Fast-growing aquatic plant nitrate transport protein GeNRT2.1 and coding gene and application thereof

A nitrate and encoding technology, applied in the direction of plant genetic improvement, application, microorganisms, etc., can solve the problem that strains cannot be transported or grown

Active Publication Date: 2016-04-13
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

YNT1 deletion mutation ( ynt1) can cause strains to fail

Method used

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  • Fast-growing aquatic plant nitrate transport protein GeNRT2.1 and coding gene and application thereof
  • Fast-growing aquatic plant nitrate transport protein GeNRT2.1 and coding gene and application thereof
  • Fast-growing aquatic plant nitrate transport protein GeNRT2.1 and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Obtaining GeNRT2.1

[0062] 1. Discovery of GeNRT2.1

[0063] 1. Extraction of total RNA and cDNA synthesis of Dwarf Pearl

[0064] Use the RNAprepPurePlant kit of TIANGEN Company to extract the total RNA of Dwarf Pearl according to the kit instructions, and obtain cDNA by reverse transcription.

[0065] 2. PCR amplification

[0066] Using the cDNA obtained by reverse transcription in step 1 as a template, PCR amplification was performed using degenerate primers H5 and H3 to obtain a PCR product. The primer sequence is as follows: H5: GGNGCNGAYCARTTYGAYG; H3: AVYTCYTCNACYTGNGTNAC. The PCR amplification procedure is: 95°C for 5 minutes; 94°C denaturation for 30 seconds; 52°C annealing for 30 seconds; 72°C extension for 1 minute, 35 cycle; 72°C, 10 minutes.

[0067] 3. Electrophoresis and sequencing

[0068] After the amplification, 1% agarose gel electrophoresis was performed to detect the PCR product, and the PCR product was connected to the T vector. The sequencing re...

Embodiment 2

[0126] Example 2. Verification of GeNRT2.1 gene function

[0127] In this example, the Δynt-Leu double mutant Hansenula polymorpha was used to verify the function of the GeNRT2.1 gene.

[0128] 1. Construction of transgenic GeNRT2.1 yeast

[0129] 1. Construction of shuttle plasmid

[0130] Insert the DNA molecule shown in sequence 4 in the sequence table between the speI and salI restriction sites of pYNR-EX vector, and keep the other sequences of pYNR-EX vector unchanged, to obtain the recombinant vector pYNR-GeNRT2.1.

[0131] 2. Linearization

[0132] The pYNR-GeNRT2.1 was digested and linearized with BstpⅠ to obtain the linearized pYNR-GeNRT2.1, 1% agarose gel electrophoresis ( Figure 4 ), recover and purify the target product, use a spectrophotometer for quantification, and reserve.

[0133] 3. Preparation of double mutant Hansenula polymorpha (△ynt-Leu) competent cells

[0134] ① Pick a single colony of △ynt-Leu double mutant Hansenula polymorpha and inoculate it into 5mLYGNH mediu...

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Abstract

The invention discloses a fast-growing aquatic plant nitrate transport protein GeNRT2.1 and a coding gene and application thereof. The protein is the protein a or the protein b or the protein c, wherein the protein a is provided with an amino acid sequence shown in the sequence 5 in a sequence table; the protein b is a fusion protein obtained by connecting labels to the end N and/or the end C of the protein shown in the sequence 5 in the sequence table; the protein c has the same function and is the protein obtained through substitute and/or deletion and/or addition of one or more amino acid residues for the amino acid sequence shown in the sequence 5 in the sequence table. A test proves that the protein GeNRT2.1 has the function of a nitrate transport protein and can improve the plant nitrogen utilization efficiency, so that the plant grows rapidly.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the fast-growing aquatic plant nitrate transporter GeNRT2.1 and its coding gene and application. Background technique [0002] Nitrogen is a basic nutrient element necessary for plant growth and development. It plays an important role in plant growth, development and morphogenesis, and it also has important significance for crop life activities and yield formation. Nitrate (NO 3 - ) Is one of the main nitrogen sources of crops, and insufficient nitrate supply will seriously inhibit the growth and development of crops. Physiological studies have shown that plants absorb NO from the soil 3 - Need a set of high-and low-affinity NO 3 - Participation in the transfer system, NO 3 - The entry of H + Gradient driven. Some NO 3 - The transport system is constitutively expressed, and some are affected by NO 3 - Induced, and with NO 3 - Negative feedback regulation [0003] The dwarf pear...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/81C12R1/78
CPCC07K14/415C12N15/815
Inventor 刘昱辉师双锋李梅董航
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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