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Brassica napus phosphorus deficiency inducible expression promoter

A Brassica napus, phosphorus-deficiency-induced technology, applied in the field of genetic engineering, can solve the problems of wasting energy, affecting plant physiological metabolism, etc.

Inactive Publication Date: 2013-07-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This not only wastes energy, but also affects the normal physiological metabolism of plants

Method used

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  • Brassica napus phosphorus deficiency inducible expression promoter
  • Brassica napus phosphorus deficiency inducible expression promoter
  • Brassica napus phosphorus deficiency inducible expression promoter

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Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0015] The cloning of embodiment 1BnPR2 promoter

[0016] (1) The gene BnPR2 was discovered and identified by the inventors from Brassica napus (Eyou long pod; Liu Dingfu et al., extra long pod mutant of Brassica napus. Journal of Hubei Agricultural University, 1994, 14 (2): 1- 5) A phosphorus-deficiency-induced expression gene isolated in 5). Under normal conditions, the gene was weakly expressed in the root of rapeseed, but not in the leaf; under the condition of phosphorus deficiency, the expression of the gene increased sharply in the root and leaf. By performing BLAST analysis on the gene sequence of BnPR2 (http: / / brassica.bbsrc.ac.uk / BrassicaDB / blast_form.html), a Chinese cabbage BAC (accession number CU695313) consistent with the sequence of BnPR2 was found in the BrassicaDB database. The following primer pairs were designed according to the BAC sequence:

[0017] PF: 5'- CCTGCAGG AATTTTAGAATGTTGTGTCG-3',

[0018] PR: 5'- TCTAGA TCTTTTGATTTGTGGTGTTGTGC-3'.

[00...

Embodiment 2

[0021] The construction of embodiment 2 plant expression vectors

[0022] To determine the expression characteristics of the BnPR2 promoter, a plant expression vector containing the BnPR2 promoter and the reporter gene GUS was constructed. The specific method is:

[0023](1) Select the clone with the correct promoter sequence in Example 1, shake the bacteria in a large amount in LB medium containing 50 μg / mL ampicillin, and extract the plasmid with a small amount of plasmid extraction kit from Dopp Company. For specific methods, refer to Dopp The company's instructions for the kit. Subsequently, the plasmid containing the BnPR2 promoter and the pBI121 vector were double-digested with restriction endonucleases Sda I and Xba I (gifted by Dai Shutao, the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, which has been used in the previous patent application of Huazhong Agricultural University. mention the carrier). The digestion system is as f...

Embodiment 3

[0026] Embodiment 3 utilizes plant expression vector P BnPR2 :GUS transformation of Arabidopsis

[0027] (1) Preparation of Agrobacterium Competent: Inoculate a single colony of Agrobacterium GV3101 (gifted by Zhang Jinzhi, Huazhong Agricultural University College of Horticulture and Forestry) in 50mL YEB culture medium (beef extract 5.0g / L, tryptone 5.0g / L, yeast extract 1.0g / L, sucrose 5.0g / L, magnesium sulfate 0.5g / L, adjust pH to 7.0 with NaOH). 28°C, shake culture at 200 rpm to OD 260 0.5 or so, ice bath for 30 minutes. Collect the cells by centrifugation at 5000 rpm for 5 minutes, discard the supernatant, and then suspend the cells with 10 mL of 0.5 mol / L NaCl. Centrifuge again, suspend the bacteria in 1mL 20mmol / L CaCl 2 in solution. Aliquot 100 μL of the bacterial solution into frozen 1.5mL centrifuge tubes, quickly freeze in liquid nitrogen, and store in a -80°C refrigerator.

[0028] (2) Freeze-thaw transformation of Agrobacterium GV3101: Take 1.0 μg of the pla...

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Abstract

The invention belongs to the technical field of plant gene engineering. A phosphorus deficiency inducible expression promoter is cloned from Brassica napus, and has a nucleotide sequence shown as 1-1459th base group in a SEQ ID NO.1 in the sequence table; the promoter is fused with a report gene GUS and transferred into Arabidopsis; and GUS histochemical analysis shows that the promoter only has activity under phosphorus deficiency conditions, and is lack of activity under normal, nitrogen deficiency, potassium deficiency, sulfur deficiency and iron deficiency conditions. In addition to application to cultivation of low phosphorus tolerant or phosphorus efficiency tolerant crop, the promoter can also be applied to research on regulation mechanism of phosphorus deficiency induced expression gene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a rapeseed BnPR2 gene promoter and its application. The invention provides a rapeseed phosphorus deficiency-induced expression promoter and its application. Background technique [0002] Phosphorus is an essential macronutrient for plant growth and development. It is not only a component of important molecules such as nucleic acids, phospholipids, and ATP in plants, but also plays a series of important roles in the regulation of energy transfer and metabolic pathways. Plants primarily use their roots to absorb phosphorus in the form of phosphate. Although the total amount of phosphorus in the soil is very rich, the bioavailability of phosphorus in the soil is very low because phosphate is easily adsorbed and fixed by soil particles and metal ions, and phosphorus deficiency often becomes a factor limiting crop yield. The application of phosphorus fertilizer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A01H5/00
Inventor 徐芳森杨广哲
Owner HUAZHONG AGRI UNIV
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