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Applications of GLDH gene of brassica campestris ssp. Chinensis makino for enhancing Vc content of Brassica norinosa

A technology of head cabbage and black cabbage, which is applied in the field of molecular biology and biology, to achieve the effect of short experiment period, small workload and strong operability

Active Publication Date: 2008-11-19
NANJING AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Wutaicai is a kind of non-heading cabbage, and there is no report on the successful transgenic of Wutaicai

Method used

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  • Applications of GLDH gene of brassica campestris ssp. Chinensis makino for enhancing Vc content of Brassica norinosa
  • Applications of GLDH gene of brassica campestris ssp. Chinensis makino for enhancing Vc content of Brassica norinosa

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0041] (1) Construction of GLDH gene overexpression vector

[0042] 1. Cloning of GLDH gene coding region sequence

[0043] Design a pair of specific primers S1 / A1 according to the full-length cDNA sequence of non-heading Chinese cabbage GLDH gene (Genbank Accession No AY899298):

[0044] S1: 5′-GC TCTAGA CGCCTGAACTAAAACAAAAATGC-3′ XbaI

[0045] A1: 5′-TCC CCCGGG CGGCTTCACTCTTCTTACAAACACT-3′SmaI

[0046] Using the leaves of non-heading Chinese cabbage'Suzhou Qing' (a local variety in Jiangsu, purchased from the market) as the material, total RNA was extracted as a template, the first strand of cDNA was synthesized by reverse transcription, and PCR amplification was performed with ExTaq enzyme to amplify the integrity of GLDH Sequence of coding region. The reaction conditions are: 94°C 3min; 94°C 1min, 45°C 1.5min, 72°C 3min, 5 cycles; 94°C 45s, 64.4°C 1min, 72°C 2.5min, 72°C, 30 cycles; 72°C 10min; 4°C storage.

[0047] In order to facilitate the construction of the vector, accor...

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Abstract

The invention relates to an application of non-heading Chinese cabbage GLDH gene in increasing Vc content in Wuta-tsai, belonging to the molecular biology and biotechnological field. A GLDH gene integrated coding region sequence is obtained by means of a RT-PCR technology so as to further construct the plant excessive expression carrier of the sequence and to introduce agrobacterium LBA4404. Technical proposals such as a culture medium-containing formula and an operation flow are optimized, and a Wuta-tsai transgenic genetic transformation system is established. An agrobacterium mediating method is adopted to convert the plant excessive expression carrier into Wuta-tsai. A transgenic plant is obtained through a kanamycin resistant sieve, and a PCR technology is adopted to complete the identification of target gene integrated Wuta-tsai genome. The application verifies that the gene plays an important role in Vc synthesis pathway, and successfully obtains the transgenic plant of excessive expressed GLDH; compared with a non-transgenic plant, the maximum GLDH gene expression of the transgenic plant is 28.5 times of that of the non-transgenic plant, and maximum plant Vc content is 1.8 times of that of the non-transgenic plant. Therefore, the application provides both theoretical foundation and practical experience for improving vegetable quality through genetic engineering in the future.

Description

1. Technical Field [0001] The invention relates to the application of non-heading Chinese cabbage GLDH gene to increase the Vc content of Wutacai plants, belongs to the field of molecular biology and biotechnology, and is dedicated to the genetically modified molecular breeding of Wutacai. 2. Background technology [0002] Vitamin C (vitamin C, hereinafter referred to as Vc) in plants, also known as L-ascorbic acid (AsA), not only has very important physiological functions for the plant's own antioxidant system, photosynthetic protection, and regulation of growth and development, but also Provide humans with a rich and convenient source of Vc (humans cannot rely on their own synthesis and storage of AsA, they need to ingest from food). At present, the natural AsA market is very broad, while the industrial synthesis of AsA has high economic costs and huge environmental energy consumption. Researchers also hoped to use microorganisms to directly convert cheap feed into AsA, but so ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/82C12Q1/68A01H1/00A01H5/00
Inventor 李英高红亮侯喜林史公军
Owner NANJING AGRICULTURAL UNIVERSITY
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