A transcription factor hvnlp2 derived from barley involved in the regulation of nitrate nitrogen and its use

A transcription factor and a technology encoding a transcription factor, applied in the field of transcription factor HvNLP2, can solve problems such as the lack of literature reports in research, and achieve the effect of improving nitrogen utilization rate and assimilation utilization efficiency

Active Publication Date: 2022-04-08
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no literature report on the genes related to nitrate nitrogen metabolism in barley using the method of reverse genetics

Method used

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  • A transcription factor hvnlp2 derived from barley involved in the regulation of nitrate nitrogen and its use
  • A transcription factor hvnlp2 derived from barley involved in the regulation of nitrate nitrogen and its use
  • A transcription factor hvnlp2 derived from barley involved in the regulation of nitrate nitrogen and its use

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: Fluorescence complementation experiment

[0062] Fluorescence complementation experiment is an important means to identify whether a gene is a nitrate-nitrogen-regulated gene. In order to determine whether HvNLP2 is a nitrate-nitrogen-regulated gene, the present invention screens out a homozygous transgenic line of HvNLP2 / nlp7-4, wherein nlp7-4 It is a mutant screened by EMS mutagenesis, in 5mM KNO 3 Grow vertically on solid medium for 5 days, observe root fluorescence ( figure 1 ).

[0063] Due to the AtNLP7 gene mutation, the fluorescence of yellow fluorescent protein induced by the nitrate nitrogen signal in the body was significantly reduced, so nlp7-4 showed a weak fluorescence phenotype. When we transformed the HvNLP2 gene into nlp7-4, the fluorescence of yellow fluorescent protein induced by the nitrate nitrogen signal in the transformed homozygous lines returned to the wild-type level of the system ( figure 1 ), indicating that HvNLP2 can play t...

Embodiment 2

[0066] Embodiment 2: Construction of HvNLP2 gene plant expression vector

[0067] Grow barley wild-type on 1 / 2 MS medium for 10 days, take whole seedlings and extract RNA, use the kit to reverse-transcribe the RNA into cDNA, use cDNA as a template, and use high-fidelity DNA polymerase to amplify the gene fragment of HvNLP2 , the primer sequences used in PCR are:

[0068] Upstream primer: 5'-TATAGTCGACATGGATGAGATTGGGACACC-3', (SEQ ID NO.3);

[0069] Downstream primer: 5'-TCTCGGTACCTCGACCGGATATTGAGCTACT-3', (SEQ ID NO.4).

[0070] The PCR product was electrophoresed in 1% agarose, and the target band was found according to the DNA Marker and the gel block was excised. Utilize the gel recovery kit (purchased from Omega Company) to recover the target DNA, carry out enzyme digestion (BamHI and SalI) to the recovered target DNA and the plant expression vector Psuper1300, and then digest the DNA fragment and vector with T 4 DNA ligase ligation. Then, the ligation product was tra...

Embodiment 3

[0076] Embodiment 3: Identification of transgenic positive plants

[0077] Pick the Agrobacterium cells stored at -80°C in 250mL LB culture medium, place them on a shaker at 28°C, and culture them with shaking for 16 hours. Agrobacterium was used to infect Arabidopsis wild-type plants by flower dipping method. After the plants are mature, collect the seeds, and spread the seeds in the water containing H + On the resistant 1 / 2 MS plate, the screened positive seedlings are the transgenic line T1 generation. The selected T1 generation seeds were transplanted into vermiculite for cultivation, and the T2 generation seeds were harvested. The T2 generation seeds in the H + The resistant 1 / 2 MS plate was cultured, and its survival rate was found to be 3 / 4. The surviving T2 plants were moved to vermiculite for culture, and the T3 generation seeds were harvested from a single plant. Seeds of the T3 generation in the H + The resistant 1 / 2 MS plates were cultured, and the homozygous ...

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Abstract

The invention discloses a transcription factor HvNLP2 derived from barley and involved in the regulation of nitrate nitrogen and its application. The full-length cDNA sequence of the gene is shown in SEQ ID NO.1. Compared with the recipient Arabidopsis thaliana, the Arabidopsis thaliana transformed with the full-length cDNA of the gene has an enhanced ability to respond to nitrate nitrogen, and the nitrate nitrogen in vivo is enhanced. The accumulation of nitrate nitrogen decreased, the activity of nitrate nitrogen reductase and amino acid content increased significantly, and the expression of genes related to nitrate nitrogen assimilation increased significantly; the protein encoded by this gene can also interact with nitrate on the promoter of the downstream target gene in cis The element NRE binds and activates its transcription, indicating that HvNLP2 plays an important role in the regulatory pathway of nitrate nitrogen signaling in barley.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a transcription factor HvNLP2 derived from barley and involved in the regulation of nitrate nitrogen and its application. Background technique [0002] Nitrogen is one of the macroelements needed for plant growth and development. The high and stable yield of modern agriculture is inseparable from the application of nitrogen fertilizer. Wheat crops are one of the main food crops in my country. They mainly need a lot of nitrogen in three growth and development stages: emergence stage, jointing stage, and filling stage. Appropriate amount of nitrogen fertilizer can promote the growth and development of wheat roots, stems, and leaves. , thereby improving photosynthetic efficiency and accumulation of nutrients; nitrogen application can also promote the differentiation and development of tillers and young panicles of plants, and is beneficial to the growth and developm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H5/06A01H5/10A01H6/20
CPCC07K14/415C12N15/8243C12N15/8261
Inventor 王勇高阳阳吕波田田齐盛东权姝璇聂振田
Owner SHANDONG AGRICULTURAL UNIVERSITY
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