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High affinity ammonium transport factor fuse gene and its use in transgenic plant

A technology that combines genes and high affinity, and is applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of reducing production costs

Active Publication Date: 2007-07-25
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there have been no literature or patent reports on transgenic crops that have successfully overexpressed nitrogen uptake-related proteins and obtained high-efficiency nitrogen uptake in soil at home and abroad.

Method used

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  • High affinity ammonium transport factor fuse gene and its use in transgenic plant
  • High affinity ammonium transport factor fuse gene and its use in transgenic plant
  • High affinity ammonium transport factor fuse gene and its use in transgenic plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Arabidopsis root-specific expression promoter AtPRP3 promoter clone

[0093] Using Arabidopsis thaliana genomic DNA as a template, the 1733bp AtPRP3 gene promoter was amplified by PCR method, and the amplified product was recovered and cloned by TA.

[0094] (1) PCR amplification of the target fragment

[0095] Specific primers were designed according to the sequence of the promoter region of the Arabidopsis thaliana root-specific expression AtPRP3 gene published in GenBank, and a BglII restriction site was introduced into the downstream primers.

[0096] Upstream primer: 5'-TAGAAGAGTTGTAACGCAAGCTGTG-3'

[0097] Downstream primer: 5'- AGATCT ACCATTGCTGAGCGCTTGGC-3' (BglII cut point introduced)

[0098] Genomic DNA of Arabidopsis thaliana was extracted according to a conventional method, and the genomic DNA was used as a template to carry out PCR amplification using the above primers to prepare AtPRP3 gene promoter fragments.

[0099] PCR reaction system: ...

Embodiment 2

[0121] Example 2: Arabidopsis high-affinity ammonium transport factor AtAMT1; 1 gene cloning

[0122] According to the sequence information (GenBank accession number X75879) of Arabidopsis thaliana AtAMT1;1 gene cDNA published in GenBank, specific primers were designed, and Bg1 II and BstE II restriction sites were introduced at the 5' end, respectively. The size of the PCR product should be 1526bp.

[0123] Upstream primer: 5'- AGATCT CAACATGTCTTGCTCGGCCA-3' (Bgl II cut point introduced)

[0124] Downstream primer: 5'- GGTCACC AAATCAAACCGGAGTAGGT-3' (BstEII cut point introduced)

[0125] Genomic DNA of Arabidopsis thaliana was extracted according to conventional methods, and AtAMT1; 1 gene was amplified by PCR using genomic DNA as a template, and then TA cloned with pUCm-T vector. The PCR reaction system, reaction conditions, recovery, cloning and sequencing of target fragments are the same as in Example 1.

Embodiment 3

[0126] Example 3: Construction of AtPRP3-AtAMT1 using pCAMBIA1301 vector; 1 fusion gene

[0127] (1) Extract vector plasmid pCAMBIA1301 from Escherichia coli (this strain can be obtained from other laboratories or purchased from reagent companies), and use EcoR I / BstE II double enzyme digestion to recover a large vector fragment.

[0128] (2) Extract the plasmid from the TA clone prepared in Example 1, digest it with Bgl II / EcoR I, and recover the AtPRP3 promoter fragment by agarose gel electrophoresis (trap method, the same as in Example 1).

[0129] (3) Extract the plasmid from the TA clone prepared in Example 2, digest it with Bgl II / BstE II, and recover the AtAMT1;1 gene fragment by agarose gel electrophoresis.

[0130] (4) The above three fragments were ligated overnight at 16° C. under the catalysis of ligase to complete the construction of the AtPRP3-AtAMT1; 1 fusion gene on the pCAMBIA1301 vector (see FIG. 1 ).

[0131] Connection system:

[0132] Reagent

...

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Abstract

Disclosed is a high affinity ammonium transport factor fuse gene and its use in transgenic plant, wherein promotors of Arabidopsis thaliana root system specific expression genes AtPRP3 and high-affinity ammonia transferring factor gene AtAMT1,1 are utilized to construct fusion gene AtPRP3-AtAMT1,1, and the fusion genes are successfully transferred into pattern plants of Arabidopsis thaliana and minitype tomatoes, making them exhibit rather strong nitrogen stress tolerant capacity.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically uses the promoter of Arabidopsis thaliana root-specific expression gene AtPRP3 and the high-affinity ammonium transport factor AtAMT1;1 gene to construct AtPRP3-AtAMT1;1 fusion gene and its application in transgenic plants Applications. Background technique: [0002] Modern agricultural production needs to apply a large amount of fertilizers to the soil, which not only requires large economic investment, but also causes pollution and damage to the geological environment, thus greatly limiting the development of modern agriculture. At the same time, the rapid growth of the world population has caused great pressure on the supply of agricultural and sideline products. The high cost, low efficiency and geological environment damage caused by changing the modern agricultural production mode have become a major issue to be solved worldwide, especially in developing countries....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/82C12N15/29C12N15/66
Inventor 王宁宁张茹林景瑜李鹏丽李晔崔孟祥
Owner NANKAI UNIV
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