Chrysanthemum bHLH transcription factor involved in anthocyanin biosynthesis and regulation

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: 2015-12-16
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 bios

Method used

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  • Chrysanthemum bHLH transcription factor involved in anthocyanin biosynthesis and regulation
  • Chrysanthemum bHLH transcription factor involved in anthocyanin biosynthesis and regulation
  • Chrysanthemum bHLH transcription factor involved in anthocyanin biosynthesis and regulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: chrysanthemum chrysanthemum Cmbhlh2 Gene clone

[0016] According to the chrysanthemum RNA-SEQ database information, screening out the regulation that may participate in chrysanthemum anthocyanoside biological synthesis BHLH Transcription factor Cmbhlh2 Partial sequence (SEQ: No.2).Application of genetelite 2 (GSP2) SEQ: NO.3, and nest genomitiax (NGSP2) SEQ: No.4, use 3'CDNA to rapid amplification (3'race) technology Cmbhlh2 The 3'UTR sequence (SEQ: NO.5).Then apply genes of genes 1 (GSP1) SEQ: No.6, and nest genomitite 1 (NGSP1) SEQ: No.7, and use 5'CDNA to rapid amplification (5'race) technology Cmbhlh2 The 5'UTR sequence (SEQ: NO.8).The self -started password (ATG) to the termination codon is obtained based on the 3 'and 5'UTR stitching Cmbhlh2 (SEQ: NO.1) full -length sequence.

[0017] The first round of PCR programs in race is 94 o C5min; 94 o C10S, 72 o C2min30s, 5 cycles; 94 o C10S, 70 o C30S, 72 o C2min30s, 5 cycles; 94 o C10S, 67 o C30S, 72 o C2min30s,...

Embodiment 2

[0020] Example 2: Chrysanthemums Cmbhlh2 Gene expression mode analysis

[0021] (1) Experimental method

[0022] Use PrimerPremier5, based on the basis Cmbhlh1 (SEQ: NO.9) and Cmbhlh2 (SEQ: NO.5) of the 3'UTR sequences designed real -time quantitative PCR (QPCR) primer combinations SEQ: No. 10 and SEQ: No.11 and SEQ: No. 12 and SEQ: No.13.Code, the length of 201BP and 192BP, respectively.Using the expression of chrysanthemum Actin gene (SEQ: No.14) as an internal reference analysis Cmbhlh1 and Cmbhlh2 The expression mode, its QPCR primer combination is SEQ: No.15 and SEQ: No.16.All QPCR primers are analyzed by melting point curve, gel electrophoresis analysis, and QPCR products re -sequencing verification.

[0023]Extract three different colorful chrysanthemums of chrysanthemum RNA, and refer to CDNA synthesis Kit (BIO-Rad, USA) instructions to synthesize CDNA.Refer to the SSOFASTEVAGREENSUPERMIXKIT (BIO-Rad, USA) instruction manual to carry out QPCR analysis, using a 20μL system:...

Embodiment 3

[0026] Example 3: Regulating target genetic activity analysis

[0027] (1) Experimental method

[0028] Applying primers to apply primer combinations SEQ: No.17 and SEQ: No.18, SEQ: No.19 and SEQ: No. 20 and SEQ: No.21 and SEQ: No. 22, respectively expanded respectively Cmbhlh1 (SEQ: NO.9), Cmbhlh2 (SEQ: NO.1) and CmmyB6 (SEQ: NO.23) full -length sequence.Select FastStartHighfidelitypcrsystem (Roche) during amplification, and the PCR system is buffer (withmgCl 2 ) 2 μL, DNTP (2.5 μm) 1.6 μL, 2 μL of upstream and downstream primers (10 μm), CDNA0.5μL, enzyme 0.5 μl, water 11.4 μl.PCR program is 95 o C2min; 95 o C30S, 55-72 o C30S, 72 o C30S-3min, 35 cycles; 72 o C6min, 16 o Chold.PCR products have been limited to the digestion of NOTI and SPEI, SALI, and APAI, NOTI, and SPEI, respectively, respectively. Cmbhlh1- SK, Cmbhlh2- SK and CMMYB6- SK.Translate the electric carrier of the expression carrier that is finally confirmed to be correctly constructed into the GV3101 :: PSOUP Agrob...

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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 present invention belongs to the field of plant molecular biotechnology and genetic engineering, involving a new chrysanthemum involved in the regulation of biosynthesis BHLH Transcription factor Cmbhlh2 (SEQ: NO.1). Background technique [0002] Chrysanthemum is one of the world's important flowers, and is the second largest cut flower in the world second only to roses.Chrysanthemum is also a traditional famous flower in my country, with a long history and deep cultural accumulation.So far, Chinese researchers have independently cultivated nearly 250 varieties of chrysanthemum cutting, of which about half of the total number of cut flowers and chrysanthemums accounted for about half of the total number of cut flowers.The color is one of the important ornamental traits of chrysanthemums. The color of the chrysanthemums and chrysanthemums is mostly yellow and white. In addition to the yellow and white system, the chrysanthemum varieties independently cul...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29A01H5/00
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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