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Fast-growing aquatic plant nitrate transporter genrt2.1 and its coding gene and application

A technology that encodes genes and nitrates, which is applied in the direction of plant genetic improvement, application, microorganisms, etc., and can solve problems such as inability to transport or grow

Active Publication Date: 2018-11-13
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 to translocate or grow at nitrate concentrations below 500μM

Method used

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  • Fast-growing aquatic plant nitrate transporter genrt2.1 and its coding gene and application
  • Fast-growing aquatic plant nitrate transporter genrt2.1 and its coding gene and application
  • Fast-growing aquatic plant nitrate transporter genrt2.1 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, the acquisition of GeNRT2.1

[0062] 1. Discovery of GeNRT2.1

[0063] 1. Total RNA extraction and cDNA synthesis of dwarf pearls

[0064] Using the RNAprep Pure Plant kit from TIANGEN Company, the total RNA of dwarf pearls was extracted according to the kit instructions, and cDNA was obtained 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 sequences are as follows: H5: GGNGCNGAYCARTTYGAYG; H3: AVYTCYTCNACYTGNGTNAC. The PCR amplification program was: 95°C pre-5min; 94°C denaturation 30s; 52°C annealing 30s; 72°C extension 1min, 35cycle; 72°C, 10min.

[0067] 3. Electrophoresis and sequencing

[0068] After the amplification was finished, 1% agarose gel electrophoresis was detected, and the PCR product was connected to the T carrier, and the sequencing results s...

Embodiment 2

[0126] Embodiment 2, verification of GeNRT2.1 gene function

[0127] In this example, the function of the GeNRT2.1 gene was verified by using the Δynt-Leu double mutant Hansenula polymorpha.

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

[0129] 1. Construction of the shuttle plasmid

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

[0131] 2. Linearization

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

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

[0134] ①Pick a single colony of △ynt-Leu double mutant Hansenu...

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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 biological technology, and in particular relates to the fast-growing aquatic plant nitrate transporter GeNRT2.1 and its coding gene and application. Background technique [0002] Nitrogen is an essential nutrient element necessary for plant growth and development, and plays an important role in plant growth, development and morphogenesis, and is also of great significance to the life activities and yield formation of crops. Nitrate (NO 3 - ) is one of the main nitrogen sources for crops, and insufficient nitrate supply will seriously inhibit the growth and development of crops. Physiological study reveals that plants absorb NO from soil 3 - A full set of high- and low-affinity NO 3 - Transport system participation, NO 3 - The entry is by H across the plasma membrane + Gradient driven. Some NO 3 - The transport system is constitutively expressed, while others are regulated by NO 3 - induced, and with N...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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