MYB transcription factor implicated in anthocyanin biosynthesis regulation

A transcription factor and biosynthesis technology, applied in the field of plant molecular biotechnology and genetic engineering, can solve problems such as restricting the development of diversification of chrysanthemum flower color quality improvement breeding industry

Active Publication Date: 2015-07-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Involved in the regulation of plant anthocyanin biosynthesis MYB Members have been identified in many plants, but in chrysanthemums involved in the transcriptional regulation of anth

Method used

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  • MYB transcription factor implicated in anthocyanin biosynthesis regulation
  • MYB transcription factor implicated in anthocyanin biosynthesis regulation
  • MYB transcription factor implicated in anthocyanin biosynthesis regulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Chrysanthemum CmMYB Gene cloning

[0021] Based on the information from the chrysanthemum RNA-Seq database, four possible involvements in the regulation of chrysanthemum anthocyanin biosynthesis were screened out MYB Transcription factor CmMYB3 , CmMYB4 , CmMYB5 with CmMYB6 ,among them CmMYB6 It already contains part of the open reading frame (ORF) sequence and 3'non-coding region (3' UTR) sequence (SEQ: NO. 1), CmMYB4 (SEQ: NO. 2) and CmMYB5 (SEQ: NO. 3) already contains part of ORF sequence and 5'UTR sequence. Application gene-specific primer 2 (GSP2) SEQ: NO. 4, SEQ: NO. 5 and SEQ: NO. 6, and nested gene-specific primer 2 (NGSP2) SEQ: NO. 7, SEQ: NO. 8 and SEQ: NO. 9, respectively obtained by 3'cDNA end rapid amplification (3' RACE) technology CmMYB3 (SEQ: NO. 10), CmMYB4 (SEQ: NO. 11) and CmMYB5 (SEQ: NO. 12) (3' UTR) sequence. Then use gene-specific primer 1 (GSP1) SEQ: NO. 13 and SEQ: NO. 14, and nested gene-specific primer 1 (NGSP1) SEQ: NO. ...

Embodiment 2

[0025] Example 2: Chrysanthemum CmMYB Gene expression pattern analysis

[0026] (1) Experimental method

[0027] Using Primer Premier 5, based on CmMYB3 (SEQ: NO. 10), CmMYB4 (SEQ: NO. 11), CmMYB5 (SEQ: NO. 12) and CmMYB6 (SEQ: NO. 1) 3'UTR sequence design real-time quantitative PCR (QPCR) primer combinations SEQ: NO. 23 and SEQ: NO. 24, SEQ: NO. 25 and SEQ: NO. 26, SEQ: NO. 27 and SEQ: NO. 28 and SEQ: NO. 29 and SEQ: NO. 30. The PCR product contains a stop codon and the length is 198 bp, 222 bp, 136 bp and 154 bp, respectively. Use the expression of the chrysanthemum actin gene (SEQ: NO. 31) as an internal reference to analyze each MYB The expression pattern of the transcription factor, its QPCR primer combination is SEQ: NO. 32 and SEQ: NO. 33. The specificity of all QPCR primers was verified by melting point curve analysis, gel electrophoresis analysis, and re-sequencing of QPCR products.

[0028] The RNA from the four stages of petal development and the tender leaves and ...

Embodiment 3

[0031] Example 3: Analysis of regulatory target gene activity

[0032] (1) Experimental method

[0033] Application of primer combinations SEQ: NO. 34 and SEQ: NO. 35, SEQ: NO. 36 and SEQ: NO. 37, SEQ: NO. 38 and SEQ: NO. 39 and SEQ: NO. 40 and SEQ: NO. 41 , Amplification CmMYB3 (SEQ: NO. 19), CmMYB4 (SEQ: NO. 20), CmMYB5 (SEQ: NO. 21) and CmMYB6 The full-length sequence of (SEQ: NO. 22) was loaded onto the pGreenII 0029 62-SK expression vector to construct a recombinant expression vector CmMYB3-SK , CmMYB4-SK , CmMYB5-SK or CmMYB6-SK . The Fast stat high fidelity PCR system enzyme is used to construct the expression vector, and the PCR system is Buffer (with Mgcl 2 ) 3 μL, dNTP (2.5 μM) 2.4 μL, upstream and downstream primers (10 μM) each 1 μL, cDNA 0.6 μL, enzyme 0.3 μL, water 21.3 μL. The expression vector that was finally confirmed to be correctly constructed was electrotransformed into GV3101::pSoup Agrobacterium competent cells, and 3 positive clones were selected, ...

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Abstract

The invention provides an MYB transcription factor CmMYB6 implicated in chrysanthemum petal anthocyanin biosynthesis regulation. The coding region sequence of the MYB transcription factor CmMYB6 contains 765 nucleotides. A CmMYB6 amino acid sequence contains a conserved R2R3 MYB domain, a [D/E]Lx2[R/K]x3Lx6Lx3R motif and an ANDV motif exist in the R3 domain, the [D/E]Lx2[R/K]x3Lx6Lx3R motif can interact with bHLH, and the ANDV motif is the characteristic motif of the MYB transcription factor implicated in anthocyanin biosynthesis regulation. The gene expression of CmMYB6 rises in the petal growth process and is significantly and positively correlated with anthocyanin synthesis the CmMYB6 can induce the activity of synthesis of a key gene CmDFR promoter from anthocyanin, and the CmMYB6 has substantially enhanced induction usefulness and can strongly induce accumulation of tobacco leaf anthocyanin during cooperative expression of the CmMYB6 and MrbHLH1. The MYB transcription factor can be used in transcription regulation of plant anthocyanin biosynthesis and plant color modification.

Description

technical field [0001] The invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a device involved in the transcription regulation of chrysanthemum petal anthocyanin biosynthesis MYB transcription factor CmMYB6 (SEQ: NO. 22). Background technique [0002] Chrysanthemum is a traditional famous flower in my country and one of the important flowers in the world. It is the second largest cut flower in the world after roses. my country has independently cultivated nearly 250 varieties of cut-flower chrysanthemums, among which the single-head cut-flower chrysanthemum accounts for about half of the total cut-flower production. Flower color is one of the important ornamental traits of chrysanthemums. The flower colors of single-head cut chrysanthemums are mostly yellow and white in their original colors. In addition to yellow and white, there are also green, blue, pink and red chrysanthemum varieties cultivated independently in m...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8261
Inventor 李方刘晓芬向理理殷学仁陈昆松
Owner ZHEJIANG UNIV
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