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A method for increasing anthocyanin content

An anthocyanin content and anthocyanin technology, applied in the field of genetic engineering, can solve the problems of down-regulation of gene expression, reduction of anthocyanin synthesis, etc., and achieve the effect of increasing the content

Active Publication Date: 2017-11-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Davison et al. (2002) found that overexpressing the HYD gene in Arabidopsis, the content of carotenoids increased and the accumulated anthocyanins decreased; Cao Hongbo (2012) found a large amount of carotenoids when studying apple callus. Accumulation will lead to a decrease in the synthesis of anthocyanins, and the expression of genes that regulate anthocyanin synthesis will be down-regulated

Method used

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  • A method for increasing anthocyanin content
  • A method for increasing anthocyanin content
  • A method for increasing anthocyanin content

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Construct the expression plasmid of citrus phytoene synthase gene CitPSY.

[0080] For details on the amplification of CitPSY and the method of constructing CitPSY expression plasmid, please refer to the literature: Zhang Jiancheng. Functional analysis of red navel orange carotenoid synthase genes (CsPSY, CsLCYb) and the effect of CrtB transgene on carotenoid biosynthesis. [Ph.D. Dissertation ]. Wuhan: Huazhong Agricultural University, 2009. The cDNA of the cloned CitPSY is 1540bp in length, contains an open reading frame of 1308bp, encodes 436 amino acids, has a 49bp untranslated region at the 5' end, and contains a 160bp non-coding sequence at the 3' end; predicted molecular weight and isoelectric point 49.5kDa and 5.86, respectively.

[0081] The navel orange carotenoid synthase gene (CsPSY) in the above-mentioned documents is named as the citrus phytoene synthase gene (CitPSY) in the description of the present invention.

[0082] The expression vector pBI-CsPSY co...

Embodiment 2

[0085] Agrobacterium tumefaciens mediates the genetic transformation of strawberry leaves, and the specific process is shown in Figure 2.

[0086] 1. Preparation of Strawberry Aseptic Test Tube Plantlet Materials

[0087] Inoculate the sterile test-tube seedlings on the rapid propagation solid medium (adding the sucrose with a final concentration of 30g / L and the agar with a final concentration of 7g / L on the basis of the rapid propagation medium for plants) for propagation and cultivation, single plant propagation, every Four explants were inoculated in each Erlenmeyer flask and placed in a light culture room for cultivation. Culture conditions: temperature 25°C, light intensity 2000lx, photoperiod 16h / d. After 2-3 months, a large number of sterile test-tube seedlings were obtained and used as experimental materials for future use.

[0088] 2. Inoculation of explants

[0089] Take the leaves grown on the medium of step 1 for about 30-40 days and have the same growth position...

Embodiment 3

[0124] Molecular biological identification of embodiment 3 transgenic strawberry

[0125] 1. Strawberry DNA extraction: Leaf DNA was extracted using the CTAB small amount method. For specific steps, see (Yunjiang Cheng, Wenwu Guo, Hualin Yi, Xiaomin Pang, Xiuxin Deng. An efficient protocol forgenomic DNA extraction from Citrus species. Plant Molecular Biology Reporter, 2003, 21:177a-177g).

[0126] 2. Identification by PCR

[0127] Example 2 was used to obtain 14 resistant seedlings screened by Kan. Using CitPSY-specific primers (fwd5'-CGGGATCCATGTCTGTTACA-3', rev5'-GGGGTACCTTAAAGCCTTACT-3') (Zhang Jiancheng. Red navel orange carotenoid synthase genes (CsPSY, CsLCYb) function analysis and crtB transgene on carotenoid biosynthesis Impact. [PhD dissertation]. Wuhan: Huazhong Agricultural University, 2009), DNA was extracted from the leaves of resistant seedlings, and PCR amplification was performed. The positive control is the Agrobacterium plasmid, and the negative control i...

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Abstract

The present invention relates to the use of citrus phytoene synthase gene for increasing the anthocyanin content of strawberries and a method for increasing the anthocyanin content of strawberry by using the citrus phytoene synthase gene, comprising the following steps: constructing citrus phytoene synthase gene Lycopene synthase gene expression plasmid; optimization of strawberry genetic transformation system; use of Agrobacterium-mediated method to transfer citrus phytoene synthase gene expression plasmid into strawberry, and obtain citrus phytoene synthase gene expression plasmid Strawberry: Molecular biological identification was carried out on the strawberry transformed with the citrus phytoene synthase gene, and the content of anthocyanin in the strawberry expressing the citrus phytoene synthase gene was detected. Utilizing the citrus phytoene synthase gene and the above-mentioned method can significantly increase the content of anthocyanins in the strawberry.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for increasing the content of strawberry anthocyanins by utilizing the citrus phytoene synthase gene (CitPSY). Background technique [0002] Strawberry (Fragaria anannassa Duch.) belongs to the perennial herbaceous plant of the genus Strawberry in the Rosaceae family. The whole genome of strawberry is relatively small, and the genome size of diploid is about 240Mbp. It does not have high requirements on the growth environment and can be planted anywhere. Compared with other fruit trees, the growth period of strawberry is relatively short, and fruits can be obtained in a short time. One of the new ways of strawberry breeding by genetic engineers, its regeneration and genetic transformation systems are relatively mature. In addition, strawberry is asexual reproduction, and after the target gene is transferred into the plant for expression, it can be preserved and mult...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12P17/06C12N9/00C12N15/84
Inventor 徐娟贾楠李绍杰吴海静田静赵甜甜
Owner HUAZHONG AGRI UNIV