阅读说明：本技术 植物氮吸收相关蛋白nar及其编码基因与应用 (Plant nitrogen absorption related protein NAR and coding gene and application thereof ) 是由 白文钦 胡明瑜 王春萍 吴红 雷开荣 于 2020-07-08 设计创作，主要内容包括：本申请提供了植物氮吸收相关蛋白及其编码基因与应用,属于分子育种及生物工程领域。本申请提供的植物氮吸收相关蛋白NAR及其编码基因,通过转基因的方式,在植物尤其是作物体内表达植物氮吸收相关蛋白NAR的基因,表达出植物氮吸收相关蛋白NAR,尤其是在根系中特异性的过表达,就能提高植物氮素尤其是在低浓度氮素环境下的氮素吸收和转运能力,提高植物尤其是作物的产出,提高经济效益；还能减少氮肥的使用,减少环境污染,还具有一定的社会效益和环境效益。(The application provides plant nitrogen absorption related protein, and a coding gene and application thereof, belonging to the field of molecular breeding and bioengineering. According to the plant nitrogen absorption related protein NAR and the coding gene thereof, the gene of the plant nitrogen absorption related protein NAR is expressed in plants, particularly crops, in a transgenic manner, the plant nitrogen absorption related protein NAR is expressed, particularly, the specific overexpression in root systems is realized, the nitrogen absorption and transport capacity of the plants, particularly in the low-concentration nitrogen environment can be improved, the yield of the plants, particularly the crops, is improved, and the economic benefit is improved; the use of nitrogen fertilizer can be reduced, the environmental pollution is reduced, and certain social and environmental benefits are achieved.)

1. The plant nitrogen absorption-related protein NAR is characterized in that the amino acid sequence of the plant nitrogen absorption-related protein NAR is shown as SEQ ID No. 1.

2. A coding gene of the plant nitrogen absorption related protein NAR of claim 1.

3. The coding gene of the plant nitrogen absorption-related protein NAR as claimed in claim 2, wherein the sequence of the coding gene is shown as SEQ ID No. 2.

4. An expression vector comprising the gene encoding the plant nitrogen uptake-associated protein NAR as claimed in claim 2.

5. A host cell expressing the plant nitrogen uptake-associated protein NAR of claim 1; or comprises the coding gene of claim 2 or 3; or comprising the expression vector of claim 4.

6. A method for improving nitrogen absorption and transport capacity of plants is characterized in that plants over-expressing plant nitrogen absorption related protein NAR in vivo are planted, and the amino acid sequence of the plant nitrogen absorption related protein NAR is shown as SEQ ID NO. 1.

7. The method for improving nitrogen uptake and transport capacity of a plant according to claim 6, wherein said plant overexpresses the NAR protein associated with nitrogen uptake in the root system.

8. The method of improving nitrogen uptake and transport capacity of a plant according to claim 6 or 7, wherein the plant is one of rice, wheat, corn, sorghum, tobacco and millet.

9. A kit for detecting nitrogen absorption and transport capacity of plants is characterized by comprising a detection primer for detecting a gene encoding nitrogen absorption associated protein NAR, wherein the detection primer comprises a forward primer and a reverse primer, the forward primer is shown as a sequence SEQ ID NO.3, and the reverse primer is shown as a sequence SEQ ID NO. 4.

10. A method for detecting nitrogen uptake transport capacity of a plant, comprising the steps of:

taking a plant sample, and extracting the genome DNA of the plant sample;

amplifying by using a forward primer shown as a sequence SEQ ID NO.3 and a reverse primer shown as a sequence SEQ ID NO. 4;

if the target gene band is obtained by amplification, the plant has better nitrogen absorption and transport capacity.

Technical Field

The application relates to the field of molecular breeding and bioengineering, in particular to a plant nitrogen absorption related protein NAR and a coding gene and application thereof.

Background

Nitrogen is an essential nutrient element for plant growth, and a large amount of nitrogen fertilizer is commonly applied worldwide to improve the yield of crops. However, nitrogen fertilizer is expensive, pollutes the environment and threatens human health. One strategy to solve this problem is to develop crops that are capable of nitrogen fixation and efficient nitrogen fertilizer utilization, which results in higher crop yields with reduced nitrogen fertilizer utilization.

At present, only a few plants such as leguminous plants have nitrogen fixation capacity, but the leguminous plants fix nitrogen through nitrogen fixation bacteria through symbiosis with the nitrogen fixation bacteria; if the crops can have strong nitrogen absorption and transportation capacity; on one hand, the use of nitrogen fertilizer can be reduced, and the yield or output of the plant can be improved to a certain extent.

Therefore, the crop has strong nitrogen absorption and transportation capability, and is a key point, a hotspot and a difficult point of the current research.

Disclosure of Invention

The application discloses a plant nitrogen absorption related protein NAR, and the amino acid sequence of the plant nitrogen absorption related protein NAR is shown in SEQ ID NO. 1.

The absorption and utilization of nitrogen by plants are often accomplished through the root system. The plant root system absorbs nitrate nitrogen and ammonium nitrogen from the soil through the mediation of nitrate transport protein. According to the nitrate transport activity of the nitrate transporter family, there are two major classes, low affinity and high affinity, among which nitrate transporter 1(NRT1), belongs to the low affinity system, and has low nitrate affinity except for NRT1.1 in arabidopsis and NRT1.1b in rice. Of the 53 NP F/NRT1 proteins of Arabidopsis, 16 proteins have been identified for their functions to date. Unlike NRT1, most members of NRT2 have high affinity activity, primarily to control plant uptake or transport of nitrate at low nitrate concentrations. In arabidopsis thaliana, 7 NRT2 proteins have been cloned and identified as being associated with nitrate transport. Interestingly, the NRT2 protein cannot transport nitrate alone and needs to be completed with the help of the helper protein NAR2, which indicates that NAR2 plays an important role in nitrogen uptake of plants.

The sequence MTMARPGAAL PLLLVVVGAC CARLAAAVHLSALGRTLIVE ASPKAGQVLH AGEDTITVTW HLNASASSVG YKALEVTLCY APASQE DRGW RKANDDLSKDKACQFRIARH AYAGGQGTLR YRVARDVPTA SYHVRAYALD A SGAPVGYGQ TAPAYYFHVA GVSGVHASLRVAAAVLSAFS IAALAFFVVV EKRRKDE of the amino acid shown in SEQ ID NO. 1.

The application discloses a coding gene for coding a plant nitrogen absorption related protein NAR.

The sequence of the plant nitrogen uptake related protein NAR (nitrate uptake related protein) is shown in SEQ ID NO.1, and the number of the coding genes for coding the plant nitrogen uptake related protein is a huge number according to the degeneracy of codons and different combination methods.

In some embodiments of the foregoing encoding gene, the encoding gene may be the sequence shown in SEQ ID NO. 2.

The sequence of the coding gene shown in SEQ ID NO.2 is ATGACGATGGCTCGTCCTGGGGCGGCTTTGCCG CTGCTGCTGGTCGTGGTCGGCGCTTGCTGCGCGCGCCTGGCGGCGGCAGTGCACCTCTC CGCGCTCGGCAGGACACTCATCGTCGAGGCGTCGCCGAAGGCCGGACAAGTCCTGCAC GCCGGCGAGGACACGATAACCGTGACATGGCACCTCAACGCGTCGGCGTCCAGCGTCG GGTACAAGGCGCTGGAGGTGACCCTCTGCTACGCGCCGGCGAGCCAGGAGGACCGCGG GTGGCGCAAGGCCAACGACGACTTGAGCAAGGACAAGGCGTGCCAGTTCAGGATCGCC CGGCATGCATACGCCGGCGGCCAGGGGACGCTCCGGTACAGGGTCGCCCGCGACGTCC CCACCGCGTCCTACCACGTGCGCGCCTACGCGCTGGACGCGTCCGGGGCGCCGGTGGG CTACGGCCAGACCGCGCCCGCCTACTACTTCCACGTCGCGGGCGTCTCGGGCGTCCACG CGTCCCTCCGGGTCGCCGCCGCCGTGCTCTCCGCGTTCTCCATCGCCGCGCTCGCCTTCT TTGTCGTCGTCGAGAAGAGGAGGAAGGACGAGTAG.

The application discloses an expression vector containing a coding gene of a plant nitrogen absorption related protein NAR.

Through the expression vector, the nitrogen uptake related protein NAR can be expressed.

The application discloses an expression vector containing a coding gene of a plant nitrogen absorption related protein NAR.

A host cell expressing a plant nitrogen uptake-associated protein NAR; or comprises a coding gene for expressing a plant nitrogen absorption related protein N AR; or an expression vector for expressing the plant nitrogen absorption related protein NAR.

The host cell can highly express the plant nitrogen uptake related protein NAR, can express the plant nitrogen uptake related protein NAR by integrating a gene coding the plant nitrogen uptake related protein NAR into the host cell genome, or directly and independently express the plant nitrogen uptake related protein NAR in the form of plasmid in the host cell.

The application discloses a method for improving nitrogen absorption and transport capacity of plants, plants a plant over-expressing plant nitrogen absorption related protein NAR in vivo, and the amino acid sequence of the plant nitrogen absorption related protein NAR is shown as SEQ ID NO. 1.

Through the overexpression of the plant nitrogen absorption related protein NAR in the plant body, the effect of the plant nitrogen absorption related protein NAR is cooperated with the plant nitrate transport protein to carry out nitrogen absorption and transport work, especially nitrogen absorption and transport work in a low-nitrogen environment, and the absorption and transport capacity of the plant nitrogen is improved.

In some embodiments of the foregoing methods of increasing the ability of a plant to assimilate and transport nitrogen, the plant overexpresses a nitrogen uptake-associated protein, NAR, in the root system.

In the embodiment, in general, the nitrogen absorbed and transported by the plant is mainly absorbed and transported by the root system, and the nitrogen in the soil environment is absorbed by the active transportation of the root system; therefore, the nitrogen absorbing, transporting and utilizing capacity of plants can be improved to a great extent by enhancing the capacity of the root system to absorb and transport nitrogen.

The fertilizer application mode can be continuously adjusted due to different fertilizer application modes, and the nitrogen absorption and transport capacity of plants can be improved to a certain extent by improving the absorption capacity of the leaf surfaces and applying the foliar fertilizer to the leaf surfaces.

In some embodiments of the foregoing method of increasing the ability of a plant to absorb and transport nitrogen, the plant is one of rice, wheat, corn, sorghum, tobacco, and millet.

In the examples, the main grain crops of human at present include rice, wheat, corn, etc., and sorghum is widely used as a superior crop for growing; tobacco is also widely planted as a commercial crop. However, in the current planting process of the crops, a large amount of chemical fertilizer is needed, and the use of the chemical fertilizer not only causes environmental pollution, but also causes the problems of soil fertility reduction, sudden hardening and the like; therefore, by improving crops such as rice, wheat, corn, sorghum, tobacco and the like, the nitrogen absorption and transport capacity of plants is improved, and particularly under the condition of low-concentration nitrogen, the nitrogen absorption and transport capacity can be better, so that the use of chemical fertilizers can be reduced, and the utilization efficiency of nitrogen is improved.

The application discloses a kit for detecting nitrogen absorption and transport capacity of plants, which comprises a detection primer for detecting a NAR gene encoding nitrogen absorption related protein, wherein the detection primer comprises a forward primer and a reverse primer, the forward primer is shown as a sequence SEQ ID NO.3, and the reverse primer is shown as a sequence SEQ ID NO. 4.

Detection primer forward primer SEQ ID NO. 3: 5'-CGATAACCGTGACATGGCACCT-3', respectively;

detection primer reverse primer SEQ ID NO. 3: 5'-CTACTCGTCCTTCCTCCTCTT-3', respectively;

the primers in the kit can be used for rapidly detecting crops, and the kit can also contain other reagents which are convenient to carry out experiments, such as DNA polymerase, dNTP and the like, and rapid detection can be realized through the reagents.

The application discloses a method for detecting nitrogen absorption and transportation capacity of plants, which comprises the following steps:

taking a plant sample, and extracting the genome DNA of the plant sample;

amplifying by using a forward primer shown as a sequence SEQ ID NO.3 and a reverse primer shown as a sequence SEQ ID NO. 4;

if the target gene band is obtained by amplification, the plant has better nitrogen absorption and transport capacity.

In the embodiment, the detection primer is used for detecting whether the crop has the gene segment, so that the nitrogen absorption and transport capacity of the plant can be judged; if the gene fragment is detected, the plant may have better nitrogen uptake and transport capacity.

Compared with the prior art, the beneficial effect of this application is: according to the plant nitrogen absorption related protein NAR and the coding gene thereof, the gene of the plant nitrogen absorption related protein NAR is expressed in plants, particularly crops, in a transgenic manner, the plant nitrogen absorption related protein NAR is expressed, particularly, the specific overexpression in root systems is realized, the nitrogen absorption and transport capacity of the plants, particularly in the low-concentration nitrogen environment can be improved, the yield of the plants, particularly the crops, is improved, and the economic benefit is improved; the use of nitrogen fertilizer can be reduced, the environmental pollution is reduced, and certain social and environmental benefits are achieved.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a graph showing the expression levels of ZmNAR2.3 at different sites in maize in example 1;

FIG. 2 is a schematic diagram showing the expression of the ZR promoter-extended GUS gene in the root system in example 2;

FIG. 3 is a graph showing the expression levels of the ZmNAR2.3 genes of 8 independent transformation events in Experimental example 2.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present application are described in further detail below with reference to examples.