Pear anthocyanin synthesis transcription factor PbMYB109 and application thereof

A technology for transcription factors and pigments, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems that need to be confirmed, the anthocyanin biosynthesis has an impact, and the PbMYB109 gene has not been reported to participate in the anthocyanin synthesis. The effect of promoting synthesis

Active Publication Date: 2018-09-28
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the fruit color of related plants is regulated by MYB transcription factors. The functions of some MYB tra

Method used

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  • Pear anthocyanin synthesis transcription factor PbMYB109 and application thereof
  • Pear anthocyanin synthesis transcription factor PbMYB109 and application thereof
  • Pear anthocyanin synthesis transcription factor PbMYB109 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Cloning and sequence comparison of embodiment 1PbMYB109 gene

[0027] 1. Design primers

[0028] Cloning primers were obtained through library screening and primer design; their specific sequences are as follows:

[0029] Clone-F1: 5'-ATGACGGCCCCAAACGACG-3'; (SEQ ID NO.3)

[0030] Clone-R1: 5'-CTAGGTAGTGGCAGCTGCTTGAAAG-3'. (SEQ ID NO.4)

[0031] 2. PCR reaction amplification

[0032] The PCR amplification reaction system included: 2 μL of Primer F (10 mmol L -1 ), Primer R (10mmol·L -1 ), Template cDNA, 29μL of ddH 2 O. 10 μL of 5×TransStart FastPfu Fly Buffer, 4 μL of 2.5mMdNTPs, 1 μL of 5×TransStart FastPfu Fly DNA Polymerase; PCR reaction conditions: 95°C for 2 min, 95°C for 20 s, 60°C for 2 s, 72°C for 2 s , 40 cycles, and a final extension of 10 min at 72°C.

[0033] 3. Purification and recovery

[0034] Then, 5 μL of the PCR product was subjected to 1.0% agarose gel electrophoresis to detect the amplification result, and the SanPrep Column PCR Product Rec...

Embodiment 2

[0040] Transient expression of embodiment 2PbMYB109

[0041] 1. Primer design

[0042] A pair of primers were designed at both ends of the sequence of the cloned PbMYB109 gene, and the primer sequences were:

[0043] Prime-F: 5'-GGCCGGTACCATGACGGCCC-3'; (SEQ ID NO.5)

[0044] Prime-R: 5'-CGGGATCCCTAGGTAGTGGCAGC-3'; (SEQ ID NO.6)

[0045] 2. PCR amplification

[0046] According to the analysis of the sequence, the expression vector pCambia1301-35SN has the enzyme cutting sites BamHI and KpnI, and through PCR amplification, the required enzyme cutting sites are respectively introduced before and after the target gene; the reaction system of the PCR amplification includes: 1.5μL Primer F (10mmol·L -1 ), 1.5μL Primer R (mmol L -1 ), 2μL Template cDNA, 32μL ddH 2 O, 10 μL 5×Prime STAR Buffer (Mg2+Plus), 4 μL dNTP; PCR reaction conditions were 98°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 58°C annealing for 5 s, 72°C extension for 1 min, 35 cycles, and finally 7...

Embodiment 3

[0074] Embodiment 3 Arabidopsis transformation

[0075] 1. Prepare 1 L of resuspended osmotic culture medium with a pH value of 5.7 for use. The culture medium includes: 1 / 2 MS medium, 500 μL Silwet L-77, 50 g of sucrose, and 0.5 g of MES.

[0076] 2. Construct the overexpression vector PCAMBIA1301-PbMYB109, and then transform it into Agrobacterium, inoculate the transformed Agrobacterium into an EP tube containing 1mL medium, cultivate for 13h, and then expand the culture to 100mL, continue Grow to OD600 =1.0 or so, enrich the cells, then add an appropriate amount of resuspended osmotic culture solution to resuspend the cells to OD 600 About 0.8.

[0077] 3. Prepare healthy Arabidopsis plants, cut them off when the plants grow terminal inflorescences, and then stimulate the occurrence of axillary inflorescences through the Agrobacterium cell suspension obtained in step 2. When more axillary inflorescences grow to 1- After transformation at a length of 10 cm, the T1 generati...

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Abstract

The invention discloses a pear anthocyanin synthesis transcription factor PbMYB109 and an application thereof. The nucleotide sequence of the transcription factor is as shown in SEQ ID NO. 1, and theamino acid sequence of protein encoded by the transcription factor is as shown in SEQ ID NO. 2. By an agrobacterium-mediated genetic transformation method, pear fruits are transiently transformed andArabidopsis thaliana is stably transformed by a PbMYB109 gene, obtained transgenic plants are subjected to phenotype identification, results indicate that the transgenic Arabidopsis thaliana has obvious anthocyanin accumulation on rosette leaves, seed pods and main stems as compared with mild types, the clonal PbMYB109 gene is a new transcription factor participating in anthocyanin synthesis, andanthocyanin synthesis can be promoted. Discovery of the transcription factor provides an important gene resource for gene engineering of exploration of anthocyanin synthesis routes and improvement offruit quality.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a method of separating and obtaining an encoding anthocyanin synthesis transcription factor PbMYB109 from the fruit of 'Hongzaosu' pear (Pyrusbretschneideri Rehd), and also relates to a pear anthocyanin synthesis transcription factor Application of PbMYB109 gene in regulating anthocyanin synthesis. Background technique [0002] Anthocyanins are water-soluble pigments that commonly exist in vacuoles in plants, and generally exist in the form of anthocyanins in plant fruits, flowers and leaves. Anthocyanins widely exist in plants, and are accumulated in many plant organs such as flowers, leaves, stems, and fruits. The accumulation of anthocyanins makes plants present a variety of colors, which can attract insects for pollination and facilitate seed formation and dispersal. At the same time, the accumulation of anthocyanins is also an important indicat...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/825
Inventor 张勇程丽娟汤浩茹陈清罗娅孙勃王小蓉马旭辉孔令灵
Owner SICHUAN AGRI UNIV
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