【作者】 陈坤；

【导师】 姜静；

【作者基本信息】 东北林业大学 ， 林学， 2024， 博士

【摘要】 PIN-FORMED（PIN）蛋白是负责将生长素（Auxin）从细胞中外排的次级转运蛋白,在细胞内呈不对称性分布,对植物器官形成和向性生长等发育过程中生长素的分布起重要作用。团队前期研究发现,白桦BpPIN3基因对叶缘发育至关重要,但其作用机制尚不知晓。为此,本研究以BpPIN3过表达、抑制表达和基因编辑株系为材料,采用组织解剖观察、内源生长素IAA的组织定位观察及含量测定等实验方法,探讨BpPIN3基因在叶片形态建成及不定根发育过程中的作用,为后续白桦分子设计育种奠定基础。主要研究结果如下:（1）白桦BpPIN3组织表达特性和应答特性分析。以1年生以及2年生两个年份白桦无性系的根、茎、叶和顶芽为材料,q RT-PCR分析显示,2年生白桦BpPIN3在各组织表达量明显高于1年生白桦,其中,2年生白桦的第1叶片中BpPIN3的相对表达量最高。以白桦幼苗为材料,分别采用100μmol·L^-1生长素（IAA）、100μmol·L^-1赤霉素（GA₃）、200μmol·L^-1脱落酸（ABA）及避光处理下,q RT-PCR分析显示,叶片组织中IAA、GA₃及避光处理均可诱导白桦叶片组织中BpPIN3上调表达;在根组织中,上述4种处理12 h后均能诱导BpPIN3的上调表达。结果表明,BpPIN3在白桦生长发育过程及IAA、GA₃和ABA信号转导途径和光响应过程中发挥重要调控作用。（2）BpPIN3转基因株系不定根的变异研究。采用农杆菌介导法获得白桦BpPIN3基因编辑株系（bppin3）,进而,以前期获得的BpPIN3过表达、抑制表达及bppin3为材料,对不定根形态特征、根的向地性及生物量等进行分析,结果显示,BpPIN3过表达株系主根变长,BpPIN3抑制表达株系主根变短,而bppin3株系的主根长度与WT株系相比无显著变化。此外,抑制表达株系和bppin3株系侧根密度增加,同时向地性缺失。生长素的组织定位分析显示,与WT株系相比,抑制表达株系生长素集中于根尖分生区,其伸长区以及成熟区的生长素分布减少。（3）BpPIN3转基因株系高生长和分枝性状的变异。以BpPIN3转基因白桦为材料,开展生长性状观察、内源激素及RNA-seq测定,结果显示,与WT株系相比,BpPIN3过表达和抑制表达株系速生期生长量及相对高生长量均显著增高,过表达株系分枝数明显增加。内源激素测定显示,顶芽中BpPIN3过表达株系IAA含量降低,CKs含量增加;而抑制表达株系IAA含量增加,CKs含量减少,腋芽中BpPIN3过表达株系IAA含量降低,抑制表达株系IAA含量增加。RNA-seq分析显示,过表达植株中IAA、细胞分裂素以及木质素相关基因的表达量发生了显著的变化。（4）BpPIN3抑制表达株系的叶片呈近轴面卷曲现象。叶型分析显示,与WT株系相比,BpPIN3过表达株系叶片形态无显著变化,而BpPIN3抑制表达株系叶片呈现出近轴面卷曲的现象。细胞观察显示,BpPIN3抑制表达株系叶片具有独特的形态特征和细胞结构,具体呈现为BpPIN3抑制表达株系叶缘区域栅栏组织细胞密度降低、排列松散。生长素含量测定显示,与WT株系相比,顶芽以及叶片中均呈现出BpPIN3过表达株系生长素减少,BpPIN3抑制表达株系生长素增加的现象。IAA定位分析发现,BpPIN3抑制表达株系叶缘区域IAA含量显著高于主叶脉区域,呈不对称分布趋势,同时BpPIN3抑制表达株系叶缘区域的栅栏组织细胞中有大量的生长素信号富集。（5）基于转录组和蛋白质组测序联合分析。测序结果显示,BpPIN3转基因株系中的差异表达蛋白和基因主要富集于光合通路、胁迫响应通路和激素信号转导途径中的IAA信号转导途径中。同时对表型相关差异基因互作分析发现BpPIN3与多个基因存在潜在的互作关系。综上所述,BpPIN3作为生长素运输载体,在白桦生长发育过程中发挥着重要作用,其表达量的变化影响着生长素的分布,生长素含量的变化进而影响着其它激素以及激素信号转导相关基因的变化,从而导致生长发育的变异。研究结果进一步揭示PIN3基因的功能提供重要参考。更多还原

【Abstract】 PIN-FORMED（PIN）proteins are secondary transporters responsible for the efflux of auxin from cells.They are asymmetrically distributed in cells and play an important role in the distribution of auxin during plant organ formation and tropic growth.Previous studies have identified the essential role of the BpPIN3 gene in leaf margin development,but its mechanism of action remains unknown.Therefore,this study utilized BpPIN3 overexpression,suppresses expression,and CRISPR/Cas9 gene editing lines to investigate the role of the BpPIN3 gene in leaf morphogenesis and adventitious root development through tissue anatomy observation,endogenous IAA tissue localization,and content determination.The findings provide a foundation for subsequent molecular design breeding of Birch.The primary findings of the study are outlined below:（1）The tissue expression and response characteristics of BpPIN3 in birch were analyzed.Materials included roots,stems,leaves,and apical buds of one-year-old and two-year-old birch clones.q RT-PCR analysis revealed significantly higher expression of BpPIN3 in various tissues of two-year-old birch compared to one-year-old birch,with the highest relative expression observed in the first leaf of two-year-old birch.Birch seedlings were subjected to treatments with 100μmol·L^-1 auxin（IAA）,100μmol·L^-1 gibberellin（GA₃）,200μmol·L^-1abscisic acid（ABA）,and darkness.The q RT-PCR analysis indicated that IAA,GA3,and darkness in leaf tissue could induce the up-regulation of BpPIN3 expression,while in root tissue,all four treatments led to an up-regulation of BpPIN3 expression after 12 h.These results demonstrate the important regulatory role of BpPIN3 in the growth and development of birch,as well as in IAA,GA3,and ABA signal transduction pathways and light response processes.（2）The investigation focused on the variation of adventitious roots in BpPIN3 transgenic lines of birch.The BpPIN3 CRISPR/Cas9 lines（bppin3）was obtained through the Agrobacterium-mediated method.Subsequently,morphological characteristics,root geotropism,and root biomass of adventitious roots were analyzed using the BpPIN3overexpression,suppresses expression,and bppin3 lines obtained in the previous period.Results revealed that the primary root of the BpPIN3 overexpression lines displayed increased length,whereas the primary root of the BpPIN3 suppresses expression lines exhibited reduced length.Conversely,the main root length of the bppin3 line did not show significant changes compared to the WT line.Furthermore,the lateral root density increased in the suppresses expression lines and bppin3 lines,with the absence of geotropism.Tissue localization analysis of auxin indicated that,in comparison with WT lines,auxin in the suppresses expression lines was concentrated in the root apical meristem,and the distribution of auxin in the elongation zone and mature zone was decreased.（3）BpPIN3 transgenic lines and variations in growth and branching traits.BpPIN3transgenic birch served as the material for observing growth traits,determining endogenous hormones,and conducting RNA-seq analysis.The findings revealed that,in comparison to the WT lines,the rapid growth and relative height growth of BpPIN3 overexpression and suppresses expression lines were significantly increased,with a notable increase in the number of branches in overexpression lines.Analysis of endogenous hormones indicated a decrease in IAA content in apical buds of BpPIN3 overexpression lines and an increase in CKs content.Moreover,the IAA content decreased in the axillary buds of BpPIN3 overexpression lines,while it increased in the suppresses expression lines.Additionally,RNA-seq analysis demonstrated substantial changes in the expression levels of IAA,cytokinin,and lignin-related genes in the overexpressing plants.（4）Adaxial curling phenomenon in BpPIN3 suppresses expression lines.Leaf analysis indicates that,in comparison to the wild-type（WT）line,there is no substantial alteration in leaf morphology in the BpPIN3 overexpression lines.Conversely,the BpPIN3 suppresses expression lines exhibit adaxial curling of the leaves.Observation of the cells revealed distinctive morphological characteristics and cellular structure in the leaves of BpPIN3suppresses expression lines,as evidenced by reduced cell density and a loosely arranged palisade tissue at the leaf margin of these lines.Hormone content analysis highlights a reduction in auxin content in both the shoot apex and leaves of the BpPIN3 overexpression lines when compared to the WT line,whereas the BpPIN3 suppresses expression lines display an increase in auxin content.Hormone localization analysis uncovers a significant asymmetrical distribution trend,with higher IAA content in the leaf margin region of the BpPIN3 suppresses expression lines compared to the main leaf vein region.Additionally,there is a notable accumulation of auxin signals in the epidermal cells of the leaf margin region in the BpPIN3 suppresses expression lines.（5）Integrated analysis of transcriptome and proteome sequencing.The sequencing results indicate.The findings unveiled that differentially expressed proteins and genes within BpPIN3transgenic lines are predominantly enriched in pathways related to photosynthesis,stress response,and IAA signal transduction.Simultaneously,the analysis of phenotype-related differential gene interactions revealed that BpPIN3 potentially interacts with multiple genes.In conclusion,BpPIN3,serving as an auxin transport carrier,significantly influences the growth and development of birch.Alterations in BpPIN3 expression impact auxin distribution,consequently influencing other hormone levels and the expression of genes involved in hormone signal transduction,leading to variations in growth and development.The findings elucidate the regulatory role of BpPIN3 in auxin distribution and hormone signal transduction-related genes,offering valuable insights for further understanding the function of the PIN3gene in woody plants.更多还原