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A method of cultivating transgenic plants with enhanced plant root development and delayed leaf senescence

A technique for the aging of plant roots and leaves, applied in the field of genetic engineering

Active Publication Date: 2019-03-08
INST OF CEREAL & OIL CROPS HEBEI ACAD OF AGRI & FORESTRY SCI +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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  • A method of cultivating transgenic plants with enhanced plant root development and delayed leaf senescence
  • A method of cultivating transgenic plants with enhanced plant root development and delayed leaf senescence
  • A method of cultivating transgenic plants with enhanced plant root development and delayed leaf senescence

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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 EST sequence of wheat GS2 Unigene Ta.24748 was spliced, 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 preliminarily sequencing. Then, primers were designed according to the obtained sequence for screening the Xiaoyan 54 BAC 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 of ...

Embodiment 2

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

[0032] 2.1 Gene cloning: The total RNA of wheat variety Xiaoyan 54 was extracted with Trizol reagent from Invetrogen Company. After DNase I (RNase-free) treatment, 4 μg was reverse-transcribed with M-MLV reverse transcriptase from Promega Company to synthesize cDNA. One strand is used as a template for PCR amplification to obtain a PCR amplification product.

[0033] The sequences of PCR primers (respectively introducing BamH I and Kpn I restriction sites into the forward primer and reverse primer) 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 (10 ...

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 BamH I and Kpn I (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 enzymes BamH I and Kpn I 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 endonucleases Pst I and BamH I, and recover a...

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Abstract

The invention discloses a method for cultivating transgenic plants with enhanced plant root development and delayed leaf senescence. The gene shown in SEQ ID NO: 1 is introduced into the target plant and stably expressed and inherited to obtain root systems compared with non-transgenic control plants. Transgenic plants with enhanced development and delayed senescence. The transgenic plants can significantly promote the growth and development of the root system, and significantly enhance the photosynthesis of the functional leaves in the later stage of growth, delay the degradation of chlorophyll in the leaves, delay the senescence of the transgenic plants, and then increase the crop yield.

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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/113C12N15/11C12N15/82A01H5/06A01H5/00A01H6/46
Inventor 胡梦芸赵学强刘茜门福圆张颖君孙丽静童依平李辉
Owner INST OF CEREAL & OIL CROPS HEBEI ACAD OF AGRI & FORESTRY SCI
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