CN106906191B - Fungus TrGDH protein for improving efficient utilization of nitrogen and application thereof - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, and discloses a fungus (A) capable of improving the efficient utilization of rice nitrogenTrichurus) Glutamate dehydrogenase geneTrGDHCloning and application of (3). The research finds that the content of the active ingredients in the active ingredients is high,Trthe in vitro positive NADP (H) enzyme activity of GDH is greater than the reverse reaction, i.e.TrGDH proteins tend to utilize NH₄ ⁺α -ketoglutaric acid is converted to glutamic acid, andTrGDH vs NH₄ ⁺Is much greater than glutamic acid. Through gene engineering technologyTrGDHThe gene is heterogeneously overexpressed into rice, so that the utilization of nitrogen by the transgenic rice is improved, the growth condition of the transgenic rice is improved, and the effective spike weight and thousand kernel weight of the transgenic rice are increased. Thus, the utilization of nitrogen in rice is improvedTrGDHThe gene can be used for cultivating new rice varieties with good agronomic characters.

Description

Fungus TrGDH protein for improving efficient utilization of nitrogen and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering. In particular, the invention relates to a method for producing riceOryza sativaL.) heterologous expression of fungiTrichurusGlutamate dehydrogenase gene of (1)TrGDHCan improve the high-efficiency utilization of nitrogen of rice and improve the growth of rice.

Background

Rice (1)Oryza sativaL.) Is one of the grain crops with the most nitrogen fertilizer consumption and is also the crop extremely sensitive to drought. According to the worldChina food and agriculture organization (FAO 2001) statistics, wherein the year of China 1995-1997 rice planting area is 31.7 multiplied by 10⁶hm²The fertilizer accounts for 20% of the rice planting area in the world, and the rice nitrogen fertilizer accounts for 37% of the total global rice nitrogen fertilizer, so that the country applying the most inorganic nitrogen fertilizers in the world is formed. The average nitrogen fertilizer dosage of the rice field in China is 180kg/hm²And the average amount of the nitrogen fertilizer is about 75 percent higher than that of the nitrogen fertilizer in the rice field in the world (Pendulong et al 2002). The reality of inefficient utilization and excessive application of nitrogen fertilizer of rice aggravates serious environmental problems such as eutrophication and air pollution; meanwhile, the rice production in China is increasingly threatened by drought. Therefore, the cultivation of new varieties of rice with high nitrogen utilization efficiency and strong drought resistance is very important for improving the grain yield in China and maintaining the sustainable development of agriculture (Bates et al 2008; Xu et al 2012).

The nitrogen source of plants is mainly derived from inorganic nitrides, such as ammonium salts (NH)₄ ⁺) And Nitrate (NO)₃ ^-). However, the main form of inorganic nitrogen source available to rice in the field is ammonium salt (NH)₄ ⁺). NH in the Rice body₄ ⁺Assimilation includes two pathways: glutamine Synthetase (GS) -glutamate synthase (golat) pathway^[8]And the Glutamate Dehydrogenase (GDH) pathway (Jiangliping et al 2002; Qiaet al 2009). NH in higher plants₄ ⁺The main pathway of assimilation is GS/GOGAT, whereas GDH may be the helper NH₄ ⁺Assimilated and involved in the balance of regulation of carbon-nitrogen metabolism in vivo (Lea and Miflin 1974; Abiko et al 2005; Qiu et al 2009.) glutamate dehydrogenase (GDH; E.C.1.1.4.1) catalytic reaction α -oxoglutarate + NH₄ ⁺+ NAD(P)H + H⁺→ glutamic acid + H₂O + NAD(P)⁺. In organisms, GDH is classified into 2 types: one is NADPH-GDH with NADPH as a coenzyme, which mainly acts on the synthesis of glutamic acid, and the other is NADH-GDH with NADH as a coenzyme, which mainly acts on the decomposition of glutamic acid (Loulakakis et al 1991; Bernard et al 2009). Hereinafter, GDH referred to in the present invention is NADPH-GDH. In higher plants, GDH gene is present, but the GDH gene is responsible for NH₄ ⁺Low affinity of (a) K _m 10 mM-80 mM), and GDH vs NH derived from fungi₄ ⁺Very high affinity of (a)K _m 0.2 mM-4.5 mM) indicating low NH of fungal GDH₄ ⁺The ammonia assimilation ability at concentration is stronger than that of higher plants (Abiko et al 2010; Zhou et al 2014). Therefore, the utilization of exogenous glutamate dehydrogenase (mainly bacteria and fungi) to improve the utilization rate of nitrogen fertilizer of plants is one of the hot spots of bioengineering research at home and abroad at present. Lightfoot et al (2001) Escherichia coli (E.coli) ((R))E. coli) Is/are as followsgdhAThe gene is expressed in tobacco, and the transgenic plant can enhance the tolerance to PPT (phosphinothricin herbicides) and obviously improve the utilization efficiency of nitrogen. Lightfoot et al (2007) Escherichia coliE.coligdhAWhen the gene is transferred into corn, the germination rate and the biological yield of a transgenic plant in the season of obvious water shortage are higher than those of a control, and the nitrogen utilization efficiency is also obviously improved, so that the nitrogen utilization efficiency can be improved and the drought tolerance of the plant can be enhanced by the efficient expression of the gene in the plant. Especially in semiarid regions, the modification of the glutamate dehydrogenase of crops has good application prospect. Bacillus licheniformis (Bacillus licheniformis) (2000) has been clonedBacillus licheniformis) Glutamate dehydrogenase gene and its expression and function were studied. The fungus Neurospora intermedia (2001) is cloned by an RT-PCR methodNeurospora intermedia) Mortierella gracilis (A) and (B)Neurospora sitophila) Neurospora crassa (A, B, C)Neurospora crassa) The glutamate dehydrogenase gene of (1). Analysis of the activity of the recombinant glutamate dehydrogenase shows that:NiGDH having higher enzymatic ActivityKmThe value is about 0.3-0.45 mmol/L. It was transformed into tobacco and found to promote the growth of tobacco at low nitrogen levels. Chlorella vulgaris (Chlorella vulgaris) by RT-PCRChlorella sorokiniana) Cloning NADPH-GDH gene, and transferring into tobacco (A), (B), (C), and (D)Nicotiana tabacumL.). Studies showed that growth rate and leaf number were significantly higher in low nitrogen medium or in low nitrogen vermiculite than control. Meanwhile, ammonium toxicity experiments show that the transgenic green leaf disks inoculated on the MS solidified culture medium have long survival time no matter under the condition of low ammonium or high ammonium,chlorophyll is high (Huangguo et al, 2002). The research results show that the exogenous NADPH-GDH has great potential and application value in improving the absorption and utilization capacity of plant nitrogen.

In the application aspect of exogenous GDH in rice, a plurality of fungal GDH genes are transferred into rice and show good application prospect. Abiko et al (2010) Aspergillus niger (A. niger)Aspergillus niger) The GDH gene is expressed in rice, and the nitrogen assimilation efficiency and the yield of the rice are obviously improved. Meanwhile, Zhou et al (2015) transform Erenia terebrata (A)Cylindrocarpon ehrenbergii) Is/are as followsCeGDHThe gene is transferred into rice to obtain transgenic plant with obviously raised nitrogen assimilation efficiency and grain yield, especially in low nitrogen field. However, Zhou et al (2014) Pleurotus abalonus (abalone)Pleurotus cystidiosus) Is/are as followsPcGDHThe gene is transferred into rice to obtain transgenic plant line with obviously raised nitrogen assimilation efficiency and glutelin content and obviously raised yield. In addition, Du et al (2014) Neurospora crassa (B.), (Neurospora crassa) Is/are as followsNcGDHWhen the gene is introduced into rice, the growth of the transgenic rice is inhibited, but the herbicide Basta resistance of the transgenic rice is obviously improved. The studies above found that the fungal GDH gene enhances nitrogen assimilation efficiency of rice, but it does not necessarily increase yield, and sometimes decreases yield. Therefore, it is still very urgent and of practical significance to screen and identify new fungal GDH genes that can enhance nitrogen assimilation efficiency and increase yield.

Disclosure of Invention

The invention aims to provide a fungusTrichurusThe glutamate dehydrogenase gene and the protein coded by the gene are namedTrGDHThe corresponding encoded protein is namedTrGDH protein.TrGDHThe sequence has a full length of 1359 bp and codes 452 amino acids.

The invention provides a glutamate dehydrogenase protein, which is named asTrGDH derived from a fungusTrichurusIs a protein having one of the following amino acid residue sequences:

1) SEQ ID No. 2 of the sequence list;

the sequence in the sequence table SEQ ID No. 2 consists of 452 amino acid residues.

TrThe gene encoding GDH also falls within the scope of the present invention.

TrGDHThe cDNA gene of (1), which may have one of the following nucleotide sequences:

1) the DNA sequence of SEQ ID No. 1 in the sequence table;

2) polynucleotide for coding SEQ ID No. 2 protein sequence in sequence table;

3) a nucleotide sequence which can be hybridized with a DNA sequence limited by SEQ ID No. 1 in a sequence table under high-stringency conditions;

4) DNA sequence with 70% over homology with the DNA sequence limited by SEQ ID No. 1 in the sequence list and coding the protein with the same function.

Sequence 1 in the sequence table consists of 1359 bases.

The high stringency conditions are those in which hybridization and membrane washing contain 0.1 XSSPE (or 0.1 XSS), 0.1 XSDS solutions at 65 ℃TrGDHExpression vectors, cell lines and host bacteria of the gene are all within the scope of the present invention.

FungiTrichurusGlutamate dehydrogenase gene of (1) (ii)TrGDH) And the encoded protein thereof belong to the scope of protection of the invention, and amplificationTrGDHPrimers for any segment of the gene are also within the scope of the invention.

Any vector capable of guiding exogenous genes to express in plants is utilized to improve the gene for efficiently utilizing the nitrogen of the riceTrGDHThe transgenic plant which can improve the high-efficiency utilization and growth of the nitrogen of the rice can be obtained by introducing the plant cells. When the gene of the present invention is constructed into a plant expression vector, any of a general promoter, an enhanced promoter and an inducible promoter may be added in front of the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plants or transgenic plant cells, vectors to be used may be processed, for example, by adding selectable markers (GUS gene, GFP, YFP, As-Red, luciferase gene, etc.) or antibiotic marker genes having resistance (hygromycin, gentamicin, etc.),Kanamycin, ampicillin, bleomycin, etc.). For the safety of transgenic plant release, no marker gene can be carried in the construction of plant expression vector, and specific PCR molecular marker screening is carried out in seedling stage. Comprising the inventionTrGDHThe expression vector of (3) can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation or gene gun, etc., and to grow the transformed plant tissues into plants. The plant host to be transformed may be either a monocotyledonous or dicotyledonous plant, such as: rice, wheat, corn, cucumber, tomato, poplar, turf grass, alfalfa and the like.

According to the inventionTrThe GDH protein heterologous expression promotes the nitrogen absorption of rice by improving the total NADP (H) -GDH enzyme activity of the rice in the rice so as to improve the growth of rice seedlings and the effective spike and thousand-grain weight of the rice in the mature period. Thus, heterologous expression of fungi by genetic engineering methodsTrichurusThe glutamate dehydrogenase in the rice can improve the high-efficiency utilization of nitrogen of the rice under the condition of low nitrogen, thereby ensuring that the rice can normally grow under the condition of low nitrogen, improving the utilization rate of nitrogen fertilizer and reducing environmental pollution. In the present inventionTrThe GDH protein has important effects on improving the efficient utilization of nitrogen of rice and further improving the yield of rice.

The invention is further explained below with reference to the drawings and the embodiments.

Drawings

FIG. 1 is a drawing ofTrPhylogenetic tree analysis of GDH proteins.

FIG. 2a is His₆-TF-TrAnd (5) purifying and identifying the GDH protein.

FIG. 2b is His₆-TF-TrAnd (3) identifying the western blot of the GDH protein.

FIG. 3a is His₆-TF-TrGDH in different NH₄Kinetic analysis under Cl concentration gradient.

FIG. 3b is His₆-TF-TrKinetic analysis of GDH at different α -oxoglutarate (2-OG) concentration gradients.

FIG. 3c shows His₆-TF-TrKinetic analysis of GDH at different L-glutamic acid concentration gradients.

FIG. 3d is His₆-TF-TrGDH and rice endogenesisOsGDH in different NH₄Comparative kinetic analysis under Cl concentration gradient.

FIG. 4a shows heterologous expressionTrGDHThe binary vector pCAMBIAL1300-TrGDH map.

FIG. 4b shows heterologous expressionTrGDHSemi-quantitative RT-PCR analysis.

FIG. 4c shows heterologous expressionTrWestern blot analysis of GDH.

FIG. 4d shows heterologous expressionTrGDHFor endogenous GDHs in rice (GDH1，GDH2，GDH3) Quantitative RT-PCR analysis of expression impact.

FIG. 5a shows different NH groups₄Overexpression of plants under Cl concentration gradientUbi::TrGDH-4AndUbi::TrGDH-13(ii) phenotypic analysis of (a).

FIG. 5b is an overexpressed plantUbi::TrGDH-4AndUbi::TrGDH-13and (4) analyzing the enzymatic activity of GDH in roots and aerial parts.

FIG. 5c shows different NH groups₄Overexpression of plants under Cl concentration gradientUbi::TrGDH-4AndUbi::TrGDH-13detecting the nitrogen content.

FIG. 5d shows different NH groups₄Overexpression of plants under Cl concentration gradientUbi::TrGDH-4AndUbi::TrGDH-13wet gravimetric analysis of (1).

FIG. 5e shows different NH groups₄Overexpression of plants under Cl concentration gradientUbi::TrGDH-4AndUbi::TrGDH-13dry weight analysis of (2).

FIG. 6 shows different NH groups₄Overexpression of plants under Cl concentration gradientUbi::TrGDH-4AndUbi::TrGDH-13and (4) analyzing the content of the seed gluten.

Detailed Description

The experimental procedures mentioned in the following examples are conventional ones unless otherwise specified.

Cloning of TrGDH Gene

The conserved region of the GDH gene was found by sequencing the GDH gene sequence of the fungal strain and degenerate primers were designed. Forward priming of degenerate primersStarting from the start codon of the open reading frame, the sequence is 5 '-ATGTCCCACCTGCCTTTCGARCCNGARTT-3'; the reverse primer of the degenerate primer is located behind the 3' end of the open reading frame and has the sequence of 5' -CCACCAGTCACCCTGGTCRTGCATNGC-3 '. By fungiTrichurusThe cDNA is taken as a template, and the cDNA is cloned by using an RT-PCR methodTrGDHA gene. A method according to Chen et al (2006) willTrGDHThe gene was cloned into Gateway entry vector pGWC and then sequenced. Sequence analysis shows that the gene is not included in NCBI database, has complete Open Reading Frame (ORF) and can be read through, is a new gene discovered by the inventor for the first time, and is named asTrGDH. Since the preferences of fungi and rice for codons are different, the aim is to make the rice have different codon usageTrGDHThe gene is better expressed in a rice plant, a codon needing to be changed is found out according to a method in a website http:// www.kazusa.or.jp/codon/, and then a structural domain of a sequence is analyzed, so that an original fungus codon in a functional domain is changed into a codon preferred by rice. Adopting design point mutation primer and using overlap PCR techniqueTrGDHThe original fungal codons in the sequence are changed into rice preferred codons, and simultaneously, the fungal growth is enhancedPcGDHAt the level of translation of the gene in transgenic rice plants, a kozark sequence (GCCACC) was added before the start codon ATG. The full-length primers for cloning are as follows:TrGDH-F：GCCACCATGTCCCACCTGCCTTTCGA，TrGDH-R: CCACCAGTCACCCTGGTCGT are provided. Adding kozark sequence and modifying rice preferred codonTrGDHThe sequence was re-cloned into pGWC vector and recombined into the engineered pCAMBIA1301 vector by Gateway LR reaction. The modified pCAMBIA1301 vector contains hygromycin resistance screening geneHPTAnd red fluorescent protein screening marker geneAsRedAnd cloning the gateway cassette sequence into the pCAMBIA1301 vector multiple cloning siteSacI andSacbetween II, 2 FLAG tag sequences were then added to the 3' end of the gateway cassette sequence (FIG. 2). The constructed pCAMBIA1301-TrGDHTransferring to EHA105 Agrobacterium by electric shock and transforming to Rice (Oryza sativa cv. Kitaake）。

Functional verification of TrGDH gene

(1)TrGDHEvolutionary tree analysis of genes

To analyzeTrGDHHomology relationship between the gene and GDH gene of other fungi and species, we collected the GDH gene of fungi,E.coliThe amino acid sequences of GDH proteins from algae, rice and Arabidopsis (Table 1: specific information for protein sequences used for evolutionary tree analysis), and construction of evolutionary trees using CLUSTAL X and MEGA 5 software. The results show that the method has the advantages of high yield,TrGDHand the prior literature reports on NH₄ ⁺Having very high affinitygdhA(Aspergillus niger),gdhA(Aspergillus nidulans) AndNiGDH(Neurospora intermedia) Has high homology to GDH endogenous to the plant (FIG. 1).

TABLE 1 specific information on protein sequences used for evolutionary Tree analysis

（2）TrProkaryotic expression and enzymatic kinetic analysis of GDH

To analyzeTrNADP (H) enzyme activity of GDH, and we constructed prokaryotic expression vector pCold-TF-TrConversion of GDHE. coli(BL 21). In vitro inducible expressionTrGDH and detection of purified protein by SDA-PAGE and identification by western blot, purified from FIGS. 2a-bTrGDH protein is unique and can be used for determining NADP (H) -GDH enzymatic activity andK _m values (fig. 3a-d, table 2). As a result of the enzyme activity analysis of GDH,Trthe enzyme activity of the GDH in the in vitro positive reaction is greater than that of the reverse reaction, indicating thatTrGDH tends to utilize NH₄ ⁺α -ketoglutaric acid is converted to glutamic acid and consisting ofK _m The value can be knownTrGDH vs NH₄ ⁺Has an affinity greater than that for glutamic acid.

TABLE 2 His₆-TF-TrKinetic parameter analysis of GDH protein on different substrates

(3)TrGDHTransgenic rice

To studyTrGDHThe influence of the gene on the utilization of the nitrogen of the rice can construct the pCAMBIA1301-TrGDH(FIG. 4 a) electroporation transfer into Agrobacterium: (A)Agrobacterium) Strain EHA 105. Then using a plasmid containing the recombinant plasmid pCAMBIA1301- TrGDHThe agrobacterium of (2) infects rice callus, and after culturing for 3 days at 28 ℃ in the dark, resistant callus and transgenic plants are screened on a selection medium containing 50 mg/L hygromycin. The hygromycin resistant plants were acclimatized for 1 week and then transplanted to the field.

(4) Screening and molecular identification of transgenic plants

Harvesting of T₀Seed of transgenic Rice (T)₁Passage), seeds were soaked in water for 2 days, transferred to a 37 ℃ incubator for germination for 3 days, and then seedlings were subjected to semi-quantitative RT-PCR and western blot analysis (fig. 4 b-c). The experimental results show that the high-temperature-resistant steel,TrGDHin transgenic linesUbi::TrGDH-4AndUbi::TrGDH-13the expression level is higher, so that the 2 strains are selected for further experimental study. In order to study exogenous sourcesTrGDHFor endogenous source of riceGDHs（GDH1,GDH2,GDH3) Influence of expression, we analyzed endogenous sources of rice by quantitative RT-PCRGDHs（GDH1,GDH2,GDH3) Expression of (4 d), and as a result, the transfer was foundTrGDHReduce the endogenous source of the riceGDHs（GDH1,GDH2,GDH3) The transcription level of (a). To determineTrWhether GDH has GDH enzyme activity in the transgenic plants, we extracted the total protein of the transgenic plants to detect the activity of NADP (H) -GDH enzyme (FIG. 5 b). The experimental result shows that the enzyme activity of the NADP (H) -GDH of the transgenic plant is greater than that of the wild type, and the enzyme activity of the positive reaction is greater than that of the reverse reaction in the transgenic plant and the wild type, which indicates thatTrThe GDH protein has functions in the transgenic plants.

(5) Phenotypic analysis of transgenic plants

The transgenic rice plant obtained in the last stepUbi::TrGDH-4AndUbi::TrGDH-13and wild type respectivelyHydroponic and field experiments were performed under homonitrogen conditions to observe the phenotype (FIG. 5 a). In hydroponic experiments, the dry weight, wet weight, nitrogen content, and gluten, etc. of transgenic plants were all better than wild type (FIG. 5c-e; FIG. 6). In the field experiment results, the effective panicle weight and thousand seed weight of the transgenic plants are higher than those of the wild type, but the maturing rate is slightly lower than that of the wild type, so that the yield of the transgenic rice is not obviously improved compared with that of the wild type (Table 3).

TABLE 3 wild type andTrGDHstatistical analysis of field agronomic traits in transgenic rice

3.TrGDHApplication of gene

In recent years, the nitrogen utilization efficiency of rice has attracted attention from the viewpoints of economic efficiency and environmental protection. At present, the research on the utilization of the nitrogen in the rice mainly focuses on the aspects of exploration of a fertilizer application method and period, research and development and application of a slow-release compound fertilizer, cultivation and use of azotobacter in rice combined engineering and the like, and certain progress is made. The transgenic technology generated in the last 80 th century makes up the defects of the conventional breeding method due to the advantages of directly modifying the genetic material of the plant at the gene level, directionally modifying the genetic character of the plant, breaking the reproductive isolation barrier between species, enriching the gene resources and the like by the transfer of the exogenous gene and has unprecedented development. The Glutamate Dehydrogenase (GDH) of rice itself is relative to NH₄ ⁺The low affinity of the rice protein determines that the utilization efficiency of the nitrogen of the rice is not high. At present, the research work at home and abroad proves that the exogenous source is passedGDHThe high-efficiency expression of the gene in the transformed plant can improve the utilization rate of nitrogen. To date, although many fungal GDH genes have been transferred into rice and have shown promising application, the fungal GDH genes do not necessarily increase yield. Therefore, it is still pressing to develop a novel fungal GDH gene that can enhance nitrogen assimilation efficiency and increase yield. We transformed the fungus by Agrobacterium-mediated genetic transformationTrichurusGDH gene of (4)（TrGDH) The rice is transferred to obtain good effects on improving the utilization of the nitrogen of the rice and improving the growth and the agronomic characters of the rice.

<110> university of Hunan

<120> fungal TrGDH protein for improving efficient utilization of nitrogen and application thereof

<160>2

<210>1

<211>1359

<212>cDNA

<213> trichoderma (Trichurus)

<400>1

ATGTCCCACCTGCCTTTCGAGCCTGAGTTCGAGCAGGCTTACAACGAGCTTGCCACCACGCTCGAGAACTCGACCCTCTTCCAGAAGCACCCCGAGTATCGCACCGCGCTCAAGGTTTCCGCCATCCCCGAGCGTGTCATCCAGTTCCGCGTTGTCTGGGAGGACGATGCGGGCAACCTGCAGGTCAACCGCGGTTACCGCGTTCAGTTCAACTCCGCGCTAGGCCCTTACAAGGGTGGCCTGCGCCTCCACCCCTCGGTCAACCTGTCTATTCTTAAGTTCCTTGGCTTCGAGCAGATCTTCAAGAATGCGCTCACTGGTCTTTCCATGGGCGGCGGCAAGGGAGGCGCCGATTTCGACCCCAAGGGCAAGAGCGACGGCGAGATCCGCCGTTTCTGCACCTCTTTCATGCGTGAGCTTGGAAAGCACATCGGTGCCGACACTGACGTGCCCGCTGGTGACATCGGTGTGGGCGGTCGCGAGATCGGCTACCTGTTCGGTGCCTACCGCAAGGACCGCAACAAGTTCGAGGGTGTCCTGACCGGCAAGGGCCTCAGCTGGGGCGGCAGCCTGATCAGGCCCGAGGCCACCGGCTACGGTCTCGTCTACTACGTTGACCTCATGCTCCAGCACATGGGCAAGGGCGGCTTCGCTGGCAAGCGCGTCGCCATCTCCGGCTCCGGCAACGTCGCCCAGTACGCGGCGCTCAAGTGCATTGAGCTCGGTGCCACCGTCGTCTCTCTCTCCGACTCCACCGGTGCCCTCGTCCTCGACGGTGCGGAGGACTCCTTCACTCCCGAGGATATCAGCGCCATCATGAGCCTCAAGGAGAAGCGCAAGGCCATCACTGAGTTCAGCGGCAACGGCAAGCACAGGTACATCGCCGGCGCCAGGCCGTGGGTGTACGTCGGCAAGGTCGACATCGCGCTCCCCTGCGCGACCGAGAACGAGGTCAGCAAGGAGGAGGCCGAGGTGCTCGTCCGCAACGGCTGCTACGTCGTCGCTGAGGGCTCCAACATGGGCTGCACCCAGGAGGCCATCGACCTCTTCGAGGCCGAGAGGCAGACCAAGGGCAGCTCCGCCATCTGGTACGCCCCCGGCAAGGCCGCCAACTGCGGTGGTGTCGCCGTCTCGGGTCTCGAGATGGCCCAGAACAGCCAGCGCCTCCGGTGGACCCGTGAGGTCGTCGACGAGAGGCTCAAGGACATCATGAAGGACGCCTTCGAGGCCGGTCTCAAGAGCGCCGCCGAGTACGTCGAGACCAAGGAGGGCGAGCTTCCTTCGCTCGTTGCCGGTAGCAACATCGCCGGCTTCATCAAGGTCGCGGAGGCTATGCACGACCAGGGTGACTGGTGGTGA

<210>2

<211>452

<212>PRT

<213> trichoderma (Trichurus)

<400>2

MSHLPFEPEFEQAYNELATTLENSTLFQKHPEYRTALKVSAIPERVIQFRVVWEDDAGNLQVNRGYRVQFNSALGPYKGGLRLHPSVNLSILKFLGFEQIFKNALTGLSMGGGKGGADFDPKGKSDGEIRRFCTSFMRELGKHIGADTDVPAGDIGVGGREIGYLFGAYRKDRNKFEGVLTGKGLSWGGSLIRPEATGYGLVYYVDLMLQHMGKGGFAGKRVAISGSGNVAQYAALKCIELGATVVSLSDSTGALVLDGAEDSFTPEDISAIMSLKEKRKAITEFSGNGKHRYIAGARPWVYVGKVDIALPCATENEVSKEEAEVLVRNGCYVVAEGSNMGCTQEAIDLFEAERQTKGSSAIWYAPGKAANCGGVAVSGLEMAQNSQRLRWTREVVDERLKDIMKDAFEAGLKSAAEYVETKEGELPSLVAGSNIAGFIKVAEAMHDQGDWW

Claims (8)

1. The rice nitrogen high-efficiency utilization protein is a protein consisting of an amino acid sequence shown as SEQ ID No. 2.

2. The gene encoding a protein for efficient utilization of rice nitrogen as claimed in claim 1.

3. The gene according to claim 2, characterized in that: the cDNA code of the gene is protein composed of amino acid sequence shown as SEQ ID No. 2.

4. The gene according to claim 3, characterized in that: the cDNA nucleotide sequence of the gene is shown as SEQ ID No. 1.

5. An expression vector, cell line or host bacterium comprising the gene of any one of claims 2 to 4.

6. A primer for amplifying the gene of any one of claims 2 to 4, which isTrGDH-F：GCCACCATGTCCCACCTGCCTTTCGA，TrGDH-R：CCACCAGTCACCCTGGTCGT。

7. Use of the gene of any one of claims 2 to 4 for breeding rice cultivars.

8. Use according to claim 7 for increasing nitrogen utilisation by crops under low nitrogen conditions.

Images