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MYB transcription factor for regulating and controlling synthesis of plant procyanidine as well as coding gene and application of MYB transcription factor

A technology of proanthocyanidins and coding genes, which is applied in plant genetic improvement, botany equipment and methods, angiosperms/flowering plants, etc. It can solve the problem of less research on the synthesis and regulation mechanism of proanthocyanidins and unclear metabolism regulation mechanism of flavonoids And other issues

Pending Publication Date: 2021-12-28
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the metabolic regulation mechanism of flavonoids in tartary buckwheat is not yet clear, and there are few studies on the synthesis and regulation mechanism of proanthocyanidins

Method used

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  • MYB transcription factor for regulating and controlling synthesis of plant procyanidine as well as coding gene and application of MYB transcription factor
  • MYB transcription factor for regulating and controlling synthesis of plant procyanidine as well as coding gene and application of MYB transcription factor
  • MYB transcription factor for regulating and controlling synthesis of plant procyanidine as well as coding gene and application of MYB transcription factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Cloning of FtMYB43 Gene CDS

[0051] The FtMYB43 gene was cloned from the cDNA of tartary buckwheat seedlings, and specific primers were designed according to the ORF of FtMYB43:

[0052] FtMYB43-F: 5'-ATGGGGAGGCCACCTTGCT-3' (SEQ ID NO.3),

[0053] FtMYB43-R: 5'-CTAGAACAAATCATGATCATTTT-3' (SEQ ID NO.4);

[0054] The material "Pinku" was used as cDNA as a template for PCR amplification to obtain the CDS sequence of the target gene.

[0055] The PCR program was 95°C for 3min; 95°C for 30s, 57°C for 30s, 72°C for 90s, 33 cycles. The PCR purified product was connected to the pTOPO-Blunt Simple blunt-end cloning vector to obtain the FtMYB43-T vector plasmid. The full-length sequence of the FtMYB43 gene was obtained after sequencing, analysis and splicing. The nucleotide sequence of the FtMYB43 gene is shown in SEQ ID No.2.

Embodiment 2

[0056] Example 2 Amino acid sequence analysis of transcription factor FtMYB43

[0057] The amino acid sequence of FtMYB43 gene was compared with Blast in the NCBI database to obtain the MYB-like protein sequence of other species. A clustering tree was constructed using MEGA6.0 software, and the isoelectric point (pl) and protein molecular weight (Mw) were predicted using the protein prediction tool website (https: / / web.expasy.org / cgi-bin / compute_pi / pi_tool) .

[0058] The length of the CDS of the FtMYB43 gene is 939bp, the encoded protein has 312 amino acids with a molecular weight of 34.8KDa, and the isoelectric point (pI) of the encoded protein is 5.13. Using NCBI's blast to analyze the conserved amino acid domain of FtMYB43, it was found that the encoded protein belongs to the MYB type transcription factor. The MYB protein sequences of other species were obtained by Blast comparison in the NCBI database, and the clustering tree was constructed using MEGA6.0 software ( f...

Embodiment 3

[0059]Example 3 Construction of FtMYB43 Gene Overexpression Vector, Transformation of Tartary Buckwheat and Expression Level Detection of Key Genes of FtMYB43 and Proanthocyanidin Synthesis Pathway in Positive Tartary Buckwheat Hairy Roots

[0060] The FtMYB43 gene is operably constructed in the expression control sequence to form a plant expression vector containing the FtMYB43 gene, including: designing homologous recombination primers, using the FtMYB43-T vector as a template, the designed primers are as follows:

[0061] OE-FtMYB43-F / R:

[0062] OE-FtMYB43-F:gacttgaactcggtatctagaATGGGGAGGCCACCTTGC;

[0063] OE-FtMYB43-R:gtcgacggtatcgataagcttCTAGAACAAATCATGATCATTTTCAAAT;

[0064] The full-length sequence of the FtMYB43 gene was amplified by PCR using the above-mentioned OE-FtMYB43-F / R primers. After digestion, recovery, and ligation transformation, the full-length sequence of FtMYB43 was inserted forward into the CaMV35S promoter of the overexpression vector, and the over...

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Abstract

The invention discloses an MYB transcription factor for regulating and controlling plant procyanidine synthesis as well as a coding gene and application of the MYB transcription factor. The MYB transcription factor FtMYB43 is cloned from tartary buckwheat, and the amino acid sequence of the MYB transcription factor FtMYB43 is shown as SEQ ID No. 1. The transcription factor FtMYB43 gene is respectively subjected to genetic transformation of buckwheat and arabidopsis thaliana to obtain transgenic buckwheat hairy roots and arabidopsis thaliana, the expression quantity of an enzyme gene of a proanthocyanidins pathway in the obtained positive arabidopsis thaliana is detected, and the content of proanthocyanidins after the FtMYB43 gene is transformed into arabidopsis thaliana for ectopic expression is detected. Test results display that the FtMYB43 gene is beneficial for improving the expression quantity of key enzyme genes of a flavonoid metabolic pathway part in hairy roots, so that the biosynthesis of procyanidine is promoted; the content of procyanidine in a transgenic line is remarkably increased, which indicates that the FtMYB43 gene participates in biosynthesis of procyanidine in an up-regulated arabidopsis thaliana plant.

Description

technical field [0001] The present invention relates to transcription factors, in particular to MYB transcription factors isolated from tartary buckwheat (Fagopyrum tataricum (L.) Gaertn) and their coding genes. The present invention further relates to their application in regulating plant proanthocyanidin synthesis, belonging to MYB transcription factors and their fields of application. Background technique [0002] Modern clinical medical observations show that tartary buckwheat products have medicinal effects such as lowering blood sugar, lowering blood fat, coronary heart disease, anti-cancer, and anti-aging. These biological activities are closely related to the antioxidant capacity of bioflavonoids in tartary buckwheat. Proanthocyanidins are important flavonoid secondary metabolites of tartary buckwheat. The multi-electronic phenolic hydroxyl structure in its molecular structure makes it have strong physiological activity and is widely used in health food, medicine, co...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/00A01H6/20A01H6/00
CPCC07K14/415C12N15/825
Inventor 张凯旋周美亮赵辉范昱丁梦琦胡永平
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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