WO2019085237A1 - Procédé d'induction de multiples couches de bandes de caspary dans une racine de plante - Google Patents

Procédé d'induction de multiples couches de bandes de caspary dans une racine de plante Download PDF

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Publication number
WO2019085237A1
WO2019085237A1 PCT/CN2017/118785 CN2017118785W WO2019085237A1 WO 2019085237 A1 WO2019085237 A1 WO 2019085237A1 CN 2017118785 W CN2017118785 W CN 2017118785W WO 2019085237 A1 WO2019085237 A1 WO 2019085237A1
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Prior art keywords
plant
kjeldahl
belt
shr
myb36
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PCT/CN2017/118785
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English (en)
Chinese (zh)
Inventor
吴双
李朋雪
于巧芝
许春苗
顾旭
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福建农林大学
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Publication of WO2019085237A1 publication Critical patent/WO2019085237A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the invention belongs to the field of agricultural biotechnology, and particularly relates to a method for inducing the formation of Kjeldahl belt in plant roots.
  • the Casparian strip is a corked and lignified banded structure of the radial and lateral walls of the cell in the endothelial layer of higher plants.
  • the corked Kjeldahl belt forms an insurmountable barrier to water and solutes, so the continuity of the apoplasts of higher plant roots is blocked here. After the water and mineral elements are absorbed by the epidermis of the root, they can be transported laterally to the vascular bundle along two pathways: one is the apoplast of the intercellular space; the other is the symplast pathway inside the cell.
  • the transcription factor SHR can promote the formation of the endothelial layer by inducing directional division of cortical endothelial stem cells.
  • the expression of SHR in cortical and ectodermal cells induces a large number of cell divisions and produces multiple layers of cells outside the vascular tissues.
  • SHR activates the transcription factor MYB36, and it has been reported that MYB36 regulates several important genes involved in the formation of the Kjeldahl belt. Therefore, it is possible to increase the cell layer having the Kjeldahl belt in the plant root by regulating the expression of SHR or MYB36.
  • the object of the present invention is to provide a method for inducing a multi-layer Kjeldahl belt in a plant root, and to realize the expansion of the Kjeldahl belt structure in the plant root, thereby achieving anti-caries and other anti-reverse effects.
  • a method for inducing a multi-layer Kjeldahl belt in a plant root which induces the formation of a multi-layered functional Kjeldahl belt in a plant root by expressing a plant transcription factor SHR or MYB36 in combination with a small peptide CIF1 or CIF2.
  • the essential genes for the formation of the Kjeldahl belt are induced by constitutive expression, inducible expression, tissue-specific expression or inducible tissue-specific expression of the plant transcription factor SHR or MYB36; synthetic small peptides are added to the plant roots expressing SHR or MYB36 CIF1 or CIF2, or endogenously expressing CIF1 or CIF2, ultimately induces the formation of multi-layered functional Kjeldahl bands in plant roots.
  • a composition in which non-endothelial cells in the plant root induce the formation of the Kjeldahl belt.
  • the composition includes the transcription factor SHORT-ROOT (SHR) (AT4G37650), or the transcription factor MYB36 (AT5G57620); also includes the synthetic polypeptide CIF1 or CIF2, or the Arabidopsis gene CIF1 (AT2G16385) and CIF2 which endogenously express the synthetic small peptide. (AT4G34600).
  • the application of multi-layer Kjeldahl belt formation in crop roots is induced in the cultivation of highly resistant crops.
  • Plants of the invention refer to Arabidopsis or other higher plants. Since SHR function is conserved among Arabidopsis and other higher plants including rice, the present invention is also widely applicable to crops other than Arabidopsis.
  • the present invention uses Arabidopsis SHR and MYB36, but the orthologous gene function of SHR and MYB36 in crops is highly conserved. Therefore, SHR and MYB36 using other higher plant sources are also covered in the invention.
  • a plant expression vector containing the above SHR or MYB36 transcription factor is provided.
  • a host strain containing the above SHR or MYB36 transcription factor gene A host strain containing the above SHR or MYB36 transcription factor gene.
  • the invention can artificially induce the formation of the Kjeldahl belt structure in the plant non-endothelial cells, and the analysis results show that the induced Kjeldahl belt structure can block the free entry of external moisture into the root.
  • the invention is of great significance for the use of biotechnology to breed highly resistant various crop varieties.
  • FIG. 1 Inducible expression vector, pG1090-XVE::SHR and pG1090-XVE::MYB36 express SHR or MYB36 in all cells in Arabidopsis roots.
  • the genes controlling the Kjeldahl band such as PER64, ESB1 and SGN3 are only expressed in the endothelial layer (represented by the reporter-linked GFP reporter system, the fluorescent signal part in the figure), in contrast, expression In the roots of SHR or MYb36, these genes are significantly up-regulated and expressed in most cells.
  • (a) expression of SHR induces expression of the Kjeldahl position-localized protein CASP1-GFP in cortical cells (cor in the figure), but exhibits a discontinuous structure (indicated by arrows in the figure).
  • CIF1 c in the figure
  • CIF2 d in the figure
  • the root expressed by SHR can exclude the red propidium iodide dye from the cortical cells (as indicated by b), indicating that the Kjeldahl band formed in this layer of cells is functional.
  • Arabidopsis root RNA was extracted according to OMEGA's E.Z.N.A.® Plant RNA Kit kit instructions. This was reverse transcribed into cDNA according to the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TRANS) instructions.
  • TRANS TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR
  • the cDNA sequences of SHR and MYB36 were searched on the TAIR website (http://www.arabidopsis.org/), and the primers SHR(+): 5'GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGATACTCTCTTTAGA3' and SHR(-):5' were designed.
  • the DH5 ⁇ competent state was prepared, the ligation product was transferred to DH5 ⁇ , positive colonies were screened with kanamycin containing 100 mg/L, and the plasmid was verified by PCR, and the positive plasmid was sent to the company for sequencing.
  • the correct plasmid and the expression vector pMDC7 were mixed, LR enzyme was added, and the target fragment carrying the SHR cDNA was ligated to the expression vector by LR reaction, transferred to DH5 ⁇ , and positive colonies were screened with 100 mg/L of spectinomycin.
  • the plasmid was verified by PCR to obtain expression vectors pG1090-XVE: SHR and pG1090-XVE: MYB36.
  • Example 2 Establishing plant expression systems
  • the sealed plate was placed in a refrigerator at 4 ° C, and after 3 days, it was placed in a tissue culture incubator at 22 ° C for light culture. About 10 days, the positive seedlings were transplanted into pots for cultivation, collected and homozygously screened.
  • the TAIR website searches for small peptide amino acid sequences, CIF1: DYGNNSPSPRLERPPFKLIPN, CIF2: DYGHSSPKPKLVRPPFKLIPN, and the company synthesizes small peptides.
  • Seedlings were transferred to 1/2 MS medium containing 10 ⁇ M small peptide (CIF1 or CIF2) and 10 ⁇ M estrogen and treated under normal light for 2 days.
  • Laser scanning confocal microscope Zeiss Observe the root under LSM880 ( Figures 1 and 2).
  • Propidium iodide is a fluorescent dye that cannot pass through the cell membrane of living cells and can only enter the roots through the intercellular space. The place where the PI solution enters will fluoresce under the microscope, due to the presence of the root layer in the mature layer.
  • the Kjeldahl belt prevents the entry of the PI solution into the vascular bundle, thereby determining which layer of Arabidopsis roots can form a functional Kjeldahl belt.
  • a drop of the prepared PI solution (1-10 ⁇ g/ml) was placed on the slide, and the Arabidopsis seedlings were picked up with tweezers, the roots were immersed in the PI solution, placed flat on the glass slides, and the coverslips were gently covered. , observed under the microscope ( Figure 2).

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé d'induction de multiples couches de bandes de Caspary dans une racine de plante. Le procédé induit une bande de Caspary dans une racine de plante à l'aide d'un facteur de transcription végétal SHORT-ROOT (SHR) ou MYB36 en combinaison avec des petits peptides dérivés de plantes CIF1 et CIF2. L'expression constitutive (à l'aide d'un promoteur 35S) ou inductive (à l'aide d'un système d'expression inductible par des œstrogènes) de SHR ou de MYB36 permet l'induction d'une pluralité de gènes impliqués dans la synthèse de bande de Caspary dans des cellules corticales et d'autres cellules tissulaires essentielles dans des racines de plante.
PCT/CN2017/118785 2017-10-31 2017-12-27 Procédé d'induction de multiples couches de bandes de caspary dans une racine de plante WO2019085237A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711040085.6A CN107557382B (zh) 2017-10-31 2017-10-31 一种植物根中多层凯氏带的诱导方法
CN201711040085.6 2017-10-31

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WO2019085237A1 true WO2019085237A1 (fr) 2019-05-09

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Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109628471B (zh) * 2019-02-22 2021-02-26 福建农林大学 一种提高豆科植物根瘤形成进而促进其生长的方法
CN114438119B (zh) * 2022-02-16 2023-10-13 广西大学 一种携带标记的多基因突变体水稻植株的制备方法及其应用
CN116949055A (zh) * 2023-04-18 2023-10-27 中国农业大学 ZmMYB36基因及其应用
CN116949059A (zh) * 2023-07-27 2023-10-27 扬州大学 ZmCASP4基因在调控根系发育和矿质元素吸收中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001090314A1 (fr) * 2000-05-24 2001-11-29 New York University Gene de racine courte, son promoteur et ses utilisations
CN103343139A (zh) * 2013-06-27 2013-10-09 杭州昂众生物科技有限公司 一种提高植物抗旱性的新方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001090314A1 (fr) * 2000-05-24 2001-11-29 New York University Gene de racine courte, son promoteur et ses utilisations
CN103343139A (zh) * 2013-06-27 2013-10-09 杭州昂众生物科技有限公司 一种提高植物抗旱性的新方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KAMIYA ET AL.: "The MYB36 Transcription Factor Orchestrates Casparian Strip Formation", PNAS, 18 August 2015 (2015-08-18), pages 10533 - 10538, XP055560214, ISSN: 1091-6490 *
LIBERMAN ET AL.: "MYB36 Regulates the Transition from Proliferation to Differentiation in the Arabidopsis Root", PNAS, vol. 112, no. 39, 7 August 2015 (2015-08-07), pages 284 - 286, XP055614713, ISSN: 1091-6490 *
TAKUYA NAKAYAMA ET AL.: "A Peptide Hormone Required for Casparian Strip Diffusion Barrier Formation in Arabidopsis Roots", SCIENCE, vol. 355, no. 6322, 20 January 2017 (2017-01-20), pages 284 - 286, XP055614714, ISSN: 0036-8075 *

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