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MYB transcription inhibition factor LrETC1 related to lycium ruthenicum anthocyanin synthesis and application thereof

A technology of transcriptional repressor and qianthocyanidin, which is applied in the field of genetic engineering and can solve problems that have not been reported

Active Publication Date: 2021-09-28
WOLFBERRY SCI INST NINGXIA ACAD OF AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MYB transcription factors play an important role in the regulation of anthocyanin metabolism. Although some research results have reported several MYB transcription factors that positively regulate anthocyanin synthesis in Lycium barbarum, but the research on inhibiting its anthocyanin synthesis MYB transcription factor has not been reported

Method used

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  • MYB transcription inhibition factor LrETC1 related to lycium ruthenicum anthocyanin synthesis and application thereof
  • MYB transcription inhibition factor LrETC1 related to lycium ruthenicum anthocyanin synthesis and application thereof
  • MYB transcription inhibition factor LrETC1 related to lycium ruthenicum anthocyanin synthesis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] For the extraction of DNA and RNA, refer to the kit instructions, and operate in a conventional manner.

[0063] Synthesis of single-stranded cDNA: Using the total RNA of Lycium barbarum fruit as a template, the single-stranded cDNA was synthesized using the experimental steps given by Takara's PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time).

[0064] Cloning of LrETC1 gene: See Table 2 for the sequences of the amplification primers. The primers were diluted to 10 μM, and the obtained cDNA was used as a template for PCR amplification reaction. The reaction system is shown in Table 3 below.

[0065] Table 2

[0066]

[0067] table 3

[0068]

[0069] After the amplification is completed, take out the PCR product and perform 1% agarose gel electrophoresis detection. After electrophoresis for about 7 minutes, observe it under a UV gel imager. If the target band is single and the size is correct, cut out the gel piece and place it in the In a 2mL ce...

Embodiment 2

[0086] The open reading frame and deduced amino acid sequence of the LrETC1 transcription factor were searched through the ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) online search tool on NCBI. The online software ProtParam (http: / / web.expasy.org / protparam / ) was used to predict the amino acid composition, protein molecular weight, theoretical isoelectric point and stability of transcription factors. The hydrophobicity and charge distribution of proteins were analyzed using the online software ProtScale (https: / / web.expasy.org / protscale / ). The transmembrane domain of LrETC1 protein was analyzed using TMHMM 2.0 (http: / / www.cbs.dtu.dk / services / TMHMM) software. The signal peptide of the protein was predicted by Cell-PLoc2.0 (http: / / www.cbs.dtu.dk / services / SignalP). Use Wolf Psort (https: / / www.genscript.com / wolf-psort.htmL) and online software (https: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2 / ) to predict proteins subcellular localization. Protein analysis using SOPMA ...

Embodiment 3

[0163] Construction of Plant Overexpression Recombinant Vector

[0164] Cloning of target gene homologous recombination fragment:

[0165] Using the cDNA solution obtained in the steps of the above examples as a template, the corresponding primers in Table 12 were used to amplify the homologous recombination fragment of the plant overexpression vector. The amplification system, detection and recovery of PCR products are the same as those in the above examples.

[0166] Table 12

[0167]

[0168]

[0169] Vector digestion reaction

[0170] The vector pCM1307 was double-digested with two restriction enzymes XbaI and KpnI; the vector pZYB9-pEAQ-HT was double-digested with two restriction enzymes SmaI and StuI. The enzyme digestion system and product recovery are the same as those in the above-mentioned examples.

[0171] Homologous recombination connection fragments and vectors, Escherichia coli DH5α transformation, bacterial liquid PCR identification and plasmid extract...

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Abstract

The invention discloses an MYB transcription inhibition factor LrETC1 related to lycium ruthenicum anthocyanin synthesis and application thereof, and belongs to the technical field of gene engineering. According to the invention, the MYB transcription inhibition factor LrETC1 participating in the anthocyanin synthesis is screened and cloned, the total cDNA of the gene is 240bp, and 79 amino acids are encoded; protein sequence database analysis shows that the transcription factor belongs to R3 type MYB; and multiple sequence alignment and evolutionary tree analysis show that the transcription inhibition factor belongs to AtCPC-like transcription inhibition factors, and is a transcription factor which is located in a cell nucleus and does not have an activation function. A LrETC1 transgenic arabidopsis thaliana strain is obtained through agrobacterium tumefaciens-mediated genetic transformation, compared with a wild type, seed coats of seeds are light brown, arabidopsis thaliana seedlings are free of pigment accumulation under the stress condition of high sucrose concentration, and the expression level of related structural genes is remarkably reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically relates to a MYB transcription inhibitor LrETC1 related to anthocyanin synthesis of Lycium barbarum black fruit and its application. Background technique [0002] Lycium ruthenicum Murry (Lycium ruthenicum Murry.) belongs to the Solanaceae (Solanaceae) Lycium genus deciduous thorny shrubs. It is rich in nutrition and has both medicine and food. It has attracted widespread attention because of its rich anthocyanins. Anthocyanins have strong antioxidant activity, which can not only help plants resist stressful environments, but also have great benefits in promoting human health. [0003] Transcription factors, also known as trans-acting factors, usually refer to a class of proteins encoded by genes, which can specifically bind to relevant cis-acting elements in the gene promoter region to activate or inhibit gene expression, thereby increasing the plant's response ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00A01H6/20C12Q1/6895
CPCC07K14/415C12N15/825C12Q1/6895C12Q2600/158C12Q2600/13
Inventor 曹有龙唐琳樊云芳秦欢安巍李婷婷陈晓军戴国礼秦垦尹跃秦小雅梁晓婕
Owner WOLFBERRY SCI INST NINGXIA ACAD OF AGRI & FORESTRY SCI
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