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Transcription factor ZmNLP4 derived from corn and application thereof

A technology of transcription factors and uses, applied in the fields of application, peptide source, genetic engineering, etc., can solve problems that cannot be solved in a short time, and achieve the increase of main root length and number of lateral roots, total biomass increase, nitrate reductase gene expression The effect of volume increase

Active Publication Date: 2018-01-19
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional genetic breeding requires a long-term process, and to a certain extent, it cannot solve this problem in a short time

Method used

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  • Transcription factor ZmNLP4 derived from corn and application thereof
  • Transcription factor ZmNLP4 derived from corn and application thereof
  • Transcription factor ZmNLP4 derived from corn and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Obtaining of transcription factor ZmNLP4

[0051] RNA was extracted from leaves of maize seedlings at the three-leaf stage and reverse-transcribed into cDNA. Using cDNA as a template, use

[0052] Upstream primer: 5'-ctgcagGTACGATCACACTAGCACCGCCAGC-3', the nucleotide sequence of which is shown in SEQ ID NO.3;

[0053] Downstream primer: 5'-ggtaccTCAACCGGAGCTTCCACAAGAACTGCCA-3', the nucleotide sequence of which is shown in SEQID NO.4;

[0054] The nucleic acid sequence of ZmNLP4 was amplified by polymerase chain reaction (PCR). The amplification program was: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds; annealing at 60°C for 2 minutes; extension at 72°C for 2 minutes; The final extension was 8 minutes, and agarose gel electrophoresis analysis was performed after the PCR was completed. The amplified fragment obtained the target gene fragment shown in SEQ ID NO.1; the encoded amino acid sequence was shown in SEQ ID NO.2.

Embodiment 2

[0055] Example 2 Construction of Overexpression Arabidopsis Vector and Its Application

[0056] Using the pBI121 vector as a template, primers 35S_F (5'-AAGCTTatggtggagcacgacactctcga-3', whose nucleotide sequence is shown in SEQ ID NO.5;) and 35S_R: (5'-AAGCTTagagatagatttgtagagagagactgg-3', whose nucleotide sequence As shown in SEQ ID NO.6;), use Pfu high-fidelity enzyme to amplify the 35S promoter sequence, PCR reaction conditions: 98°C pre-denaturation for 4 minutes; then 98°C for 30s, 58°C for 30s, 72°C for 1min, 25 cycles; Final extension at 72°C for 8 min; since the HindIII restriction site was added to the primers 35S_F and 35S_R, the PCR amplified product was digested with HindIII, and then ligated into the plant expression vector pCAMBIA1300, which was also digested with HindIII, and then digested and sequenced Identify the clone that the 35S promoter is connected in the correct direction, and construct a plant overexpression vector containing the 35S promoter; then us...

Embodiment 3

[0059] Embodiment 3 function verification

[0060] The strain that is cultivated on the basis of the seeds of the transgenic plants that are transferred to the ZmNLP4 gene obtained in Example 2 in KNO 3 The phenotypes of MS plants grown on solid medium at concentrations of 0.2mM, 2.5mM, and 5mM were analyzed. From the result ( Figure 2-Figure 7 ) It can be seen that the growth of the transgenic Arabidopsis plant expressing the ZmNLP4 gene is significantly better than that of the recipient plant, and the biomass (including fresh weight and dry weight) of the transgenic Arabidopsis plant expressing the ZmNLP4 gene is significantly increased compared with the recipient plant; Under 1 / 2MS culture conditions, the amino acid content and total nitrogen content of the transgenic Arabidopsis plants expressing the ZmNLP4 gene were significantly increased compared with the recipient plants; and the main root length and the number of lateral roots of the transgenic Arabidopsis plants ex...

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Abstract

The invention relates to the technical field of plant genetic engineering and provides a transcription factor ZmNLP4 derived from corn and application thereof. A nucleotide sequence of the transcription factor is shown as SEQ ID NO.1, and a coded amino acid sequence of the transcription factor is shown as SEQ ID NO.2. Compared with a receptor Arabidopsis, the Arabidopsis in which the transcriptionfactor is transferred has the advantages that the total biomass is obviously increased, the content of in-vivo amino acids and total nitrogen is increased, the main root length and quantity of lateral roots are obviously increased, the yield per plant is obviously increased, and the expressions of nitrate transporters, nitrate reductase genes and nitrite reductase genes are obviously increased.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, and provides a transcription factor ZmNLP4 derived from maize and its application. Background technique [0002] Nitrogen is one of the important mineral nutrients to maintain plant growth and biological output, and it participates in the synthesis of nucleic acid, protein, lipid, chlorophyll and other biological macromolecules in the organism. Nitrogen deficiency significantly limits plant growth and development (Krouk et al., 2010, Current Opinion in Plant Biology 13, 265 272). Therefore, nitrogen fertilizers are widely used in agricultural production to increase crop yields and increase the cost of agricultural production. Every year, about 8.5-9.0 million tons of nitrogen fertilizers are applied to the soil globally, but 50-70% of them are not absorbed by plants and are lost to the environment, causing serious ecological and environmental problems such as eutrophication and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
Inventor 王勇齐盛东曹怀荣孙梦伟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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