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Volume 31 Issue 4
Jul.  2019
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Structure, Expression and Function Analysis of Class Ⅰ KNOX Genes in Populus

  • Corresponding author: SONG Xue-qin, xqsong@caf.ac.cn
  • Received Date: 2016-08-27
  • Objective The expression of Populus class Ⅰ KNOX genes during the regeneration of adventitious shoot and adventitious root as well as in the vascular cambium were analyzed to reveal the function of class Ⅰ KNOX genes in woody plants meristem formation and differentiation. Method The nucleic acid and amino acid sequence of Populus class Ⅰ KNOX genes were obtained through blast analysis using Arabidopsis STM protein sequence as query in the genome of Populus trichocarpa. The phylogenetic tree was constructed according to the full length protein sequences of class Ⅰ KNOX genes from Arabidopsis and Populus. The intron/exon structure and domain composition were presented along the phylogenetic tree. The regeneration of adventitious bud and adventitious root using leaf and stem explants from 84 K (Populus alba×P. glandulosa) was used to simulate the shoot and root apical meristem initiation and differentiation, respectively. Quantitative real-time PCR was carried out to analyze the expression of Populus class Ⅰ KNOX genes during the regeneration of adventitious bud/root and in the vascular cambium related region. Result Ten class Ⅰ KNOX genes were found in the genome of P. trichocarpa through sequence alignment analysis. According to the phylogenetic relationship and gene structure similarity, class Ⅰ KNOX genes from Arabidopsis and Populus could be divided into three groups. Arabidopsis KNAT2 and KNAT6 along with their Populus homolog genes belong to group 1, Arabidopsis STM and BP along with their Populus homolog genes belong to group 2. Group 3, to be noticed, was unique to Populus. Through investigating the expression alteration of Populus class Ⅰ KNOX genes during the regeneration of adventitious buds, it was found that group 1 genes showed increased expression during the transition from bud primordium to adventitious bud, while group 2 and group 3 genes demonstrated higher expression during the transition from meristem to bud primordium. As for the regeneration of adventitious root, group 1 genes showed increased expression in the stage when root primordium differentiate to adventitious root, while group 2 and group 3 genes demonstrated higher expression in the adventitious root formation stage. In addition, all Populus class Ⅰ KNOX genes had an expression in the vascular cambium, and the expression of group 1 gene PtKNAT2/6b and group 2 genes ARK1 and ARK2 were especially high. Conclusion Group 3 is a new group occurred during the evolution of class Ⅰ KNOX genes from A. thaliana to P. trichocarpa, which was along with group 1 and group 2 to participate in the regulation of different stages of meristem formation and differentiation. Most importantly, PtKNAT2/6b, ARK1 and ARK2 show high expression in the vascular cambium, which may play important roles in vascular cambium activity maintenance and xylem differentiation.
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    Kerstetter R, Vollbrecht E, Lowe B, et al. Sequence analysis and expression patterns divide the maize Knotted1-like homeobox genes into two classes[J]. Plant Cell, 1994, 6(12):1877-1887. doi: 10.1105/tpc.6.12.1877
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    Laura Ragni, Enric Belles-Boix, Markus G. Interaction of KNAT6 and KNAT2 with BREVIPEDICELLUS and PENNYWISE in Arabidopsis inflorescences[J]. Plant Cell, 2008, 20(4):888-900. doi: 10.1105/tpc.108.058230
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    Li EY, Bhargava A, Qiang W Y, et al. The class Ⅱ KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus[J].New Phytologist, 2012, 194(1):102-115. doi: 10.1111/j.1469-8137.2011.04016.x
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    Liu Lijun, Matthew Zinkgraf, Andrew Groover. The Populus ARBORKNOX1 homeodomain transcription factor regulates woody growth through binding to evolutionarily conserved target genes of diverse function[J]. New Phytologist, 2015, 205(2):682-694. doi: 10.1111/nph.13151
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    Andrew T. Groover, Shawn D. Mansfield, et al. The Populus homeobox gene ARBORKNOX1 reveals overlapping mechanisms regulating the shoot apical meristem and the vascular cambium[J]. Plant Molecular Biology, 2006, 61(6):917-932. doi: 10.1007/s11103-006-0059-y
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Structure, Expression and Function Analysis of Class Ⅰ KNOX Genes in Populus

    Corresponding author: SONG Xue-qin, xqsong@caf.ac.cn
  • State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

Abstract:  Objective The expression of Populus class Ⅰ KNOX genes during the regeneration of adventitious shoot and adventitious root as well as in the vascular cambium were analyzed to reveal the function of class Ⅰ KNOX genes in woody plants meristem formation and differentiation. Method The nucleic acid and amino acid sequence of Populus class Ⅰ KNOX genes were obtained through blast analysis using Arabidopsis STM protein sequence as query in the genome of Populus trichocarpa. The phylogenetic tree was constructed according to the full length protein sequences of class Ⅰ KNOX genes from Arabidopsis and Populus. The intron/exon structure and domain composition were presented along the phylogenetic tree. The regeneration of adventitious bud and adventitious root using leaf and stem explants from 84 K (Populus alba×P. glandulosa) was used to simulate the shoot and root apical meristem initiation and differentiation, respectively. Quantitative real-time PCR was carried out to analyze the expression of Populus class Ⅰ KNOX genes during the regeneration of adventitious bud/root and in the vascular cambium related region. Result Ten class Ⅰ KNOX genes were found in the genome of P. trichocarpa through sequence alignment analysis. According to the phylogenetic relationship and gene structure similarity, class Ⅰ KNOX genes from Arabidopsis and Populus could be divided into three groups. Arabidopsis KNAT2 and KNAT6 along with their Populus homolog genes belong to group 1, Arabidopsis STM and BP along with their Populus homolog genes belong to group 2. Group 3, to be noticed, was unique to Populus. Through investigating the expression alteration of Populus class Ⅰ KNOX genes during the regeneration of adventitious buds, it was found that group 1 genes showed increased expression during the transition from bud primordium to adventitious bud, while group 2 and group 3 genes demonstrated higher expression during the transition from meristem to bud primordium. As for the regeneration of adventitious root, group 1 genes showed increased expression in the stage when root primordium differentiate to adventitious root, while group 2 and group 3 genes demonstrated higher expression in the adventitious root formation stage. In addition, all Populus class Ⅰ KNOX genes had an expression in the vascular cambium, and the expression of group 1 gene PtKNAT2/6b and group 2 genes ARK1 and ARK2 were especially high. Conclusion Group 3 is a new group occurred during the evolution of class Ⅰ KNOX genes from A. thaliana to P. trichocarpa, which was along with group 1 and group 2 to participate in the regulation of different stages of meristem formation and differentiation. Most importantly, PtKNAT2/6b, ARK1 and ARK2 show high expression in the vascular cambium, which may play important roles in vascular cambium activity maintenance and xylem differentiation.

  • TALE(Three Amino Acid Loop Extension)蛋白因其在第一和第二螺旋结构中含有3个额外的氨基酸而得命,存在于所有的真核生物中,植物中TALE家族包含2个亚家族:KNOX(KNOTTED-like homeobox)和BELL(BELL-like homeobox)[1]。植物KNOX家族中首次被克隆得到的成员是玉米(Zea mays L.)中的Knotted1(KN1)基因,因其突变体叶片上产生结状物凸起而得名[2]。随着拟南芥(Arabidopsis thaliana L.)、烟草(Nicotiana tabacum L.)、水稻(Oryza sativa L.)等植物中KN1同源基因被克隆,这类基因被归为KNOX家族。根据基因结构与表达模式方面的差异,KNOX基因家族被分为2个亚类:Ⅰ类KNOX和Ⅱ类KNOX[1]

    拟南芥中Ⅰ类KNOX包含4个成员,分别是:SHOOTMERISTEMLESS(STM)、BREVIPEDICELLUS(BP)、KNOTTED-like from Arabidopsis thaliana 2(KNAT2)以及KNAT6[3]。Ⅰ类KNOX主要参与顶端分生组织(SAM)建成与功能维持过程,同时也参与调控侧生器官的形态发生[4]STM主要在SAM中表达,其主要通过调控赤霉素与细胞分裂素的合成和降解途径,维持SAM中高细胞分裂素浓度和较低赤霉素含量,从而维持SAM中持续分裂分化的能力,stm突变体表现为SAM缺失[5]KNAT2主要参与心皮的形成,而与KNAT2结构极为相似的KNAT6则参与调控侧根的形态建成过程。另外,KNAT2与STM在胚胎分生组织功能维持及边界建立中存在功能上的冗余[6]BPSTM共同参与SAM建成过程的调控,但BP除了影响SAM建成外还调控花序轴的形态建成,bp突变体中花梗与花序轴之间的夹角显著大于野生型,而knat2knat6双突变能够使bp突变体花梗形态向野生型恢复,因此,在花序生长过程中,BP通过抑制KNAT2和KNAT6表达,从而保证花序轴上花梗正常的形态发生[7]

    拟南芥中Ⅱ类KNOX基因包括4个成员,分别是:KNOTTED-like from Arabidopsis thaliana 3(KNAT3)、KNAT4KNAT5以及KNAT7,Ⅱ类KNOX基因表达模式相对广泛,且功能多样[8]KNAT3KNAT4负调控侧根形成,KNAT7则与BELL家族的BLH6形成二聚体进而调控次生细胞壁形成[9]。拟南芥中已证明在过表达Ⅱ类KNOX基因后,Ⅱ类KNOX与BELL家族成员选择性结合形成二聚体,抑制SAM活性,同时影响叶片形态建成[10]

    综上所述,Ⅰ类KNOX在植物分生组织建成及功能维持中起关键作用,是植物分生组织启动和器官发生的重要调控因子。但目前杨树中仅完成了对拟南芥STMBP同源基因ARBORKNOX1(ARK1)、ARBORKNOX2(ARK2)功能的初步研究,认为它们参与调控木本植物形成层的分化,而杨树中Ⅰ类KNOX其他成员的功能尚不清楚[11-12]。本文对拟南芥与杨树中Ⅰ类KNOX成员的蛋白结构、进化关系等进行比较分析,同时利用RT-PCR方法分析杨树Ⅰ类KNOX基因在不定芽、不定根形成过程中的表达,探讨杨树Ⅰ类KNOX在分生组织建成及器官分化过程中的作用,为杨树Ⅰ类KNOX基因的功能解析提供参考。

1.   材料与方法
  • 本实验所用实验材料均取自84K。

  • 选取处于相同生长状态的84K组培苗的第3片展开叶,将叶片主叶脉切断,置于分化培养基,诱导不定芽的发生。分别在0、2、4、6、8、10、14、16、18、20 d截取叶片伤口部位及后期诱导产生的不定芽。取材时间固定在11:00 am,所取材料均置于液氮中保存。

  • 转接相同生长状态的84K组培苗于生根培养基中,分别在0、2、3、4、5、6、7、8、15、17 d截取茎段伤口部位及后期诱导产生的不定根。取材时间固定在11:00 am,取材后将样品置于液氮中保存。

  • 将3年生84K剥皮,剥皮后在树皮上轻轻划取薄薄的一层松软组织,主要包含了形成层组织及未成熟木质部,并迅速将其置于液氮中保存。

  • 以拟南芥Ⅰ类KNOX基因STM的蛋白序列在毛果杨基因组(POPGENIE, http://popgenie.org)中进行BLAST分析,获取杨树Ⅰ类KNOX基因的核酸及氨基酸序列;通过Clustalw2.0软件对拟南芥和杨树的Ⅰ类KNOX基因的氨基酸序列进行比对分析。利用Mega6.0软件采用基于遗传距离的邻近法构建拟南芥和杨树的Ⅰ类KNOX基因的系统进化树。根据拟南芥和杨树的Ⅰ类KNOX基因序列信息,通过GSDS2.0(http://gsds1.cbi.pku.edu.cn/)在线分析杨树Ⅰ类KNOX基因的内含子与外显子结构,并绘制基因结构图。

  • 不定芽与不定根形成过程中不同时期的RNA提取与第一链cDNA合成分别采用RNeasy Plant Mini Kit(QIAGEN)与PrimeScriptTM RT Reagent Kit(TaKaRa)完成,具体方法参见试剂盒使用说明。qRT-PCR采用SYBR Premix Ex TaqTM Ⅱ Kit(TaKaRa)在Roche LightCycle 480 Ⅱ仪器上完成。杨树Ⅰ类KNOX基因定量引物(表 1)使用在线引物设计软件Primer3(http://frodo.wi.mit.edu/primer3/input.htm)设计得到。

    引物名称
    Primer Name
    引物序列
    Primer Sequence
    ARK1-rtF ATTGGTGGAGCAGGCATTAC
    ARK1-rtR CATCCATCACCACAAACTGC
    ARK2-rtF TGGACTGCCAAAAGGTAGGA
    ARK2-rtR GTCTTGTAAGCTCCTCACGGTA
    PtSTMb-rtF TTCTGCTCATCAGCATCACC
    PtSTMb-rtR CCAGTCGCAGTGACAGTGTT
    PtKNAT2/6a-rtF CGCAGATTGCACGTTTCTTA
    PtKNAT2/6a-rtR AGGCCTTTCAAGATCGGATT
    PtKNAT2/6b-rtF CCTACTTGGATGGTGGGATG
    PtKNAT2/6b-rtR CAGCAAATTGCAGGTTCTCA
    PtKNAT2/6c-rtF AACGAGGATCGAGAGCTGAA
    PtKNAT2/6c-rtR ATCAGCTTCCGTTGGGTATG
    PtKNAT2/6d-rtF GCTTCAATGGTGGTGGAGTT
    PtKNAT2/6d-rtR ATCGTTCTCTTCCCGGATTT
    PtKNLPa-rtF GTGCACCTCCAGAAATGGTT
    PtKNLPa-rtR CGTCAAACGGCTTGGATAAT
    PtKNLPb-rtF CTTGAAGCCATAGGCAGAGG
    PtKNLPb-rtR TCAAGAACGTAGCAGCCTCA
    PtKNLPc-rtF GGGAGGTTGAAGCATCTGAA
    PtKNLPc-rtR TCGCTTTCTCCTCTTCCGTA
    PtACTIN-rtF AAACTGTAATGGTCCTCCCTCCG
    PtACTIN-rtR GCATCATCACAATCACTCTCCGA

    Table 1.  Primers used for qRT-PCR analysis.

2.   结果与分析
  • 以拟南芥STM蛋白序列在毛果杨基因组中进行同源序列比对,得到杨树10个Ⅰ类KNOX基因。利用Mega6.0软件构建了杨树和拟南芥Ⅰ类KNOX基因的系统进化树(图 1A),并根据系统进化树中两物种间相对应的基因关系,对杨树中Ⅰ类KNOX成员进行命名,其中,2个与AtSTM同源的基因,分别命名为PtSTMaPtSTMbPtSTMa即为之前报道的杨树ARK1基因[11];与AtBP同源的基因仅1个,命名为PtBPPtBP与之前报道的杨树ARK2基因为同一基因[12];与AtKNAT2AtKNAT6同源的基因有4个,分别命名为PtKNAT2/6aPtKNAT2/6bPtKNAT2/6cPtKNAT2/6d;另外,还有3个基因没有与拟南芥相对应的同源基因,将其顺次命名为KNOTTED-like from Populus a(PtKNLPa)、PtKNLPbPtKNLPc。同时参考基因结构图(图 1B),将杨树中Ⅰ类KNOX分为3组:组1、组2、组3。组1为KNAT2KNAT6类基因,组2为STMBP类基因,组3为杨树所特有的Ⅰ类KNOX基因。

    Figure 1.  Phylogenetic tree and gene structure of Class Ⅰ KNOX from Arabidopsis and Populus

    杨树中多数Ⅰ类KNOX基因长度在3 kb以上,均长于拟南芥中Ⅰ类KNOX基因。组1成员中,PtKNAT2/6a包含6个外显子,比同组中的其它基因多1个外显子。组2基因中,ARK1PtSTMb含3个内含子,而同组中的其他基因均含有4个内含子(图 1B表 2)。这些基因内含子、外显子结构的变化表明了在杨树Ⅰ类KNOX进化过程中发生了外显子获得与内含子丢失事件。

    基因名称
    Gene name
    基因号
    Locus
    染色体上位置
    Genomic position
    亚细胞定位预测
    POSRT prediction
    氨基酸数
    Protein length
    内含子数
    introns
    ARK1 Potri.011G011100.1 Chr11:844486-848858- N: 14 373 3
    ARK2 Potri.002G113300.1 Chr02:8461980-8468320 + N: 14 368 4
    PtSTMb Potri.004G004700.1 Chr04:304576-308944 + N: 14 369 3
    PtKNAT2/6a Potri.008G188700.1 Chr08:13039428-13047773 - N: 12, C: 1 341 5
    PtKNAT2/6b Potri.010G043500.1 Chr10:7430322-7440628 + N: 10, C: 2 309 4
    PtKNAT2/6c Potri.012G087100.1 Chr12:11401140-11409183 - N: 14 340 4
    PtKNAT2/6d Potri.015G079100.1 Chr15:10400171-10408171 + N: 14 347 4
    PtKNLPa Potri.013G008600.1 Chr13:557144-561807 + N: 12, M: 1 320 4
    PtKNLPb Potri.005G014200.1 Chr05:1100485-1105673 + N: 14 317 4
    PtKNLPc Potri.005G017200.1 Chr05:1390283-1394969 + N: 13 316 4
    注:N:细胞核;C:细胞质;Ch:叶绿体;M:线粒体。
    N: nucleus: C: cytoplasm; Ch: chloroplast; M: mitochondria.

    Table 2.  Detailed information about Class Ⅰ KNOX genes from Populus

    KNOX家族成员由MEINOX、ELK与homebox KN保守结构域组成,MEINOX结构域位于KNOX蛋白N端,由KNOX1和KNOX2两个亚结构域组成。通过多重序列比对及Ⅰ类KNOX蛋白质结构域分析(图 1B)发现,杨树与拟南芥中Ⅰ类KNOX蛋白结构相似,均含有以上4个结构域:KNOX1、KNOX2、ELK和homebox KN结构域,说明Ⅰ类KNOX进化历程中蛋白质结构相对保守。

  • 不定芽与不定根的再生过程能够模拟SAM与RAM的发育过程,根据杨树不定芽与不定根再生过程中形态学观察(图 2),将此过程分三个阶段:分生组织形成阶段、芽(根)原基形成阶段、不定芽(根)形成阶段,其中,不定芽再生过程中06 d为分生组织形成阶段(图 2A),7~11 d为芽原基形成阶段(图 2B),12~20 d为不定芽形成阶段(图 2C);在不定根再生过程中04 d为分生组织形成阶段(图 2D),5~6 d为根原基形成阶段(图 2E),7~17 d为不定根形成阶段(图 2F)。

    Figure 2.  Regeneration of adventitious shoots and adventitious roots

    不定芽发生过程中,杨树Ⅰ类KNOX成员的表达量在不定芽形成的3个阶段中均表现出短暂的上调趋势,但上调时间与上调幅度存在差异,因此,将总的变化趋势分为以下3种(图 3):(1)芽原基形成时期表达量出现上调趋势,此类基因包括组1的PtKNAT2/6d、组2的ARK1PtSTMb及组3的PtKNLPaPtKNLPb(图 3A);(2)组1的PtKNAT2/6a表达量在芽原基分化产生不定芽的关键时期特异上调,而在不定芽发生过程的其他阶段表达量较低且无显著变化(图 3B);(3)不定芽发育前期表达量较低且变化不大,而在不定芽形成后表达量多次呈现上调的变化趋势,此类基因主要有组1的PtKNAT2/6bPtKNAT2/6c、组2的ARK2与组3的PtKNLPc(图 3C)。

    Figure 3.  Expression alteration of Class Ⅰ KNOX members during the regeneration of adventitious shoot

    在不定根的形成过程中,根据Ⅰ类KNOX基因表达分析,可总结为2种变化趋势(图 4):(1)表达量在根原基分化产生不定根的关键期出现短暂的上调,包括组1的PtKNAT2/6aPtKNAT2/6bPtKNAT2/6d和组3的PtKNLPb(图 4A);(2)表达量在分生组织与根原基形成阶段不断下调,而在不定根形成后期呈现上调的趋势,包括组1的PtKNAT2/6c、组2的ARK1ARK2PtSTMb和组3的PtKNLPaPtKNLPc(图 4B)。

    Figure 4.  Expression alteration of Class Ⅰ KNOX members during the regeneration of adventitious root

    为考察杨树Ⅰ类KNOX基因与形成层这一木本植物特有的分生组织之间的关系,对杨树Ⅰ类KNOX基因在形成层区域(形成层+未成熟木质部)中的表达量进行了检测,结果(图 5)显示:Ⅰ类KNOX的成员在形成层区域中均有一定的表达,其中,PtKNAT2/6bARK2ARK1表达量明显高于其他成员,表明PtKNAT2/6b、ARK2及ARK1可能参与调控形成层活动或木质部分化过程。上述结果与ARK1ARK2调控形成层细胞分化活性的结论相一致[11-12]

    Figure 5.  Expression level of Class Ⅰ KNOX members in the cambium of populus

3.   讨论
  • 本文对杨树Ⅰ类KNOX的10个成员进行了分析,包括系统进化树构建、基因与蛋白质结构及其在分生组织中的表达等。通过蛋白质结构分析发现,杨树中所有Ⅰ类KNOX成员蛋白结构相对保守,均由KNOX1、KNOX2、ELK与homebox KN 4个保守结构域组成[13]。由杨树与拟南芥Ⅰ类KNOX基因蛋白结构的相似性可知,Ⅰ类KNOX在功能上可能具有高度保守性[14-15]。依据进化关系及基因结构将杨树Ⅰ类KNOX成员分为3个组:组1、组2、组3。拟南芥Ⅰ类KNOX成员包含组1和组2,而杨树中除了包含与拟南芥中相对应的组1和组2外,还包括进化关系较远的组3。组3的产生表明在杨树进化过程中KNOX家族出现了基因扩张,这种扩张可能为KNOX基因参与调控杨树不同分生组织的分化提供了更多选择。

    木本植物Ⅰ类KNOX的研究较匮乏,在杨树中仅完成了ARK1ARK2功能的初步研究。Andrew等首次在杨树中克隆得到拟南芥STM的同源基因ARK1,发现ARK1主要在形成层区域表达,并通过对ARK1转基因材料分析,发现ARK1参与调控杨树次生生长过程中形成层分化、次生壁合成过程[11]。Du等克隆了拟南芥BP的同源基因ARK2,通过原位杂交实验发现其在SAM和形成层区域特异表达,ARK2转基因杨树表型与组织学分析证明了ARK2参与调控形成层发育与木质部细胞的分化过程[12]。正如拟南芥中STMBP在SAM发育的调控中各有侧重,杨树中ARK1ARK2对形成层及木质部分化的调控同样存在差异。为分析杨树Ⅰ类KNOX成员的功能,本研究利用不定根和不定芽诱导体系模拟杨树顶端分生组织形态建成过程,发现杨树Ⅰ类KNOX不同组别在茎端或根端分生组织形成过程中表达量发生明显变化,且在茎和根两种不同的顶端发生过程的同一阶段的表达量不同。如不定芽形成过程中,组1基因多在发育的中后期芽原基分裂分化产生不定芽的过渡期高表达,而组2与组3成员则在芽原基形成阶段表达量明显提高,提示SAM形成过程中组1基因主要参与不定芽形成及侧生器官分化过程,而组2和组3基因则主要在分生组织分化产生芽原基的过程发挥作用。在不定根形成过程中,组1基因多在根原基分化产生不定根的过渡期高表达,组2与组3基因则多在发育后期不定根形成阶段上调表达,提示RAM形成过程中组1成员主要参与调控分生组织分化产生根原基的过程,组2和组3成员则侧重于发育后期不定根形态建成过程。

    由杨树Ⅰ类KNOX成员在形成层区域(形成层与未成熟的木质部)的表达分析,发现组1成员PtKNAT2/6b与组2成员ARK1ARK2表达量明显高于其他成员,并且结合以上成员在不定芽与不定根形成过程中的表达分析发现,ARK1ARK2主要参与原基形成及后期原基内分生组织细胞进一步分化产生不定芽(根)过程,这与之前ARK1ARK2参与调控形成层细胞分化活性的结论一致[11-12]PtKNAT2/6b在形成层相关区域表达量最高,与其在根原基分化产生不定根的关键时期高表达的结果一致,共同说明了PtKNAT2/6b可能在分生组织细胞分化过程中发挥作用。而与PtKNAT2/6b结构最相似的PtKNAT2/6a虽未在形成层相关区域表现出较高的表达量,但在不定芽与不定根形成过程中芽(根)原基分化产生不定芽(根)的关键时期均表现出特异的上调,提示PtKNAT2/6a可能与芽(根)原基分化功能的开启有关。组3作为杨树特有的Ⅰ类KNOX,其成员均在形成层相关区域表达,结合在不定芽(根)发生过程中组3成员的特异表达,推测PtKNLPaPtKNLPbPtKNLPc也参与顶端分生组织发生过程中不同阶段的调控,可知杨树Ⅰ类KNOX进化过程中,通过增加其特有的KNOX成员对分生组织的分化进行调控。另外,拟南芥Ⅰ类KNOX基因成员之间还存在复杂的调控关系,如BP通过抑制KNAT2KNAT6的表达,从而调控花序轴上花梗正常的形态发生[7]。杨树Ⅰ类KNOX成员在不定根和不定芽诱导发生的不同阶段出现动态、差异的表达,成员之间可能存在着调控关系,例如BP的同源基因ARK2KNAT2的同源基因PtKNAT2/6b之间是否存在与拟南芥中同样的调控关系,尚需要进一步研究。

4.   结论
  • 本研究确认了杨树中10个Ⅰ类KNOX成员,通过进化树构建及基因结构分析将其分为3组,根据其在不定根与不定芽发生过程及其在形成层区域的表达分析,推测杨树Ⅰ类KNOX不同成员在茎顶端、根顶端及形成层等不同分生组织分化中的作用,为深入研究木本植物中Ⅰ类KNOX的功能提供了线索,为揭示它们在木本植物生长、发育中的作用,特别是木材形成中的调控机制奠定了基础。

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