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Nitrate nitrogen regulating and control gene ZmNRG2.7 from corn and purpose thereof

A technology for regulating genes, zmnrg2.7, applied in genetic engineering, plant gene improvement, application, etc., can solve problems such as corn research that has not been reported, and achieve the effect of improving nitrogen use efficiency

Active Publication Date: 2019-08-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As one of the four major food crops in the world, maize has a high nitrogen requirement, but so far the NO in maize 3 - Regulatory gene research has not been reported

Method used

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  • Nitrate nitrogen regulating and control gene ZmNRG2.7 from corn and purpose thereof
  • Nitrate nitrogen regulating and control gene ZmNRG2.7 from corn and purpose thereof
  • Nitrate nitrogen regulating and control gene ZmNRG2.7 from corn and purpose thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: Fluorescence complementation experiment

[0059] Fluorescence complementation experiment is an important means to identify whether a gene is a nitrate-nitrogen-regulated gene. In order to determine whether ZmNRG2.7 is a nitrate-nitrogen-regulated gene, the present invention screens out a homozygous transgenic line of ZmNRG2.7 / nrg2-3, Among them, nrg2-3 is the mutant obtained by EMS mutagenesis screening, in 5mM KNO 3 Grow on solid medium for 5 days and observe the fluorescence ( figure 1 ).

[0060] Due to the mutation of the AtNRG2 gene, the fluorescence of yellow fluorescent protein induced by the nitrate nitrogen signal in the body was significantly reduced, so nrg2-3 showed a weak fluorescence phenotype. When we transformed the ZmNRG2.7 gene into nrg2-3, the fluorescence of yellow fluorescent protein induced by the nitrate nitrogen signal in its transformed homozygous lines returned to the wild-type level of the system ( figure 1 ), indicating that ...

Embodiment 2

[0063] Embodiment 2: Construction of ZmNRG2.7 plant expression vector

[0064] The wild-type B73 maize seedlings normally grown to the three-leaf stage were grown on 1 / 2 MS medium for 7 days, and the whole seedlings were taken and genomic DNA was extracted. Using the extracted DNA as a template, high-fidelity DNA polymerase was used to amplify the gene fragment of ZmNRG2.7, and the primer sequences used in PCR were:

[0065] Upstream primer: 5'-TTTACTAGTATGGGTTGCTCCAACTCTAAAGT-3', the nucleotide sequence of which is shown in SEQ ID NO.3;

[0066] Downstream primer: 5'-TTTGGTACCCGAGGAAATAGCAGGTGAAGCT-3', the nucleotide sequence of which is shown in SEQ ID NO.4. 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 (SpeI and KpnI) to the recovered target DNA and plant expression v...

Embodiment 3

[0072] Embodiment 3: Identification of transgenic positive plants

[0073] 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 nitrate nitrogen regulating and control gene ZmNRG2.7 from corn and a purpose thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1; the coded amino acid sequence is shown as SEQ ID NO.2. The expression of the gene is strongly induced by the nitrate nitrogen. Compared with receptor Arabidopsis, Arabidopsis transferred by the gene has the advantages that thenitrate nitrogen response capability is enhanced; the internal nitrate nitrogen accumulated quantity, the nitrate nitrogen reductase activity, the amino acid content and the full nitrogen content areobviously improved; the nitrogen element utilization rate of plants can be obviously improved.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a nitrate nitrogen regulation gene ZmNRG2.7 derived from corn and its use in improving the assimilation and utilization of nitrate nitrogen. Background technique [0002] Nitrogen is one of the most demanded mineral elements in the growth and development of maize. It is the main component of its living matter and has various functions in the growth and development of maize. During the growth and development of maize, nitrogen deficiency is easy to exist, so maize is also called "nitrogen indicator plant". When maize is deficient in nitrogen, it will show thin and weak plants, yellow leaves, delayed development, and reduced yield ( Yu Lei et al., 2011). Nitrogen is one of the most effective regulators of photosynthetic efficiency in maize, which can affect the size of photosynthetic leaf area, the length of photosynthetic duration and leaf senescence. Nitrogen ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8243C12N15/8251
Inventor 王勇齐盛东朱明月
Owner SHANDONG AGRICULTURAL UNIVERSITY
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