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Method for culturing root-growth-enhanced leaf-ageing-delayed transgenic plant

A technique for transgenic plants and plant roots, applied in the field of cultivation of transgenic plants

Active Publication Date: 2016-02-03
河北省农林科学院粮油作物研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no relevant technical reports on the technical solutions of enhancing the expression of GS in plants to enhance root system development and delay plant senescence in the late growth period at home and abroad.

Method used

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  • Method for culturing root-growth-enhanced leaf-ageing-delayed transgenic plant
  • Method for culturing root-growth-enhanced leaf-ageing-delayed transgenic plant
  • Method for culturing root-growth-enhanced leaf-ageing-delayed transgenic plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, the discovery of TaGS2 protein and its coding gene

[0029] Firstly, the full-length was spliced ​​according to the EST sequence of wheat GS2UnigeneTa.24748, and GS2 gene primers were designed according to its conserved sequence, and Xiaoyan 54 genomic DNA was used as a template to screen out primers suitable for genome amplification, and the amplified products were initially sequenced. Then design primers according to the obtained sequence for screening the Xiaoyan 54BAC library. The screened BAC monoclonal plasmid was sequenced, and finally a TaGS2 gene was obtained, distributed on the A genome of wheat, named TaGS2-A1.

[0030] Primers were designed according to the genome sequence of the TaGS2-A1 gene, and the target fragment was amplified using the cDNA of wheat (wheat cultivar Xiaoyan 54) as a template. After sequencing, a cDNA sequence (SEQ ID NO: 1) of about 1.3 kb was obtained, which contained a complete ORF; it encoded a polypeptide consisting...

Embodiment 2

[0031] Embodiment 2, the cloning of TaGS2-A1 gene and the isolation of its promoter

[0032] 2.1 Gene cloning: Extract the total RNA of wheat variety Xiaoyan 54 with Trizol reagent from Invetrogen Company, after DNaseI (RNase-free) treatment, take 4 μg and use M-MLV reverse transcriptase from Promega Company to carry out reverse transcription to synthesize cDNA. The chain is used as a template for PCR amplification to obtain PCR amplification products.

[0033] The sequences of PCR primers (forward primer and reverse primer respectively introducing BamHI and KpnI restriction sites) are as follows:

[0034] Forward primer: 5′-AGTGGATCCATGGCGCAGGCAGTGGTGCCGGCGATG-3′ (SEQ ID NO: 4);

[0035] Reverse primer: 5'-TCAGGTACCTCATACCTTCAGCGCCAGCTTCTTG-3' (SEQ ID NO: 5).

[0036] PCR reaction system: 39.5 μl of ultrapure water, 5 μl of 10×PCR buffer, 2 μl of template, 1 μl of forward and reverse primers with a concentration of 10 μM, 0.5 μl of KOD enzyme (5u / μl), and 1 μl of dNTPs (1...

Embodiment 3

[0046] Embodiment 3, the construction of recombinant expression vector

[0047] Construction of a recombinant expression vector for TaGS2-A1 overexpression in wheat: First connect the TaGS2 gene to the Ubiquitin promoter of the expression vector pUBI:cas, then replace the Ubiquitin promoter with the TaGS2-A1 promoter, and finally construct a Sub+gene recombinant expression vector pTaGS2::TaGS2.

[0048] The specific process is as follows:

[0049] 3.1. Digest pMD18-TaGS2 with restriction endonucleases BamHI and KpnI (incomplete digestion), and recover a fragment of about 1.3kb (including the complete ORF sequence of TaGS2-A1).

[0050] 3.2. Digest pUBI:cas with restriction endonucleases BamHI and KpnI to recover the vector backbone.

[0051] 3.3. Ligate the small fragment recovered in step 1 with the vector backbone recovered in step 2 to obtain recombinant plasmid A (pUBI-TaGS2).

[0052] 3.4. Digest pMD18-pTaGS2 with restriction enzymes PstI and BamHI, and recover a fra...

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Abstract

The invention discloses a method for culturing a root-growth-enhanced leaf-ageing-delayed transgenic plant. The gene shown as SEQ ID NO:1 is introduced into a target plant and is stably expressed and inherited, so that the anti-ageing transgenic plant with growth-enhanced root is obtained compared with a non-transgenic contrast plant. Growth of root of the transgenic plant is obviously promoted, also the functional leaf photosynthesis is obviously enhanced at the late growth stage, leaf chlorophyll degradation is delayed, ageing of the transgenic plant is delayed, and therefore crop output is improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for cultivating transgenic plants that enhance the growth and development of plant roots and delay leaf senescence. Background technique [0002] Wheat is one of the most important food crops in the world, and its total area, total output and total trade volume all rank first in food crops. The increase of wheat production plays an extremely important role in world food security. Nitrogen is an essential nutrient element for crops. Since the 1980s, the input of nitrogen fertilizer in wheat production has become one of the powerful measures to increase its yield. However, the input of a large amount of nitrogen fertilizer not only reduces the nitrogen use efficiency, but also brings A series of environmental problems such as waste of resources and eutrophication of water bodies threaten human health and ecological environment safety. At present, people make ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/113C12N15/11C12N15/82A01H5/06A01H5/00
Inventor 胡梦芸赵学强刘茜门福圆张颖君孙丽静童依平李辉
Owner 河北省农林科学院粮油作物研究所
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