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Anthocyanin biosynthesis in carrot plants

A carrot and plant technology, applied in angiosperms/flowering plants, microorganisms, plant peptides, etc., can solve problems such as unclear regulatory genes

Pending Publication Date: 2022-02-25
OTERRA AS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regulatory genes controlling transcription of anthocyanin pathway structural genes in carrots are unknown

Method used

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  • Anthocyanin biosynthesis in carrot plants
  • Anthocyanin biosynthesis in carrot plants
  • Anthocyanin biosynthesis in carrot plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Cloning of coding sequences of DcMYB90 and DcEGL1 and construction of transformation vectors

[0115] The open reading frames (ORFs) of DcMYB90 (MYB 90-like; NCBI Gene ID: LOC108221186) and DcEGL1 (EGL1; NCBI Gene ID: LOC108210744) were deciphered from the corresponding Arabidopsis spp. Identification of homologous genes. use figure 1 The primers listed, by using High-Fidelity DNA polymerase (NEB, U.K.) was used to perform PCR to amplify the coding sequence from the cDNA synthesized from the purple tissue inside and outside the main root of "Night Bird". Purify the PCR fragment from the gel and clone into pCR following the manufacturer's instructions TM -Blunt vector (ThermoFisher Scientific, CA, USA). Three clones for each gene were sequenced by Sanger sequencing using standard M13 primers. Positive clones were named pTOPO-M90(A-C) and pTOPO-E1(A-C).

[0116] Under the CaMV 35S promoter, the Agrobacterium transformation vector containing the DcMYB90 and DcEGL...

Embodiment 2

[0118] Simultaneous expression of DcMYB90 and DcEGL1 causes purple pigmentation in orange carrots

[0119] The orange carrot cultivar "Danvers 126" (Daucus carotasubs. sativus; 2n=2x=18 ). After 6-10 weeks of culture on selective medium, pink / purple spots started to appear on the transformed calli. Colored calli were selected and transferred to fresh plates every 4 weeks. After 2-3 rounds of transplanting culture, part of the callus has completely turned purple. The purple callus was transferred to regeneration medium for embryo and shoot development. The regenerated plants were acclimated and transferred to the greenhouse where they were grown in 2L pots with peat. Taproots were harvested 3 months after transfer to the greenhouse, referred to as 3-month-old taproots. Taproots of 6 individual transgenic plants were harvested showing variable pigmentation from almost completely purple to almost completely orange. Individual plants with two almost completely purple taproot...

Embodiment 3

[0123] Transgenic T expressing DcMYB90 and DcEGL1 0 Identification and Identification of Anthocyanins in Plant Taproots

[0124] Sample Preparation

[0125] About 40 g of 3-month-old single taproots were coarsely ground (the rest were stored in a -80°C freezer for RNA extraction); then in 3% H2 SO 4 in (1 / 1, w / w) Homogenize in a two-speed commercial mixer (VWR-Bie & Berntsen, Herlev, Denmark). The homogenate was then mixed with 70% ethanol (1 / 2, w / w), vortexed, and incubated for 1 hour at room temperature. The extracts were spun at 4500 rpm for 20 min and the supernatant was used for further analysis.

[0126] Determination of the total content of monomeric anthocyanins

[0127] Total monomeric anthocyanins (TMA) in taproot samples were determined by a slightly modified pH differential method. The supernatant was diluted (1:20) with 0.2M KCL-HCL (pH 1 ) solution, and the absorbance was recorded between 350 and 700 nm using a UV-Vis spectrophotometer (Thermo Scientific E...

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Abstract

The present invention provides transgenic carrot plants comprising at least one heterologous DNA sequence encoding: (a) a promoter of a transcription factor gene; or (b) a transcription factor geneoperably linked to a promoter; wherein the heterologous DNA sequence increases the expression of at least one gene encoding a sinapic acid glucosyltransferase (USAGT). The transcription factor gene is preferably DcMYB90 (SEQ ID NO:1) or DcEGL1 (SEQ ID NO:2) or a sequence having at least 80% identity to the sequence of SEQ ID NO:1 or at least 80% identity to the sequence of SEQ ID NO:2, wherein the sequence encodes a transcription factor protein increasing the expression of sinapic acid glucosyltransferase (USAGT) having the sequence of SEQ ID NO:4.

Description

technical field [0001] The present invention relates to transgenic carrot plants comprising at least one gene encoding a transcription factor or a heterologous DNA sequence encoding a transcription factor gene operably linked to a promoter, which is expressed in the transgenic plant and increases at least one gene encoding a mustard Expression of acid glucosyltransferase (USAGT) gene. The sequence of USAGT can be SEQ ID NO:4, or a sequence at least 85% identical to SEQ ID NO:4. The present invention also relates to the DNA sequences encoding the transcription factor genes DcMYB90 (SEQ ID NO: 1) and DcEGL1 (SEQ ID NO: 2), and transgenic carrot plants comprising the corresponding DNA sequences operably linked to heterologous promoters. The transgenic plants of the present invention may be used in a method of preparing an anthocyanin (anthocyanin)-containing composition, which method may comprise producing a transgenic carrot plant of the present invention and isolating the anth...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/54C12N15/29A01H5/06A01H6/06A23L5/43
CPCC12N15/825C12N9/1051C12Y204/0112C07K14/415A23L5/43C12N2830/60A23V2002/00A23V2250/2104A23V2200/044A01H5/06C12N15/82C12N15/8216
Inventor 什里坎特·夏尔马朱塞佩·狄奥尼西奥印格·巴斯泰德·霍尔梅比亚内·乔恩嘉德亨里克·布林奇-佩德森
Owner OTERRA AS
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