CN111363750A

CN111363750A - Cucumber nitrate transport protein NPF7.2 gene, protein and expression method

Info

Publication number: CN111363750A
Application number: CN202010200978.8A
Authority: CN
Inventors: 张文娜; 张嘉丽
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2020-07-03

Abstract

The invention discloses a cucumber NPF7.2 gene, the sequence of which is shown in SEQ ID No. 1. The invention also discloses a cucumber NPF7.2 gene coded protein, the sequence of which is shown in SEQ ID No. 2. The cucumber NPF7.2 has a full length of 1806bp, encodes 601 amino acids 1, has a protein molecular mass of 66.6kD, a theoretical isoelectric point of 7.5, is a stable protein, contains 12 transmembrane helical regions, a large hydrophilic ring exists between the 6 th and 7 th transmembrane domains, and the protein is a hydrophilic protein. The invention also discloses the space-time expression of the cucumber NPF7.2 gene. The expression of the protein mainly in roots and flowers is obtained through real-time fluorescence quantification, and the fact that the protein NPF7.2 is probably not only involved in the transport of nitrate but also can be a signal receptor for sensing the nitrate is shown.

Description

Cucumber nitrate transport protein NPF7.2 gene, protein and expression method

Technical Field

The invention relates to a cucumber NPF7.2 gene, protein and a gene expression method, belonging to the field of molecular biology.

Background

Nitrogen (N) is one of the essential elements of life, is an important component constituting substances such as chlorophyll, protein and the like, has important significance for maintaining the life activities of organisms, and is also a key element for limiting the crop yield. Unlike other elements, which cannot be released from rock species into the soil, in agricultural systems, high yield of crops depends on the application of large amounts of nitrogen fertilizer. However, research shows that, since the middle of the last century, the application rate of nitrogen fertilizer in China has been increased and decreased from 60% to 28.3% in 2008, and the nitrogen fertilizer utilization efficiency has been slowly improved in the last decade, but the application rate is only about 30% and is lower than the average level in the world (ERSMAN J W.et al, 2008). The large application of nitrogen fertilizers not only causes serious economic losses, but also brings about a series of serious environmental problems, such as: water and soil eutrophication, soil acidification, ozone layer cavities and the like. The cucumber [ (Cucumissativus L) ] is one of the important cultivated vegetable crops in China, the cucumber is favored by water and fertilizer, the requirement on the fertilizer, especially nitrogen fertilizer, is large, and the excessive application of the nitrogen fertilizer also causes the reduction of the quality and the edible safety of the cucumber fruit. Therefore, the method has important theoretical and practical significance for improving the utilization rate of the nitrogen fertilizer, reducing the application amount of the nitrogen fertilizer and ensuring the crop yield.

In order to adapt to the space-time change of the supply concentration of the environmental nitrate nitrogen, a series of genes are evolved from plants, and the genes are classified according to gene families, and the four gene families are mainly as follows: nitrate, the NPF family of peptide transporters (also known as the NRT1 family), the NRT2 family of nitrate-associated transporters 2 family, the chloride channel family (LCA/H), and the slow anion channel family (SLAC/SLAH). The nitrate nitrogen and polypeptide transporters (NPFs, also known as NRT: nitrate nitrogen transporter) family is huge, consisting of 53 and 93 NPF genes in Arabidopsis and rice, respectively (Huang N C et al 1996). Studies in arabidopsis have shown that the nitrate transporter NPF7.2 is primarily responsible for transport of nitrate nitrogen while mitigating cadmium stress (Li J y.et al, 2010). Studies on nitrogen use efficiency have mainly focused on the model plants arabidopsis and rice. For the cucumber, which is a horticultural plant, nitrogen utilization efficiency is studied more, but most of the studies are focused on the physiological aspect, and the studies are less on the molecular level, and although the NRT2.1 gene of cucumber has been reported previously, NPF family is less reported in cucumber.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a cucumber NPF7.2 gene sequence, a protein structure and a gene expression method.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:

cucumber NPF7.2 gene, the sequence of which is shown in SEQ ID No. 1.

Furthermore, the total length of the cucumber NPF7.2 gene sequence is 1806bp, 601 amino acids are coded, the mass of a protein molecule is 66.6kD, the theoretical isoelectric point is 7.5, the number of negative charge amino acid residues is 50, the number of positive charge residues is 51, the instability coefficient is 27.98, the cucumber NPF gene sequence is stable protein, the fat coefficient is 96.04, and the total average hydrophilicity is 0.23.

The sequence of the protein coded by the cucumber NPF7.2 gene is shown as SEQ ID No. 2.

Further, the positions of the transmembrane helix regions of the proteins are respectively in 1-47, 71-82, 106-.

The invention also discloses an expression method of the cucumber NPF7.2 gene, which comprises the following steps:

(1) synthesizing primer sequences NPF7.2F and NPF7.2R of cucumber NPF7.2 gene, which are respectively shown as SEQ ID No.3 and SEQ ID No. 4;

(2) amplifying the full length of the cucumber NPF7.2 gene sequence by using a real-time fluorescent quantitative PCR method, and recovering a reaction product by 1.5 percent agarose gel electrophoresis;

(3) the product was recovered and ligated into the pMD18-T vector and transformed into E.coli DH5 α.

Further, the real-time fluorescent quantitative PCR reaction system is 20 μ l, wherein 2 × Taq10 μ l, NPF7.2F 0.5 μ l, NPF7.2R 0.5.5 μ l, template 1 μ l, ddH₂O8 mu l; the PCR reaction conditions are as follows: 3min at 95 ℃, 30s at 57 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃.

Further, the cucumber NPF7.2 gene is mainly expressed in roots and flowers.

Drawings

The invention has the following drawings:

FIG. 1 is the PCR amplification electrophoresis picture of cucumber NPF7.2 gene.

FIG. 2 is the spatiotemporal expression of cucumber NPF7.2 gene.

FIG. 3 is a diagram of the transmembrane structure of cucumber NPF7.2 protein.

Detailed Description

The present invention is described in further detail below with reference to figures 1-3.

Example 1

The cucumber NPF7.2 gene expression method comprises the following steps:

(1) synthesizing primer sequences NPF7.2F and NPF7.2R of cucumber NPF7.2 gene, wherein the sequence of NPF7.2F is shown as SEQ ID No.3, the sequence of NPF7.2R is shown as SEQ ID No.4,

(2) the full length of the cucumber NPF7.2 gene sequence is amplified by using a real-time quantitative PCR method, and the reaction system is 20 mu l, wherein 2 × Taq10 mu l, NPF7.2F 0.5 mu l, NPF7.2R0.5 mu l, template 1 mu l and ddH₂O8 mu l; the PCR reaction conditions are as follows: 3min at 95 ℃, 30s at 57 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃. The reaction product was recovered by 1.5% agarose gel electrophoresis, and the PCR amplification electrophoresis chart is shown in FIG. 1;

Example 2

Respectively extracting RNA of cucumber roots, stems, leaves, flowers and fruits, and performing reverse transcription to obtain cDNA (complementary deoxyribonucleic acid) and then performing fluorescent quantitative PCR (polymerase chain reaction) analysis;

first, extraction of RNA

The RNA extraction adopts a Huayuanyang kit, and the specific extraction steps are as follows:

(1) to a sample contained in a 2ml centrifuge tube, 1000. mu.l of cell lysate A was added, followed by grinding for 30 seconds using an electric tissue grinder.

(2) Add 300. mu.l of deproteinized solution and 200. mu.l of chloroform solution to the centrifuge tube, shake the tube on a shaker for 30s and mix the solution.

(3) Centrifugation is carried out at 12000rpm for 5-10min at room temperature, and cell debris with a thickness of about 5-10mm is disrupted between the two phases. The supernatant (no more than 700. mu.l) was transferred to another clean 1.5ml centrifuge tube.

(4) Adding the same volume of the rinsing liquid C, fully reversing and uniformly mixing. Adding the obtained mixture into the same centrifugal adsorption column twice, centrifuging at 12000rpm for 1min after each addition, and pouring off the waste liquid in the collecting tube.

(5) Add 500. mu.l of washing solution D to the adsorption column, centrifuge at 12000rpm for 1min at room temperature, discard the waste liquid, and repeat this operation.

(6) RNA elution: transferring the centrifugal adsorption column into an RNase-Free 1.5ml centrifugal collection tube, adding 30 mul of RNA eluent F into the center of the tube, standing at room temperature for 3-5min, and centrifuging at 12000rpm for 1min to obtain the solution in the centrifugal tube, namely RNA.

Synthesis of cDNA

The RNA obtained was thawed on ice, DEPC treated 0.1ml centrifuge tubes were removed, and the following reagents were added to ice:

the reverse transcription procedure comprises a first stage of reaction at 42 deg.C for 15min, a second stage of reaction at 95 deg.C for 3min, and then terminating the synthesis reaction on ice, and storing the product at-20 deg.C.

Third, fluorescent quantitative PCR analysis

The fluorescent quantitative PCR takes TUb as an internal reference gene, and the reaction system of the fluorescent quantitative PCR is shown in the following table

The results are shown in FIG. 2, which indicates that it is expressed mainly in roots and flowers.

Example 3

Hydrophilicity analysis of the amino acid sequence encoded by cucumber CsNPF7.2 was performed using TMHMM-2.0(http:// www.cbs.dtu.dk/services/TMHMM-2.0/), see FIG. 3, and the results showed that there were 12 putative transmembrane domains, of which the difference between the 6 th and 7 th domains was large, conforming to the topology of the NPF family members.

Those not described in detail in this specification are within the skill of the art.

<110> university of agriculture in China

<120> cucumber nitrate transport protein NPF7.2 gene, protein and expression method

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Claims

1. Cucumber NPF7.2 gene, the sequence of which is shown in SEQ ID No. 1.

2. The cucumber NPF7.2 gene as claimed in claim 1, wherein the full length of the cucumber NPF7.2 gene sequence is 1806 bp.

3. The cucumber NPF7.2 gene-encoded protein of claim 1, which has the sequence shown in SEQ ID No. 2.

4. The protein of claim 3, wherein said protein corresponds to a number of amino acids of 601, a molecular weight of 66594.04, a theoretical isoelectric point of 7.5, a number of negatively charged amino acid residues of 50, a number of positively charged residues of 51, a destabilization factor of 27.98, a stable protein, a fat factor of 96.04, and an overall average hydrophilicity of 0.23.

5. The protein of claim 3, wherein the positions of the transmembrane helix regions of the protein are respectively in the ranges 1-47, 71-82, 106-.

6. An expression method of cucumber NPF7.2 gene is characterized by comprising the following specific steps:

7. The method for expressing the NPF7.2 gene in cucumber of claim 6, wherein the real-time fluorescent quantitative PCR reaction system is 20 μ l,2 × Taq10 μ l, NPF7.2F 0.5 μ l, NPF7.2R0.5 μ l, template 1 μ l, ddH₂O8 mu l; the PCR reaction conditions are as follows: 3min at 95 ℃, 30s at 57 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃.

8. The method for expressing cucumber NPF7.2 gene as claimed in claim 6, wherein the cucumber NPF7.2 gene is mainly expressed in roots and flowers.