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林业科学 ›› 2020, Vol. 56 ›› Issue (3): 184-192.doi: 10.11707/j.1001-7488.20200319

• 研究简报 • 上一篇    下一篇

马尾松PmPIN1基因的克隆及功能分析

武星,胡兴峰,陈佩珍,孙晓波,吴帆,季孔庶*   

  1. 南京林业大学 林木遗传与生物技术省部共建教育部重点实验室 南方现代林业协同创新中心 南京 210037
  • 收稿日期:2019-03-12 出版日期:2020-03-01 发布日期:2020-04-08
  • 通讯作者: 季孔庶
  • 基金资助:
    “十三五”国家重点研发计划项目课题(2017YFD0600304);江苏高校优势学科建设工程(PAPD)

Cloning and Functional Analysis of PmPIN1 Gene from Pinus massoniana

Xing Wu,Xingfeng Hu,Peizhen Chen,Xiaobo Sun,Fan Wu,Kongshu Ji*   

  1. Key Laboratory of Forest Genetics&Biotechnology of Ministry of Education Co-Innovation Center for Sustainable Forestry in Southern China Nanjing Forestry University Nanjing 210037
  • Received:2019-03-12 Online:2020-03-01 Published:2020-04-08
  • Contact: Kongshu Ji

摘要:

目的: PIN1基因通过调控生长素极性运输的方式在植物根系生长发育中发挥作用。对马尾松PmPIN1基因进行全长克隆和功能分析,有助于在分子水平揭示马尾松的生根机制。方法: 运用PCR和RACE技术成功克隆马尾松PmPIN1基因cDNA序列;利用生物信息学软件分析PmPIN1基因的核苷酸序列及其编码蛋白的氨基酸序列,并构建系统进化树;通过实时荧光定量分析PmPIN1基因在10年生马尾松幼根、1年生枝条、新叶、花中的表达量差异;利用农杆菌侵染法将PmPIN1基因转入烟草获得转基因植株,比较转基因烟草与转pCAMBIA-1302空白载体烟草之间的表型差异;用激光共聚焦显微镜观察PmPIN1-GFP荧光蛋白在转基因烟草根中的表达模式;酶联免疫法测定野生型和转基因烟草的根、根茎结合处及叶中的生长素含量;用不同浓度的生长素极性运输抑制剂1-N-萘基邻氨甲酰苯甲酸(NPA)处理野生型与转基因烟草,分析烟草根、根茎结合处、叶中的生长素含量变化;利用PEG介导法将16318-hGFP-PmPIN1重组载体转化至拟南芥原生质体中进行亚细胞定位分析。结果: PmPIN1基因全长2 914 bp,包含2 085 bp的ORF,编码的蛋白由695个氨基酸组成,N端与C端均有膜运输蛋白。系统进化树显示马尾松与油松在发育树同一支上。通过实时荧光定量发现PmPIN1在不同组织中的表达量差异达到极显著,1年生枝条与幼根中表达量较高,花中最低。农杆菌介导法转化烟草获得的转基因烟草,较转空载烟草株高增长,生根量增加且根系变长。激光共聚焦显微镜观察得出转基因烟草根部中间有明显绿色荧光富集现象。酶联免疫法测定结果表明转基因烟草根、根茎结合处生长素含量高于野生型,且叶较根和根茎结合处减少。不同浓度NPA处理后,野生型烟草根中生长素含量变化达极显著,且生长素含量随NPA处理浓度的增加而减少;处理浓度为10 nmol·L-1时,叶中生长素含量最高;处理浓度为2 nmol·L-1时,根茎结合处生长素含量增加较多。不同浓度NPA处理后,转基因烟草不同部位生长素含量变化均未达到极显著。亚细胞定位分析表明PmPIN1蛋白主要分布于细胞膜上。结论: 马尾松PmPIN1与油松PIN1亲缘关系最近。PmPIN1属于膜蛋白。PmPIN1转基因烟草可能提高生长素由地上部位向地下部位的运输能力,减少NPA对生长素含量变化的影响,表明PmPIN1可能调控生长素的极性运输;PmPIN1-GFP绿色荧光富集在根部中间部位,PmPIN1在幼根中表达量较高,转基因烟草较转空载植株发根量多,根长长。研究结果表明PmPIN1基因可能在根系发育中发挥重要作用。

关键词: 马尾松, PmPIN1基因, 转基因, 功能分析

Abstract:

Objective: The PIN1 gene plays a role in the growth and development of plant roots by regulating the polar transport of auxin. In this study, the full-length cloning and functional analysis of the PmPIN1 gene of Pinus massoniana was carried out in order to reveal the molecular mechanism of rooting for cutting propagation of P. massoniana. Method: The cDNA sequence of PmPIN1 gene of P. massoniana was successfully cloned by PCR and RACE. Bioinformatics software was used to analyze the nucleic acid sequence, amino acid sequence and construct a phylogenetic tree of PmPIN1 gene. The expression of PmPIN1 gene in young roots, annual branch, young leaf and flowers of 10-year-old P. massoniana was analyzed by real-time polymerase chain reaction. The PmPIN1 gene was overexpressed in tobacco(Nicotiana tabacum)by Agrobacterium infection method to obtain transgenic plants, and the phenotypic differences between transgenic plants and pCAMBIA-1302 blank vector were compared, and PmPIN1-GFP fluorescent protein was observed in transgenic tobacco roots by laser scanning confocal microscope. The expression of auxin in roots, rhizomes and leaves of transgenic tobacco and wild tobacco was determined by enzyme-linked immunosorbent assay (ELISA). Treatment of wild-type and transgenic tobacco with different concentrations of auxin polar transport inhibitor (1-N-naphthylphthalamic acid, NPA), the changes of auxin content in tobacco roots, rhizome junctions and leaves were analyzed. 16318-hGFP-PmPIN1 recombinant vector was transferred to Arabidopsis thaliana protoplasts by PEG-mediated method for subcellular localization analysis. Result: The PmPIN1 gene is 2 914 bp in length and contains a 2 085 bp ORF, which encodes a protein consisting of 695 amino acids, with a membrane transport protein at the N-terminus and C-terminus. The phylogenetic tree showed that P. massoniana and P. tabulaeformis are on the same branch of the development tree. The difference in expression of PmPIN1 in different tissues was found to be extremely significant by real-time polymerase chain reaction, high expression in annual branch and young roots, lowest in flowers; Transgenic tobacco obtained by Agrobacterium rhizogenes-mediated transformation had higher growth, increased rooting and longer root length compared with a blank vector. Laser confocal microscopy showed that there was obvious green fluorescence enrichment around the root of transgenic tobacco. ELISA was used to determine the content of auxin in the roots and rhizomes of transgenic tobacco, and the content was found higher than that in wild type, and less in leaves than in roots and rhizomes. After treatment with different concentrations of NPA, the auxin content of wild type tobacco roots changed extremely significantly, and the auxin content decreased with the increase of NPA treatment concentration. When the treatment concentration was 10 nmol·L-1, the auxin content in leaves was the largest. When the concentration was 2 nmol·L-1, the content of auxin in the rhizome junction increased more. After treatment with different concentrations of NPA, the changes of auxin content in different parts of transgenic tobacco did not reach extremely significant. Subcellular localization analysis indicated that PmPIN1 protein was mainly distributed on the cell membrane. Conclusion: PmPIN1 of P. massoniana and PIN1 of P. tabulaeformis had a higher affinity. PmPIN1 belongs to membrane protein. PmPIN1 transgenic tobacco increases the transport capacity of auxin from aboveground to underground. Transgenic tobacco reduces the effect of NPA on auxin content, indicating that PmPIN1 likely regulates the polar transport of auxin. PmPIN1-GFP green fluorescence was enriched in the middle of the root, PmPIN1 was highly expressed in young roots. Transgenic tobacco had more roots and longer roots than transgenic plasmid. The above findings suggest that the PmPIN1 gene plays an important role in root development.

Key words: Pinus massoniana, PmPIN1 gene, transgenic, functional analysis

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