Chrysanthemum bhlh transcription factor involved in the regulation of anthocyanin biosynthesis

A technology of biosynthesis and transcription factors, applied in the field of plant molecular biotechnology and genetic engineering, can solve the problems that limit the diversification of the chrysanthemum flower color quality improvement and breeding industry.

Active Publication Date: 2018-04-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Involved in the regulation of plant anthocyanin biosynthesis wxya Members have been identified in many plants, but the members involved in the transcriptional regulation of anthocyanin biosynthesis in chrysanthemum wxya Members have not been reported so far, which greatly limits the development of chrysanthemum color quality improvement breeding and industrial diversification

Method used

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  • Chrysanthemum bhlh transcription factor involved in the regulation of anthocyanin biosynthesis
  • Chrysanthemum bhlh transcription factor involved in the regulation of anthocyanin biosynthesis
  • Chrysanthemum bhlh transcription factor involved in the regulation of anthocyanin biosynthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Chrysanthemum CmbHLH2 gene cloning

[0016] Based on the information of the chrysanthemum RNA-Seq database, the genes that may be involved in the regulation of chrysanthemum anthocyanin biosynthesis were screened out. wxya transcription factor CmbHLH2 Partial sequence (SEQ: NO. 2). Using gene-specific primer 2 (GSP2) SEQ: NO. 3, and nested gene-specific primer 2 (NGSP2) SEQ: NO. 4, obtained by using 3' rapid amplification of cDNA ends (3' RACE) technology CmbHLH2 The 3' non-coding region (3'UTR) sequence (SEQ: NO. 5). Then apply gene-specific primer 1 (GSP1) SEQ: NO. 6, and nested gene-specific primer 1 (NGSP1) SEQ: NO. 7, using 5' cDNA end rapid amplification (5' RACE) technology to obtain CmbHLH2 The 5' non-coding region (5' UTR) sequence (SEQ: NO. 8). From the start codon (ATG) to the stop codon was spliced ​​according to the resulting 3' and 5' UTRs. CmbHLH2 (SEQ: NO. 1) full-length sequence.

[0017] The first round of PCR program in RACE is ...

Embodiment 2

[0020] Example 2: Chrysanthemum CmbHLH2 Gene expression pattern analysis

[0021] (1) Experimental method

[0022] With Primer Premier 5, based on CmbHLH1 (SEQ: NO. 9) and CmbHLH2 The 3'UTR sequence of (SEQ: NO. 5) was designed respectively for real-time quantitative PCR (QPCR) primer combinations SEQ: NO. 10 and SEQ: NO. 11 and SEQ: NO. 12 and SEQ: NO. 13, and the PCR products contained termination The codons are 201 bp and 192 bp in length, respectively. The expression of chrysanthemum actin gene (SEQ: NO. 14) was used as an internal reference for analysis CmbHLH1 with CmbHLH2 16。 Expression pattern, its QPCR primer combination is SEQ: NO. 15 and SEQ: NO. 16. The specificity of all QPCR primers was verified by melting point curve analysis, gel electrophoresis analysis and QPCR product resequencing.

[0023] The petal RNAs of three chrysanthemum varieties with different flower colors were extracted, and cDNA was synthesized according to the instructions of the cDNA ...

Embodiment 3

[0026] Example 3: Analysis of regulatory target gene activity

[0027] (1) Experimental method

[0028] Application primer combination application primer combination SEQ: NO.17 and SEQ:NO.18, SEQ:NO.19 and SEQ:NO.20 and SEQ:NO.21 and SEQ:NO.22, respectively amplified CmbHLH1 (SEQ: NO. 9), CmbHLH2 (SEQ: NO. 1) and CmMYB6 (SEQ: NO. 23) full-length sequence. The FastStart High Fidelity PCR system (Roche) was used for amplification, and the PCR system was Buffer (with Mgcl 2 ) 2 µL, dNTP (2.5 µM) 1.6 µL, upstream and downstream primers (10 µM) 2 µL each, cDNA 0.5 µL, enzyme 0.5 µL, water 11.4 µL. PCR program is 95 o C 2 min; 95 o C 30s, 55-72 o C30s, 72 o C 30 s-3 min, 35 cycles; 72 o C 6 min, 16 o C hold. The PCR products were digested by restriction endonuclease combinations Not I and Spe I, Sal I and Apa I, Not I and Spe I, respectively, and loaded onto the pGreenII 0029 62-SK expression vector to recombine into an expression vector CmbHLH1- SK, CmbHLH2- SK an...

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Abstract

The invention provides a chrysanthemum bHLH transcription factor (CmbHLH2) involved in anthocyanin biosynthesis and regulation. A nucleotide sequence of the chrysanthemum bHLH transcription factor is shown as SEQ: NO.1. When the CmbHLH2 and a CmMYB6 coordinate to perform instantaneous over-expression in tobacco leaves, anthocyanin accumulation can be strongly induced to change the original green color of the leaves into a red color. Therefore, by means of transgenosis, over-expression of the CmbHLH2 in a plant is achieved or expression of the CmbHLH2 in the chrysanthemum plant is inhibited so that anthocyanin synthesis enhanced and inhibited transgenic plants can be obtained respectively, and the colors and luster of the transgenic plants can change with anthocyanin synthesis change.

Description

technical field [0001] The invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a novel chrysanthemum involved in the regulation of anthocyanin biosynthesis wxya transcription factor CmbHLH2 (SEQ: NO. 1). Background technique [0002] Chrysanthemum is one of the most important flowers in the world, and it is the second largest cut flower in the world after roses. Chrysanthemum is also a traditional famous flower in my country, with a long history and profound cultural heritage. So far, Chinese researchers have independently cultivated nearly 250 varieties of cut-flower chrysanthemums, among which single-head cut-flower chrysanthemums account 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 the yellow and white colors, there ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29A01H5/12A01H6/82
CPCC07K14/415C12N15/8222
Inventor 殷学仁李方向理理刘晓芬陈昆松
Owner ZHEJIANG UNIV
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