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Spider plant root system nitrate transport protein CcNPF5.2, and coding gene and application thereof

A nitrate transport and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem that the strain cannot be transported or grown.

Active Publication Date: 2018-08-24
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 Can cause strains to fail to translocate or grow at nitrate concentrations below 500 μM

Method used

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  • Spider plant root system nitrate transport protein CcNPF5.2, and coding gene and application thereof
  • Spider plant root system nitrate transport protein CcNPF5.2, and coding gene and application thereof
  • Spider plant root system nitrate transport protein CcNPF5.2, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Obtaining CcNPF5.2 protein and its coding gene

[0043] 1. Nitrate transporter gene cloning

[0044] 1. Use TIANGEN's RNAprep Pure Plant kit, extract total RNA from Chlorophytum roots according to the kit instructions, and obtain cDNA by reverse transcription. The reverse transcription system is shown in Table 2. The reverse transcription system was set to 65°C for 10min; the reaction was stopped rapidly at room temperature on ice, 1μl Rnase was added, and the reaction was extended at 42°C for 2h and at 70°C for 15min.

[0045] Table 2 Reverse transcription system

[0046]

[0047] 2. By comparing the amino acid sequences of nitrate transporters such as Prunus gracilis, wild rice, Arabidopsis thaliana, and rice, a pair of degenerate primers were designed according to the conserved region, consisting of primer L5 and primer L3;

[0048] Primer L5: 5'-ACNGAYGTNGARGARGTL-3';

[0049] Primer L3: 5'-CATNCCYTTNGGRCAYTC-3'.

[0050] 3. Using the cDNA obtained in step 1 as a t...

Embodiment 2

[0086] Example 2. Functional verification of CcNPF5.2 protein and its coding genes

[0087] This example uses the deltaynt-Leu double mutant Hansenula polymorpha to verify the function of the CcNPF5.2 protein and its coding gene.

[0088] 1. Construction of genetically modified yeast

[0089] 1. Replace the fragment between the salI and speI restriction sites of vector pYNR-LEU2 with the double-stranded DNA molecule shown in sequence 4 of the sequence table to obtain the shuttle plasmid pYNR-CcNPF5.2.

[0090] 2. The shuttle plasmid pYNR-CcNPF5.2 obtained in step 1 was digested and linearized with Bstp1, and the target product was recovered and purified by 1% agarose gel electrophoresis to obtain linearized fragments.

[0091] 3. Preparation of transgenic △ynt-Leu double mutant Hansenula polymorpha, the specific steps are as follows:

[0092] (1) Pick a single colony of △ynt-Leu double mutant Hansenula polymorpha, inoculate it into 5mLYGNH liquid medium, and cultivate overnight at 37°C a...

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Abstract

The invention discloses a spider plant root system nitrate transport protein CcNPF5.2, and a coding gene and application thereof. In the present invention, an encoding gene CcNPF5.2 of a nitrate transport protein is cloned from the root of a spider plant, and the gene is introduced into a delta ynt-Leu double mutant hansenula polymorpha to obtain transgenic CcNPF5.2 yeast. Experiments prove that the CcNPF5.2 protein has a function of the nitrate transport protein, the nitrogen utilization efficiency of plants can be improved and plants can grow fast.

Description

Technical field [0001] The invention relates to a Chlorophytum root system nitrate transporter CcNPF5.2 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 the most important source of nitrogen for most crops, and insufficient nitrate supply will severely 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 NO3 - 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 - The assimilation is negative feedback regulation. [0003] Chlorophytum is a perennial evergreen plant ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N15/81C12N1/19A01H5/00C12R1/78
CPCC07K14/415C12N15/815C12N15/8243
Inventor 刘昱辉朱思慧李梅谭杰
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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