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Gene PpRd for regulating fruit flesh cyanin synthesis and application thereof

A technology of anthocyanins and pulp, applied in the field of plant genetic engineering and biology, can solve problems such as the inability to apply peach pulp genetic breeding, achieve the effects of improving breeding efficiency, easy realization, and saving breeding time

Active Publication Date: 2015-04-22
WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cross-breeding needs to clarify the loci of key variations, so MYB transcription factors still cannot be applied to the genetic breeding of peach pulp color traits

Method used

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  • Gene PpRd for regulating fruit flesh cyanin synthesis and application thereof
  • Gene PpRd for regulating fruit flesh cyanin synthesis and application thereof
  • Gene PpRd for regulating fruit flesh cyanin synthesis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Extraction and determination of anthocyanins.

[0054] Take about 1 gram of peach pulp sample or 0.1 gram of tobacco sample, grind it with liquid nitrogen, and suspend it in 5 milliliters of precooled hydrochloric acid methanol (0.1% hydrochloric acid volume fraction) for 24 hours. Centrifuge at 3000g for 10 minutes, collect the supernatant, and evaporate to dryness in vacuum at 30°C on a rotary evaporator. The residue was resuspended with acidified deionized water (1.18 mM HCl) and transferred to a preconditioned CNWBOND LC-C18SPE column. Anthocyanins were eluted with 1 ml of methanol, filtered through a 0.22-micron membrane filter, and loaded for detection by high-performance liquid chromatography.

Embodiment 2

[0055] Embodiment 2: Real-time fluorescent quantitative PCR detects the relative content of gene transcripts

[0056] Approximately 0.2 g of the sample was ground with liquid nitrogen for RNA extraction. For RNA extraction, the plant polyphenol polysaccharide total RNA extraction kit from Beijing Zhuangmeng Company was used. Use 1.2% agarose gel to check the quality of RNA. RNA concentration was detected with a NanoDrop instrument. The first-strand cDNA was synthesized using the PrimeScript Reverse Transcriptase Kit (Bao Biology). The real-time fluorescence quantitative PCR system is 20 microliters, containing about 100 nanograms of cDNA, 0.2 micromole of each primer and 10 microliters of 2×SYBR premix Ex Taq TM (Bao Bio). The amplification program was as follows: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of amplification (95°C for 5 seconds, 60°C for 34 seconds). Each amplification well was replicated three times. The peach gene TEF2 was used as an ...

Embodiment 3

[0061] Example 3: Dual-luciferase reporter system detects the ability of transcription factors to activate promoters

[0062] Young N. benthamiana leaves were used as injection material. Activate Agrobacterium (GV3101) containing 35S-driven transcription factor vector or plant promoter vector containing pGreen 0800 in LB medium containing rifampicin and corresponding vector resistance at 28°C to OD 600nm The value is about 0.8, the bacteria are collected by centrifugation, and resuspended in the infection solution (10 mmol MgCl 2 , and 100 micromolar acetosyringone). Use a 1 ml sterile needle-free syringe to inject the infection solution containing bacteria into the leaves of N. benthamiana. Luciferase activity was detected after 2-3 days. The kit used was DLAR-2 kit (Targeting Systems, USA).

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Abstract

The invention discloses a gene PpRd for regulating fruit flesh cyanin synthesis and application thereof, relating to plant gene engineering and biotechnology. The gene PpRd nucleic acid sequence is disclosed as SEQ ID NO.1, and the protein sequence coded by the gene PpRd is disclosed as SEQ ID NO.2. The application of the PpRd gene is implemented by (1) designing a molecular marker WPS23 according to the sequence of the PpRd gene, and applying the molecular marker WPS23 in marker auxiliary screening in breeding; and (2) application of expression protein sequence and gene functions: detecting and screening cyanidin content related phenotype according to the expression level of the gene, or enhancing or lowering the gene expression level by genetic transformation to obtain the expected flesh color character. The invention obtains a key gene for cyanin accumulation. When the PpRd gene is used for marker auxiliary selection to instruct hybridization breeding of peaches, the screening can be performed in advance, thereby saving the breeding time and enhancing the breeding efficiency.

Description

technical field [0001] The present invention relates to plant genetic engineering and biotechnology, in particular to a gene PpRd that regulates the synthesis of anthocyanins in fruit pulp and its application; in particular, it relates to the cloning, genetic analysis, functional verification and Application; PpRd can regulate the expression of other transcription factors that regulate anthocyanin synthesis, thereby affecting the accumulation of anthocyanin in pulp; it also involves the screening of molecular markers for fruit tree cross breeding, so as to apply the gene in fruit tree breeding. Background technique [0002] Peach was cultivated in China about 4,000 years ago, and then spread to Europe along the Silk Road during the Roman period, and now it has become one of the important economic fruit trees in temperate regions (W.yu-lin.Peach growing and germplasm in China.Acta Horticulturae 173:51 -5). According to the color of the pulp, peach varieties can be divided in...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12Q1/68
Inventor 韩月彭周晖王鲁谷超
Owner WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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