Nitrate reductase gene BcNR of Chinese cabbage

A technology of head cabbage and reductase, which is applied in the field of molecular biology and biology, and can solve problems such as difficult to obtain satisfactory results

Inactive Publication Date: 2008-10-22
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Regarding measures to reduce nitrate content, predecessors have proposed many methods, such as economical and rational application of nitrogen fertilizers, improvement of vegetable ecological conditions, selection of appropriate harvesting periods, appropriate storage and processing, and pre-eating treatment, etc., but the application of these methods The effect is affected by many internal and external factors, and it is difficult to achieve satisfactory results in production

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  • Nitrate reductase gene BcNR of Chinese cabbage

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Embodiment Construction

[0027] (1) Cloning of BcNR gene cDNA

[0028] The non-heading Chinese cabbage variety 'Suzhouqing' (a local variety in Jiangsu, purchased from the market) was selected as the material. After the cotyledons were flattened, total RNA was extracted from the leaves according to the instructions of the Simply P Total RNA extraction kit from Bioflux Company, and 2 μg of total RNA was taken to synthesize cDNA. The cDNA product was digested with RNase.

[0029] According to the conserved nucleotide sequences in Arabidopsis NIAI and NR Brassica napus, design three pairs of specific primers NRF1 / NRR1: SEQ ID NO.3 / SEQ ID NO.4, NRF2 / NRR2: SEQ ID NO.5 / SEQ ID NO.6, NRF3 / NRR3: SEQ ID NO.7 / SEQ ID NO.8, use LA Taq enzyme to carry out conventional polymerase chain reaction (PCR), amplify the BcNR gene segmentally, the amplification program is: 94°C Pre-denaturation for 5 minutes, denaturation at 94°C for 30 sec, renaturation at 55°C for 30 sec, extension at 72°C for 1 min and 30 sec, after 35 ...

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Abstract

The invention relates to clone and application of a loose leaved Chinese cabbage nitratase gene BcNR, belonging to the molecular biology and biotechnology field. Total RNA is extracted from a loose leaved Chinese cabbage leaf and then 2 gamma of the total RNA is reversely transcribed into cDNA; three pairs of special primers are designed according to conservative nucleotide sequences in an NIAI (mouseearcress) and an NR (cabbage type rape) for routine polymerase chain reaction (PCR); PCR products are respectively connected to pMD18-T vectors; DH5alpha cells are transformed and sequencing is performed; full-length cDNA is obtained through quick amplification of 3' terminals and 5' terminals; a sense expression vector is further constructed and the NIAI is transformed. The transgenic NIAI has high nitratase transcription level. The genetic transformation vegetable can adjust the content of nitrate in the vegetable and improve the output and the quality and have very important economic benefit and social benefit.

Description

1. Technical field [0001] The invention relates to the cloning and application of a non-heading cabbage nitrate assimilation key enzyme gene BcNR cDNA, which belongs to the field of molecular biology and biotechnology. 2. Background technology [0002] Plants absorb NO from the environment 3 - , need to go through two assimilation steps: firstly, NO 3 - reduced to nitrite nitrogen (NO 2 - ), and then by nitrite reductase (Nitrite reductase, NiR) NO 2 - reduced to NH 4 + , in order to further participate in the synthesis of amino acids and proteins. NR is NO 3 - The first enzyme in the assimilation step is also the rate-limiting enzyme in the entire assimilation process and plays a key role in plant nitrogen metabolism. The enzyme is a homopolymeric multi-subunit protein, each subunit contains three prosthetic groups: flavin adenine dinucleotide (FAD), heme (heme) and a molybdenum-containing factor (Moco), each prosthetic group A base is a redox center. In highe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/06C12N15/10C12N15/82A01H1/00A01H5/00
Inventor 侯喜林孙菲菲
Owner NANJING AGRICULTURAL UNIVERSITY
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