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
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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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