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Anthocyanin synthesis related proteins and application in regulating anthocyanin content of plants

A technology for anthocyanin content and related proteins, which is applied in the application field of regulating and controlling plant anthocyanin content

Active Publication Date: 2020-03-03
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

With the in-depth development of functional genomics of Medicago truncatula, some genes related to anthocyanin synthesis pathway and family transcription factors such as MYB and WD40 have been cloned in Medicago truncatula. However, due to the special physiological and structural characteristics of legumes, the expression patterns and biological functions of the above genes are different from those reported in the dicotyledonous model plant Arabidopsis, indicating that the synthesis of anthocyanins between different species and metabolic regulatory pathways are genetically diverse

Method used

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  • Anthocyanin synthesis related proteins and application in regulating anthocyanin content of plants
  • Anthocyanin synthesis related proteins and application in regulating anthocyanin content of plants
  • Anthocyanin synthesis related proteins and application in regulating anthocyanin content of plants

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Experimental program
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Effect test

Embodiment 1

[0114] Embodiment 1, the discovery of RH1 and its coding gene RH1 gene

[0115] 1. Mutant acquisition and phenotype analysis

[0116] In The Nobel Foundation, by screening the Tnt1 insertion mutant library of Medicago truncatula, a mutant with abnormal anthocyanin synthesis was isolated and obtained. The mutant was numbered NF7599 in the Tnt1 database, and it mainly manifested in the specific accumulation of anthocyanins on leaves. The phenotype of red heart was presented, and the mutant was named red heart1(rh1). The mutant was crossed with wild-type Medicago truncatula R108 to obtain F 1 Expressed as an intermediate type with a lighter red heart, F 2 Three phenotypes were isolated from the population: wild type, intermediate type with lightened red heart and red heart phenotype, and the segregation ratio was 1:2:1 (p=0.754>0.05). It shows that for wild-type R108, the red heart trait of rh1 mutant is controlled by a semi-dominant single gene.

[0117] The leaf phenotype o...

Embodiment 2

[0138] Example 2, Functional Verification of RH1 Gene

[0139] 1. Construction of CRISPR / Cas9 genome editing vector

[0140] Using pMtU6-Cas (Meng et al., 2017, Targeted mutagenesis by CRISPR / Cas9system in the model legume Medicago truncatula, Plant Cell Rep (2017) 36:371–374) as a template, using the primer pair MtU6-F1 and MtU6-R1 Perform PCR amplification to obtain the MtU6 promoter; use primer pair 1 (composed of 40-sgRNA-F1 primers and R2 primers) and primer pair 2 (composed of 40-sgRNA-F2 primers and R2 primers) for PCR amplification, respectively, Obtain two DNA fragments encoding the sgRNA targeting the target fragment and the scaffold respectively, that is, the sgRNA1-scaffold fragment and the sgRNA2-scaffold fragment. Using the mixture of MtU6 promoter and sgRNA1-scaffold fragment as a template, PCR amplification was performed using a primer pair (composed of MtU6-F1 and R2) to obtain pMtU6::sgRNA1-scaffold fragment; using MtU6 promoter and sgRNA2-scaffold fragment ...

Embodiment 3

[0168] Embodiment 3, the anthocyanin content of Medicago truncatula of transfection RH1 gene increases

[0169] This example further illustrates that the RH1 gene can increase the anthocyanin content of Medicago truncatula by introducing the RH1 gene into wild-type Medicago truncatula R108. The specific method is as follows:

[0170] 1. Construction of recombinant vector

[0171] Using the genomic DNA of the mutant rh1 as a template, PCR amplification was carried out with primers 40-PRO-EcoRI-F and 40-3'UTR-PstI-R to obtain the DNA fragment shown in sequence 3 of the sequence table; the pCAMBIA2300 The DNA fragment between the EcoRI and PstI recognition sequences was replaced with the DNA fragment shown in Sequence 3, and the resulting recombinant vector was named pCAMBIA2300-RH1pro::RH1. pCAMBIA2300-RH1pro::RH1 can express the protein shown in sequence 1 in the sequence listing. The primer sequences used are:

[0172] 40-PRO-EcoRI-F:5'-ccatgattacgaattcACTCGTATGTGGGGATTTTGA-3...

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Abstract

The invention discloses an anthocyanin synthesis related protein and its application in regulating anthocyanin content of plants. The anthocyanin synthesis related protein disclosed by the invention is as shown in the following A1), A2) or A3): A1) a protein with its amino acid sequence being sequence 1; A2) a protein obtained by substitution and / or deletion and / or addition of one or several aminoacid residues of the amino acid sequence as shown in the sequence 1 in the sequence table and having the same functions; or A3) a fusion protein obtained by connecting a tag at the N-terminal or / andC-terminal of A1) or A2). The anthocyanin synthesis related protein of the invention can regulate the anthocyanin content of plants, can be used to cultivate plants with increased or decreased anthocyanin content, and can also be used to regulate the color of plants.

Description

technical field [0001] The invention relates to anthocyanin synthesis-related protein and its application in regulating plant anthocyanin content in the field of biotechnology. Background technique [0002] Anthocyanins are water-soluble flavonoids widely present in plants. They are one of the main pigments that constitute the color of petals and fruits, and play an important role in plant physiology. At the same time, anthocyanins are also natural plant antioxidants that can protect plants. It can protect the human body from the damage of free radicals and other harmful substances, and has many benefits to human health. [0003] Medicago truncatula is an emerging model plant of legumes, which has good synteny with important legume crops and pastures such as soybean and alfalfa. With the in-depth development of functional genomics of Medicago truncatula, some genes related to anthocyanin synthesis pathway and family transcription factors such as MYB and WD40 have been clone...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/54
CPCC07K14/415C12N15/825
Inventor 林浩王崇楠季文楷牛丽芳
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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