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Tobacco nitrate reductase nia2 Promoter, its expression vector and its application

A technology of eukaryotic expression vector and reductase is applied in the field of molecular biology to achieve the effect of improving plant nitrogen efficiency

Active Publication Date: 2021-07-09
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, it has not yet been clearly determined that the NIA gene promoter region Signal-responsive components are urgently needed for research NIA Gene promoter regulatory elements and the presence or absence of signal response element, for further verification its The research on the signal response function lays a foundation, which is of great significance for improving plant nitrogen efficiency, saving nitrogen fertilizer consumption, and developing resource-saving and environment-friendly agriculture.

Method used

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  • Tobacco nitrate reductase <i>nia2</i> Promoter, its expression vector and its application
  • Tobacco nitrate reductase <i>nia2</i> Promoter, its expression vector and its application
  • Tobacco nitrate reductase <i>nia2</i> Promoter, its expression vector and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Obtaining a promoter fragment

[0045] Find key enzymes of tobacco nitrogen metabolism in NCBI database NIA2 The ATG upstream sequence of the gene intercepts the base sequence of 1105bp. For the specific sequence, see SEQ ID NO.1. Design primers, use the tobacco genome as a template, and obtain fragments by PCR amplification technology. The primer sequence of the amplified fragments:

[0046] The upstream primer is NIA2-F: TACATACAAGGGCGCGAATAA;

[0047] The downstream primer is NIA2-R: AGATTATTCTAAAAAAAGAATATGAATG;

[0048] Result: see figure 1 After obtaining the 1105bp target fragment, the vector was cloned and connected by TA, sequenced and compared, and it was consistent with the sequence provided in the database.

Embodiment 2

[0049] Example 2: Promoter Acquisition of modified fragments of signal response elements

[0050] According to the inventor's previous research and prediction, a possible Signal response element, the sequence is: GACCCTACGGGCGTAAAAAG, the element is in NIA2 The position in the gene promoter is -335bp~-316bp.

[0051] Modify the element, such as figure 2 As shown, the promoters for deleting this element and the promoters for doubling (4-fold) this element were respectively constructed, tobacco nitrate reductase NIA2 Promoter, possible The schematic diagram of the construction of the recombinant vector for the modified promoter of the signal response element is shown in Fig. 3 .

[0052] When amplifying the modified promoter, the upstream and downstream primers are the same as those used in Example 1.

[0053] Results: After obtaining the target fragment of the deleted and doubled promoter, the vector was connected by TA cloning, sequenced and compared, and the base se...

Embodiment 3

[0054] Embodiment 3: Construction of promoter recombinant expression vector

[0055] (1) Use the pCAMBIA-NPT-GUS vector with Eco RI / Hind After Ⅲ enzyme digestion, cut off the 35S promoter, and recover the large fragment for later use;

[0056] (2) The promoter sequences on various T vectors sequenced correctly in Example 1 and Example 2 were used Eco RI / Hind After Ⅲ digestion, recovery and purification, the target fragment was ligated into the digested pCAMBIA-NPT-GUS vector with T4 DNA ligase;

[0057] (3) Transform the ligation product into Escherichia coli E. coli In the DH5α competent state, positive recombinants were identified by colony PCR method, so that NIA2 The promoter is fused with the GUS gene;

[0058] (4) Carry out PCR identification on the recombinant plasmid, and after confirming that the sequence size is correct, extract the plasmid for later use, and obtain the plant recombinant expression vector pCAMBIA-NPT-NIA2–P-GUS expressing the GUS gene....

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Abstract

The invention discloses a tobacco nitrate reductase NIA2 Promoter, its expression vector and its application. Should NIA2 The nucleotide sequence of the promoter is: the sequence shown in SEQ ID NO.1; or on the basis of retaining the core promoter sequence shown in SEQ ID NO.1, the regulation is removed or doubled NIA2 An expressed key element highly similar to the signal response element sequence; or a nucleic acid sequence with equivalent functions derived from the sequence shown in SEQ ID NO.1. a kind of NIA2 Eukaryotic expression vectors for plant genetic transformation with promoter sequences. a kind of NIA2 The engineering bacteria used for plant genetic transformation of the promoter sequence. NIA2 The application of the promoter in promoting the expression of the target gene, the target gene is GUS Gene. The invention is to verify NIA2 The research on the response function of promoter nitric acid signal has laid a foundation and has broad prospects for improving plant nitrogen efficiency, saving nitrogen fertilizer consumption and developing resource-saving and environment-friendly agriculture.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a tobacco nitrate reductase NIA2 Promoter, its expression vector and its application. Background technique [0002] Nitrogen is an essential macronutrient element for plant growth. It participates in the composition of protein, DNA / RNA and other biological macromolecules. It plays an important role in protein synthesis, enzyme function, pigment and plant secondary metabolism. element". [0003] Nitrate nitrogen and ammonium nitrogen are the two main nitrogen sources for crops. Among them, nitrate nitrogen can induce and regulate the expression of more than 1,000 genes. Due to the constant change of external nitrogen nutrition, plants need to constantly adjust their own growth and internal metabolic process to accommodate fluctuations in available nitrogen nutrients, during which nitrate ( ) is a key signaling molecule for plants to sense changes in external nitrogen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N1/21A01H5/00A01H5/10A01H6/82
CPCC12N9/0044C12N15/8238C12Y107/99004
Inventor 杨惠娟史宏志丁松爽王景赵莉周炎张玉宁
Owner HENAN AGRICULTURAL UNIVERSITY
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