Nitrogen efficient fusion gene SA and application thereof

A gene fusion and high-efficiency technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of excessive nitrogen fertilizer application and low nitrogen use efficiency, and achieve the effects of promoting vegetative growth, improving remobilization efficiency, and increasing yield

Pending Publication Date: 2018-11-16
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problems of low nitrogen utilization efficiency and excessive application of nitrogen fertilizer in the planting process of crops, and to pro

Method used

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  • Nitrogen efficient fusion gene SA and application thereof
  • Nitrogen efficient fusion gene SA and application thereof
  • Nitrogen efficient fusion gene SA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Cloning of Soybean "Origin" Organ-Specific Promoters

[0053] Using soybean genomic DNA as a template, the 1800bp promoter was amplified by PCR method, and the amplified product was recovered and cloned by TA.

[0054] (1) PCR amplification of the target fragment

[0055] Specific primers were designed according to the soybean "source" organ-specific promoter sequence, the sequences of which are shown in SEQ ID No.2 and SEQ ID No.3, wherein the downstream primer introduces the Nco I cutting point.

[0056] Genomic DNA of soybean was extracted according to CTAB method, and the genomic DNA was used as a template to carry out PCR amplification with the above primers to prepare gene promoter fragments.

[0057] PCR reaction system:

[0058]

[0059] PCR reaction program:

[0060] 94°C for 5 minutes;

[0061] 94°C for 30 seconds, 58°C for 110 seconds, 25 cycles;

[0062] 72°C for 10 minutes;

[0063] (2) Cloning of target fragments and identification of p...

Embodiment 2

[0073] Example 2: Cloning of soybean autophagy key genes

[0074] First, using soybean cDNA as a template, the PCR method was used to amplify a 360bp functional gene, and the amplified product was recovered and TA cloned.

[0075] (1) PCR amplification of the target fragment

[0076] Design specific primers according to the sequence of known key genes of soybean autophagy, the sequences are as shown in SEQ ID No.4 and SEQID No.5, Nco I restriction site is introduced in the upstream primer, BstP I enzyme is introduced in the downstream primer cut site.

[0077] The quasi-soybean RNA was extracted by the Trizol method and reverse-transcribed into cDNA. Using the cDNA as a template, PCR amplification was carried out using the above primers to prepare gene fragments.

[0078] PCR reaction system:

[0079]

[0080] PCR reaction program:

[0081] 94°C for 5 minutes;

[0082] 94°C for 30 seconds, 58°C for 30 seconds, 25 cycles;

[0083] 72°C for 10 minutes;

[0084] (2) Clo...

Embodiment 3

[0094] Embodiment 3: utilize pCAMBIA3301 carrier to construct SA fusion gene

[0095] (1) The vector plasmid pCAMBIA3301 (purchased from a reagent company) was extracted from corresponding Escherichia coli engineering bacteria, and the large vector fragment was recovered by double digestion with Hind III / BstP I.

[0096] (2) Extract the plasmid from the TA clone prepared in Example 1, digest it with Hind III / Nco I, and recover the promoter fragment by agarose gel electrophoresis.

[0097] (3) Extract the plasmid from the TA clone prepared in Example 2, digest it with Nco I / BstP I, and recover the functional gene fragment by agarose gel electrophoresis.

[0098] (4) The above three fragments were ligated overnight at 16° C. under the catalysis of ligase to complete the construction of the expression vector SA-pCAMBIA3301.

[0099]

[0100] (5) Transform Escherichia coli DH5α competent cells with the ligation mixture, the method is the same as in Example 1.

[0101] (6) selec...

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Abstract

The invention relates to a nitrogen efficient fusion gene SA and application thereof. One fusion gene is constructed by way of manually splicing a source organ specific promoter and an autophagic keygene. The fusion gene is named SA, the sequence of which is as shown in SEQ ID NO.1. A plant binary expression vector can be constructed by means of the SA fusion gene and a target plant can be converted. The invention provides an embodiment in soybeans, verifying that after the SA fusion gene is transferred to the soybeans, the autophagic mediated nutrient substance remobilizing efficiency in thesource organ of transgenic soybeans can be improved specifically, and the tolerance of the transgenic soybeans to low nitrogen and low nutrient stress is improved obviously; meanwhile, the output ofthe transgenic soybeans planted in a low nitrogen condition is improved obviously. In addition, the fusion gene can be also transferred to other crops to obtain transgenic novel varieties which utilize nitrogen efficiently. The fusion gene SA has important meaning in reducing application of chemical fertilizers agriculturally, lowering the production cost and protecting the environment.

Description

【Technical field】: [0001] The invention belongs to the field of plant genetic engineering, in particular to construct a fusion gene SA by artificially splicing a "source" organ-specific promoter and a key gene of cell autophagy and its application in transgenic plants. The transgenic plant of the fusion gene has the traits of improving nitrogen utilization efficiency and yield. 【Background technique】: [0002] As the world's population continues to increase, people's demand for food is also increasing, but the arable land area is limited. Therefore, increasing crop production is an important way to avoid food crises. The means usually used to increase crop yields is mainly to increase the application of chemical fertilizers, which will not only increase production costs, but also cause pollution to the environment. Therefore, breeding new crop varieties that can efficiently utilize limited nutrients in the soil and produce high yields is of great significance to solving the...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N15/66A01H5/00A01H6/54
CPCC07K14/415C12N15/66C12N15/8222C12N15/8271
Inventor 王宁宁刘生王丹夏铜梅靳洁莉
Owner NANKAI UNIV
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