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作物学报 ›› 2022, Vol. 48 ›› Issue (2): 353-366.doi: 10.3724/SP.J.1006.2022.14006

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆PIN-Like (PILS)基因家族的鉴定、表达分析及在根瘤共生固氮过程中的功能

董衍坤1(), 黄定全2, 高震2, 陈栩2,*()   

  1. 1福建农林大学资源与环境学院, 福建福州 350002
    2福建农林大学海峡联合研究院园艺植物生物学及代谢组学研究中心, 福建福州350002
  • 收稿日期:2021-01-11 接受日期:2021-04-26 出版日期:2022-02-12 网络出版日期:2021-05-18
  • 通讯作者: 陈栩
  • 作者简介:E-mail: dongyk1124@163.com
  • 基金资助:
    本研究由国家重点研发计划“七大农作物育种”专项课题作物器官发育与养分高效利用的互作机制项目资助(2016YFD0100705)

Identification, expression profile of soybean PIN-Like (PILS) gene family and its function in symbiotic nitrogen fixation in root nodules

DONG Yan-Kun1(), HUANG Ding-Quan2, GAO Zhen2, CHEN Xu2,*()   

  1. 1College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2Haixia Institute of Science and Technology, Horticultural Plant Biology and Metabolomics Center, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2021-01-11 Accepted:2021-04-26 Published:2022-02-12 Published online:2021-05-18
  • Contact: CHEN Xu
  • Supported by:
    This study was supported by the National Key Research and Development Program of China “Seven Major Crop Breeding” Special Topic Crop Organ Development and Nutrient Efficient Use Interaction Mechanism(2016YFD0100705)

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摘要：

植物激素生长素在植物的生长发育过程中发挥了至关重要的作用, 它的稳态和浓度梯度建立控制了几乎所有器官的极性建成。生长素在特定细胞中合成、运输、感知以及代谢降解建立了符合器官发育的生长素浓度梯度。在豆科植物中, 根与土壤微生物互作形成了根瘤这一特殊的器官, 进行生物固氮。然而, 生长素稳态控制生物固氮的功能还未知。拟南芥中的研究表明, PIN-Like (PILS)蛋白协助调节的细胞内生长素稳态, 并介导下游细胞核内的生长素信号传递。本研究以大豆作为研究模型, 在大豆基因组中鉴定获得19个PILS家族基因(GmPILS), 不均匀分布于大豆10条染色体上。GmPILS在大豆9种组织部位中表现出多种表达模式, 且具有明显的组织表达特异性。GmPILS1e和GmPILS1f在根瘤菌体区域富集表达, 使用人工微RNA沉默(artificial microRNA interference, amiRNAi)下调GmPILS1e和GmPILS1f在根瘤的表达, 导致根瘤的固氮酶活性上升, 而过量表达GmPILS1f导致根瘤的固氮酶活性下降, 因此GmPILS1e和GmPILS1f可能参与大豆固氮酶活性的调节。这些结果为进一步解析大豆GmPILS家族基因的功能和作用机制奠定了基础, 同时也为结瘤固氮在农业育种中的应用提供了有价值的基因资源。

关键词: 大豆, PIN-Like (PILS)基因家族, 根瘤, 共生固氮

Abstract:

Plant hormone auxin plays a vital role in the growth and development of plants. Auxin homeostasis and concentration gradient establishment control the polar formation of almost all organs. The synthesis, transportation, perception, and metabolic degradation of auxin in specific cells establish a concentration gradient of auxin in accordance with organ development. In legumes, roots interact with soil microorganisms to form a special organ called nodules, which is used for biological nitrogen fixation. However, the function of auxin homeostasis control of biological nitrogen fixation is unknown. Studies showed that PIN-Like (PILS) proteins in Arabidopsis helped to regulate intracellular auxin homeostasis and mediate auxin signal transmission in the downstream nucleus. In this study, 19 PILS family genes (GmPILSs) were identified in soybean genome and distributed unevenly on 10 chromosomes of soybean. GmPILSs exhibited a variety of expression patterns in nine tissue parts of soybean, and had obvious specificity of tissue expression. GmPILS1e and GmPILS1f were enriched and expressed in the rhizobia region, and the expression of GmPILS1e and GmPILS1f in nodules was down-regulated by artificial microRNA interference (amiRNAi), resulting in the increase of nitrogenase activity in the nodules. However, the overexpression of GmPILS1f leaded to the decrease nitrogenase activity in root nodules, GmPILS1e and GmPILS1f might participate in the regulation of soybean nitrogenase activity. These results lay the foundation for further analysis of the function and mechanism of soybean GmPILS family genes, and also provide valuable genetic resources for the application of nodulation and nitrogen fixation in agricultural breeding.

Key words: Glycine max, PIN-Like (PILS) gene family, nodule, symbiotic nitrogen fixation

表1

GmPILS基因家族基本信息"

基因名称
Gene name		 基因ID
Gene ID		 氨基酸数量
No. of amino acids		 内含子数量
No. of introns		 5'-3'坐标
5'-3' corrdinates
GmPILS1a Glyma.10G189100 313 6 Chr10: 42227422-42231792
GmPILS1b Glyma.20G201600 400 10 Chr20: 43857079-43862374
GmPILS1c Glyma.10G189000 400 10 Chr10: 42220046-42225237
GmPILS1d Glyma.20G201700 259 8 Chr20: 43863679-43868413
GmPILS1e Glyma.07G113100 418 9 Chr07: 11724201-11741130
GmPILS1f Glyma.07G113200 418 11 Chr07: 11780634-11792223
GmPILS1g Glyma.03G113600 424 10 Chr03: 32066847-32080424
GmPILS1h Glyma.16G114900 297 6 Chr16: 25456379-25485759
GmPILS1i Glyma.11G088600 415 10 Chr11: 6695030-6700783
GmPILS1j Glyma.01G156200 415 9 Chr01: 49361352-49366442
GmPILS1k Glyma.09G195600 414 10 Chr09: 42022526-42029030
GmPILS1l Glyma.16G115500 414 9 Chr16: 25622503-25630816
GmPILS2a Glyma.09G116100 440 1 Chr09: 26549730-26551878
GmPILS2b Glyma.19G072900 445 1 Chr19: 26101897-26103958
GmPILS5a Glyma.11G087300 419 10 Chr11: 6547702-6555660
GmPILS5b Glyma.01G157700 419 10 Chr01: 49570825-49577993
GmPILS5c Glyma.09G196900 409 9 Chr09: 42160775-42168544
GmPILS6a Glyma.09G271100 414 10 Chr09: 48791789-48796501
GmPILS6b Glyma.18G218300 414 10 Chr18: 50525980-50531206

图1

GmPILS基因家族的基因结构 黄色框代表外显子, 黑色线条代表不同GmPILS基因的内含子。"

图2

GmPILS基因在大豆染色体中的分布"

图3

GmPILS、AtPILS和OsPILS蛋白序列的系统进化树"

图4

GmPILS基因组织表达模式 A: GmPILS基因组织表达模式热图, 从Phytozome数据库中获取GmPILS表达量数据(FPKM)。B: GmPILS1e、GmPILS1f、GmPILS1i、GmPILS1j和GmPILS5a基因组织表达模式热图, GmPILS表达量数据来自qPCP结果。方框内颜色显示大豆PILS基因表达水平。"

图5

GmPILS1e、GmPILS1f、GmPILS1i、GmPILS1j和GmPILS5a在根瘤中的组织化学定位 从左到右分别对应根瘤发育的起始、扩张发育和成熟阶段。标尺为200 µm。"

图6

GmPILS1e和GmPILS1f的亚细胞定位 A~D: 使用烟草叶片进行pro35S:GFP-GmPILS (绿色)或pro35S:GmPILS-GFP (绿色)和pro35S:HDEL-Tdtoamto (红色, 作为内质网定位标记)混合菌液的共转化。基于白色斜线生成了共定位信号轮廓图。标尺为10 μm。"

图7

GmPILS1e和GmPILS1f参与大豆根瘤的固氮酶活性调控 A~G: 在接种根瘤菌21 d 后, 对结瘤的大豆复合植株进行表型分析。A: Mock、GmPILS1e 1f-RNAi#1和GmPILS1e 1f-RNAi#2毛根根瘤对半横切的拍照结果, 标尺为200 µm。B: 利用荧光定量PCR检测Mock、GmPILS1e 1f-RNAi#1和GmPILS1e 1f-RNAi#2毛根根瘤的表达, 以GmELF1b基因作为内参基因。C, D: 检测相同重量Mock、GmPILS1e 1f-RNAi#1和GmPILS1e 1f-RNAi#2毛根根瘤的固氮酶活性。E: 统计Mock, GmPILS1e 1f-RNAi#1和GmPILS1e 1f-RNAi#2毛根根瘤横切的菌体区域的面积占比。F: 利用荧光定量PCR检测Mock和35S::GmPILS1f 毛根根瘤的表达, 以GmELF1b基因作为内参基因。G: 检测Mock和35S::GmPILS1f 毛根根瘤的固氮酶活性。*、**、***分别表示在0.05、0.01和0.001水平差异显著; ns: 无显著性差异。"

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