belongs to GRAS protein family can interact with each other in nucleus. Conclusion This study indicates that ThSHR3 plays an important role in regulating the development of adventitious roots of Taxodium hybrid 'Zhongshanshan 406'.
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Cloning, Expression and Protein Interaction of ThSHR3 Genes in Taxodium hybrid ‘Zhongshanshan406’

    Corresponding author: HUA Jian-feng, jfhua@cnbg.net
  • Jiangsu Engineering Research Center for Taxodium Rich. Germplasm Innovation and Propagation; Institute of Botany, Chinese Academy of Sciences, Nanjing 210014, Jiangsu, China

Abstract:  Objective To obtain the SHORT-ROOT 3 gene (ThSHR3) from Taxodiumhybrid 'Zhongshanshan 406' by cloning and to identify and analyze its characteristics and related functions based on proteome and transcriptome data of adventitious roots of Taxodium hybrid 'Zhongshanshan406'. Method The full length of ThSHR3 gene was cloned by RACE technology and the bioinformatics characteristics were analyzed by bioinformatics software. Semi-quantitative PCR and real-time quantitative PCR were used to detect the expression characteristics of ThSHR3. The subcellular localization of ThSHR3 protein was confirmed by transient expression of protoplasts, and the protein interaction of ThSHR3 was verified by bimolecular fluorescence complementation (BiFC) technique. Result The authors assembled a full-length of ThSHR3 gene, which consists of 2 019 bp nucleotide sequence, containing a 1 446 bp open reading frame (ORF) encoding 482 amino acid proteins. ThSHR3 protein has conserved GRAS domains in the C-terminal, such as LHRI, VHIID, LHRII, PFYRE and SAW. Phylogenetic analysis showed that ThSHR3 belongs to the SHR subfamily. ThSHR3 gene showed a gradually increased expression pattern in the development of adventitious root. Transient expression analysis of protoplasts showed that ThSHR3 protein was located in the nucleus. Further BiFC experiment revealed that ThSHR3 protein and ThSCR protein which also belongs to GRAS protein family can interact with each other in nucleus. Conclusion This study indicates that ThSHR3 plays an important role in regulating the development of adventitious roots of Taxodium hybrid 'Zhongshanshan 406'.

  • 中山杉(Taxodium hybrid ‘Zhongshanshan’)是江苏省中国科学院植物研究所从落羽杉属树木杂交组合中选育出的,是具有一定超亲性状的优良无性系总称。中山杉具有生长迅速、抗逆性强、景观性好、成材率高等特点,已在我国沿海防护林建设、公路及城乡绿化、农田林网和滩涂造林等方面得到广泛应用[1]。目前,中山杉无性系主要通过嫩枝扦插的方法进行繁殖,近些年林业工作者一直致力于通过中山杉扦插技术改良来提高中山杉的生根率,研究表明:泥炭土、珍珠岩及沙壤土组成的混合基质有利于大多数中山杉无性系生根[2]。植物生长调节物质的浓度对中山杉扦插生根率影响显著,2 000 mg·L−1 IAA和2 000 mg·L−1 NAA混合液处理的中山杉无性系插条,生根率和生根数量都显著提升。然而,在中山杉长期选育过程中发现,不同无性系间生根能力差异显著,即使优化扦插技术,中山杉302的平均生根率(57.50%)仍显著低于中山杉118(83.93%)和406(87.14%)[3-4],且随着无性系生理年龄的增长,早期选育出的无性系生根力出现衰退现象[1],中山杉的生根机理亟需深入研究。

    SHR(SHORT-ROOT)是植物GRAS家族中与根系发生及形态建成密切相关的一个分支,对根尖干细胞微环境的特化和维持起着关键作用,它调控根皮层、内皮层初始细胞及初始细胞子细胞的垂周和平周不均等分裂,影响植物根的辐射式生长模式[5-7]。拟南芥shr突变体植株根尖分生组织的基本组织子细胞不能发生不对称平周分裂,仅产生一层类似皮层的细胞[8]shr突变体植株表现出主根生长减弱,侧根数量减少,植株矮小,子叶颜色深暗等一系列表型。过量表达SHR基因,根尖形态变化显著,基本组织发生大量的平周分裂,产生多个细胞层[9]。同时,SHR基因是一个功能进化比较保守的基因,目前已在玉米(Zea mays Linn.)[10]、水稻(Oryza sativa Linn.)[11]、毛果杨(Populus trichocarpa Torr. & Gray.)[12]、辐射松(Pinus radiata D.Don)[13]等植物中发现的SHR家族成员均和根的生长发育息息相关。研究表明:SHR转录功能的行使,通常需要依靠其下游另一个GRAS家族成员SCR(SCARECROW)[14]。SCR与SHR形成SCR/SHR复合体共同激活下游基因表达,SCR/SHR复合体直接作用于CYCD6.1(D-type cyclin 6.1)蛋白,CYCD6.1是细胞发生平周分裂的标志性蛋白,保证根尖细胞会在早期和晚期分别自发地进行2次平周分裂,产生完整的基本组织[15-17]。国内外学者对于不同植物SHR基因的挖掘以及SHR对根部细胞分裂调控机制的解析一直处于探索之中,本研究在前期已获得2个中山杉SHR基因(ThSHR1, ThSHR2)的基础上[18],新筛选到1个在中山杉406不定根生长期高表达的基因ThSHR3,而ThSHR3的生物学功能尚不清楚,拟通过生物信息学手段对其进行系统进化分析,探索其在中山杉不定根发育过程中表达模式,对ThSHR3蛋白进行亚细胞定位以及互作研究,推测其可能存在的信号通路。以期为中山杉以及落羽杉属植物不定根发育的分子机理研究提供新的理论依据。

1.   材料和方法
  • “中山杉406”的嫩枝扦插试验于2019年7月中旬开展于江苏省中国科学院植物研究所中山杉苗圃内。插穗长度约10~15 cm,扦插基质为珍珠岩和泥炭土,体积比例为1:1,光周期为14 h的光照周期和10 h的暗周期。根据中山杉406插穗基部明显的形态学改变,在4个时间点取样:0 d皮层休眠期(S0),21 d愈伤组织形成期(S1),35 d初生根形成期(S2)和56 d根系生长期(S3)[18]。其中,皮层休眠期和愈伤组织形成期的取样部位为插穗基部皮层组织(0.3 cm左右),初生根形成期和根系生长期的取样部位为根系。

    RNeasy Plant Mini Kit及Plant Genomic DNA Kit购自QIAGEN公司;PrineScript @ RTase逆转录试剂盒、3′-Full RACE和5′-FullRACE试剂盒、LAtaq酶、rtaq酶均购自TaKaRa公司;载体构建使用的GATEWAY技术及相关试剂均来自Invitrogen公司。

  • 使用RNeasy Plant Mini Kit试剂盒提取中山杉406根总RNA,确保RNA浓度为1 ng·μL−1,并通过2%琼脂糖凝胶电泳检测RNA的完整性。使用PrineScript @ RTase逆转录试剂盒合成中山杉406根cDNA,根据前期不定根转录组数据,筛选到605 bp的目的基因ThSHR3的片段(CL8931.Contig2),通过PCR扩增验证目的基因序列,PCR扩增体系如下:cDNA模板1.0 μL,TaKaRa LA Taq (5 U·μL−1) 0.5 μL,10 × LA PCR Buffer (Mg 2+ Free) 5.0 μL,MgCl2 (25 mmol·L−1)5.0 μL,dNTP Mixture(各2.5 mmol·L−1)8.0 μL,正向引物(10 μmol·L−1) 2.0 μL,反向引物(10 μmol·L−1) 2.0 μL,加超纯水补至50.0 μL。PCR扩增程序为:94℃ 3 min;94℃ 30 s,56℃ 30 s,72℃ 2 min,35个循环;72℃ 10 min。进一步参照TaKaRa公司3′和5′-Full RACE试剂盒说明进行巢氏PCR扩增,完成ThSHR3基因全长克隆,其中,涉及到的引物参考(表1)。将扩增片段序列进行序列比对、拼接,最终获得了ThSHR3基因的全长cDNA序列。利用BioXM软件预测ThSHR3基因的开放阅读框(ORF),进一步通过PCR验证。

    引物名称 
    Primer 
    引物序列(5′-3′)   
    Primer sequences (5′–3′)   
    功能  
    Function  
    扩增长度/bp
    Amplified fragment length
    3GSP1 AAGGTGTTCAATCAGAAGAGATTTA 3′-巢氏PCR引物1 1 998
    3GSP2 ATTAACTGCGAGTCATG 3′-巢氏PCR引物2
    5GSP1 TGAACGAATTGCATT 5′-巢氏PCR引物1 1 232
    5GSP2 TCAGATGCTTTGTAGTAGTTTGCTATG 5′-巢氏PCR引物2
    ThSHR3-qPCR F TGGAGGAGAGCTTTT 定量及半定量前引物 147
    ThSHR3-qPCR R CTCGCAGCCGCGCAG 定量及半定量后引物
    APRT-F TCCACAGGTTCTTGAATCGCT 内参基因前引物 106
    APRT-R TGACTTGAGCCTCATTCGCTC 内参基因后引物
    ThSHR3-ORF F ATGGATAGATTGTTTACCTCCAG 编码阅读框前引物 1 446
    ThSHR3-ORF R CAAGCAAGGCTTCCAGGCGGAA 编码阅读框后引物

    Table 1.  Primer sequences

  • 利用美国国家生物信息中心(NCBI)在线BLAST软件比对分析ThSHR3的DNA和蛋白质序列。使用在线程序Expasy Protparam计算蛋白质的理论等电点(pI)、分子量(MW)和氨基酸组成。通过PROSITE以及GORIV二级结构检测程序分别预测蛋白质的结构域及二级结构。使用ClustalX2软件将ThSHR3的蛋白序列与其他植物已公布的SHR蛋白序列进行序列多重比对。利用NLStradamus程序预测ThSHR3蛋白是否具有核定位信号。通过MEGA 7.0软件,选用最大似然法构建系统发育树,自举检测1 000次。

  • 以中山杉406不定根发育的4个时间点S0~S3为材料,根据已验证的cDNA序列设计定量引物(表1),以中山杉的APRT基因为内参基因[19],采用半定量PCR和荧光定量PCR分别对ThSHR3进行表达分析检测。半定量PCR反应体系及程序参照rtaq酶说明书(https://www.takarabiomed.com.cn/)。选择Analitik Jena qTOWER2.2 PCR系统(Biometra,德国)进行荧光定量RT-PCR(qPCR)。反应程序设定为:50℃ 2 min;95℃ 10 min;40个循环:95℃ 15 s,60℃ 1 min;通过从60℃到95℃加热扩增产物,获得熔解曲线。反应体系共20 μL,其中包括2 μL稀释后的cDNA,10 μL FastStart Universal SYBR Green Master(Rox,德国),10 pmol正向引物,10 pmol反向引物以及灭菌去离子水,每个样品设3次技术重复,采用2−ΔΔCT 法进行相对定量的分析[20]

  • 表达的载体构建利用Gateway Technology(Invitrogen,美国)技术,先将不包含终止密码子的ThSHR3 ORF序列连接到入门载体pCR8/GW/TOPO上,再使用LR酶将入门载体连接到包含绿色荧光蛋白(GFP)标签的目的载体p2GWF7上。同理,使用相同的方法构建ThSHR3及ThSCR的蛋白互作载体,目的载体为含有黄色荧光蛋白(YFP)标签的pUC-SPYNE、pUC-SPYCE。最终重组表达载体有:35S::ThSHR3-GFP、ThSHR3-YFPN、ThSHR3-YFPC、ThSCR-YFPN、ThSCR-YFPC。利用PEG介导转化法将测序正确的融合表达载体导入杨树叶肉原生质体中[21]。利用BX51荧光显微镜(Olympus,日本)观测样品中荧光表达情况。

2.   结果与分析
  • 根据中山杉不定根发育的转录组及蛋白组数据,利用RACE巢式PCR扩增,拼接得到ThSHR3基因的全长序列。结果表明:ThSHR3基因的cDNA全长为2 019 bp,包含1个1 446 bp的开放阅读框(ORF),5′端非编码翻译区(UTR)长度为354 bp,3′端非编码翻译区(UTR)长度为219 bp。ThSHR3基因编码482个氨基酸残基。Ex-pasyProtparma预测ThSHR3分子量和等电点分别为66.082和5.17。GOR IV的分析结果显示:ThSHR3含有30.03%α螺旋(Hh),15.77%延伸链(Ee),54.19%的无规则卷曲(Cc),不含有beta转角(Tt)。NLStradamus程序预测ThSHR3蛋白主要定位在细胞核上。Prosite分析表明:ThSHR3蛋白具有保守的GRAS结构域。

  • 将ThSHR3的蛋白质序列与TAIR中的拟南芥AtSHR(At4g37650.1)蛋白序列和杨树PeSHR1、 PeSHR2、PeSHR3蛋白序列[22],中山杉ThSHR1(MF045148)、ThSHR2(MF045149)蛋白序列进行多重序列比对。结果表明:ThSHR蛋白的N端不保守,而C端相对比较保守,和其他物种的SHR蛋白一样,ThSHR蛋白包括GRAS家族成员特有的LHRI、VHIID、LHRII、PFYRE和SAW基础序列(图1)。

    Figure 1.  Homologous sequence alignment of ThSHR3 protein

    将ThSHR3的氨基酸序列与其他物种中已公布的45个较典型的GRAS蛋白的氨基酸序列进行系统进化树分析。结果表明:GRAS蛋白家族可划分为具有不同的特征的SHR、DELLA、PATl、SCL9、SCR、LAS/SCL18、SCL4/7、HAM的8个分支[9]图2)。ThSHR3被划分到SHR分支,和其他物种的SHR蛋白聚为一类,ThSHR3和中山杉ThSHR1亲缘关系最近,同源性均为81%,和松科植物PmSHR(QCU71495.1)及辐射松PrSHR(ABW20412.1)的亲缘关系也较近(图2)。

    Figure 2.  Phylogenetic tree based on ThSHR3 protein sequence

  • 本研究利用半定量PCR和荧光定量PCR方法,检测ThSHR3基因在中山杉406不定根皮层休眠期、愈伤组织形成期、初生根形成期和根系生长期4个时期的动态表达情况,内参基因均为APRT基因。结果表明:ThSHR3基因在中山杉不定根4个不同发育阶段均有表达。ThSHR3表达呈现持续上升的表达模式,在根系生长期表达量最高,在皮层休眠期表达量最低,最高表达量是最低表达量的28.4倍,且半定量PCR和荧光定量PCR结果基本一致(图3)。

    Figure 3.  Expression pattern of ThSHR3 gene in adventitious root formation in Taxodium hybrid ‘Zhongshanshan 406’ at different developmental time points

  • 本研究以绿色荧光蛋白作为报告基因,在前期已经成熟的杨树叶肉原生质体瞬时表达体系的基础上[21],将中山杉重组融合表达载体导入杨树叶肉原生质体中。经过18 h,23℃暗培养,观察可得GFP标签在488 nm蓝光激发下产生了509 nm的绿色荧光,阳性对照35S::GFP在细胞核、细胞质、细胞膜等区域中都产生了明显的绿色荧光信号,35S::ThSHR3-GFP融合蛋白仅在细胞核区域产生绿色荧光,表明中山杉ThSHR3基因所编码的蛋白质定位于细胞核,这和前期在线程序预测结果一致,也符合其作为转录因子的特性(图4)。

    Figure 4.  Subcellular-localization of ThSHR3 in Populus mesophyll protoplasts

  • 双分子荧光互补(BiFC)是目前用于检测细胞体内蛋白质互作的一项成熟技术,本研究利用BiFC技术检测中山杉的ThSHR3与ThSCR蛋白的互作情况,前期已经证明ThSHR3及ThSCR亚细胞定位结果均定位在细胞核[23]。3次重复性实验结果显示:ThSHR3-YFPN与ThSCR-YFPC、ThSHR3-YFPC与ThSCR-YFPN这2个组合的YFP荧光蛋白重新恢复活性,在细胞核处观察到清晰的荧光信号,2组对照组合均无荧光信号产生(图5),表明ThSHR3与ThSCR在细胞核发生了互作。

    Figure 5.  ThSHR3 and ThSCR interaction by BiFC

3.   讨论
  • 根系是植物长期适应陆地生境而形成的重要器官,根系从土壤中吸收水分和矿质营养,并对植物起机械支撑作用[24]。2000年,Helariutta等首次在拟南芥根中发现了转录因子SHR[8],证明其参与根尖辐射形态建成,在基本组织细胞平周分裂形成过程中发挥着的重要作用,进而其他物种的SHR基因陆续被挖掘研究[25]。本研究成功克隆获得了中山杉406 ThSHR3基因全长。经过氨基酸序列比对和系统进化分析发现,ThSHR3和中山杉ThSHR1ThSHR2高度同源,和其他物种的SHR基因一起划分到SHR分支,且具有植物GRAS蛋白家族特有的结构域,因而推测ThSHR3可能参与中山杉406不定根的发育。

    木本模式植物杨树的SHR分支基因包含3个成员:PeSHR1PeSHR2PeSHR3,其表达存在着2种模式,PeSHR1PeSHR2基因表达量在根的发育过程中呈逐渐上升趋势,根发育到4 周时表达量最高,而PeSHR3基因在根中的表达模式为先上升后下降[22]。中山杉3个SHR基因的表达特性在不定根不同发育阶段是否也存在着差异?研究表明:ThSHR1呈逐渐上升的表达趋势,在根的生长期表达量最高,而ThSHR2表达量先上升再下降,在初生根形成期表达量最高[23],中山杉ThSHR3ThSHR1的表达趋势基本一致,且聚类分析发现二者的同源性较高,推测二者的功能可能更相近。SHR基因家族成员不同的表达模式,暗示着它们可能在根系发育过程中行使着不同的生物学功能,但也不排除它们之间存在功能冗余性,有待通过遗传转化等方法进行深入探究。

    染色体免疫共沉淀及酵母双杂交实验表明,拟南芥中SCR与SHR互作区域在LHRI-VHIID-LHRII基序之间[15]。水稻的SHR与SCR蛋白也存在着互作,然而水稻的2个SHR基因OsSHR1OsSHR2,仅发现OsSHR1可与OsSCR发生互作[11]。目前,关于SHR和SCR及其互作基因调控根部细胞不均等分裂的研究,主要集中在SHR/SCR/RBR/CYCD6-1-CDK这信号通路中。SHR/SCR/RBR/CYCD6-1-CDK是一条双稳信号回路,细胞周期蛋白CYCD6及其依赖性激酶1-CDK共同磷酸化RBR(RING Between RING)蛋白,进而影响SCR蛋白活力,同时CYCD6转录活性又受到SHR-SCR复合物共同的转录调控,此通路中SHR-SCR复合物的活性适中,才能保证根部细胞不均等分裂有序完成[15]。胥猛等利用BiFC技术对杨树SHR/SCR/RBR/CYCD6-1-CDK信号通路基因的互作进行研究,发现仅PeSHR1与PeSCR、PeSHR1与PeCYCD6、PeSCR与PeCYCD6三个组合发生了互作[2226]。中山杉SHR-SCR信号通路的研究尚未开展,本研究对中山杉中新挖掘出的ThSHR3进行了蛋白互作研究,BiFC实验发现中山杉ThSHR3可以和ThSCR发生明显互作,推测ThSHR3可能和ThSCR存在于同一信号通路中,通过形成ThSHR3/ThSCR复合体来行使转录功能;而ThSHR分支上的其它成员之间以及ThSHR1、ThSHR2与ThSCR的互作情况仍然未知,具体的作用机制仍需通过分子生物学手段进一步深入解析。

4.   结论
  • 本研究从中山杉406扦插苗不定根中克隆获得了ThSHR3基因,该基因编码的蛋白具有GRAS家族成员特有的保守结构域,且从属于SHR亚家族。ThSHR3基因在中山杉皮层休眠期、愈伤组织形成期、初生根形成期、根系生长期4个时期中呈逐渐上升的表达趋势。双分子荧光互补实验显示:ThSHR3蛋白和ThSCR蛋白存在着明显互作,而SHR-SCR信号通路在植物根系发育过程中发挥着关键调控作用,表明ThSHR3为中山杉不定根发育相关的重要转录因子,为进一步探究中山杉以及落羽杉属树木不定根发育的分子机理提供了理论基础。

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