CN109734785A - It is a kind of from the protein and its encoding gene of kuh-seng and application - Google Patents

Abstract

The embodiment of the invention discloses a kind of protein, the protein is selected from following (a1) or (a2): (a1): protein of the amino acid sequence as described in SEQ ID NO.1；(a2): by the substitution and/or deletion and/or addition of one or several amino acid residues and being nod factor receptor protein as derived from (a1) by amino acid sequence shown in SEQ ID NO.1.The embodiment of the present invention passes through the kuh-seng NFP gene that obtains in the transcript profile of kuh-seng root, the gene covers kuh-seng nfp mutant by carrier, so that the clover nfp mutant of covering has Noduling ability, kuh-seng NFP albumen can be used as nod factor receptor, it is applied in recipient plant, to improve the nitrogen fixing capacity of recipient plant.Legume symbiosis nitrogen-fixing efficiency is being improved, is reducing the use aspect of chemical fertilizer with good application prospect.

Description

It is a kind of from the protein and its encoding gene of kuh-seng and application

Technical field

The present embodiments relate to biological gene technical fields, and in particular to a kind of protein and its volume from kuh-seng Code gene and application.

Background technique

Pulse family kuh-seng category (Sophora) has about 50 kinds, wherein having been reported that can be 20 kinds with dross.Document closes at present It is seldom in the detailed research of kuh-seng platymiscium and rhizobium symbiosis.Sophora alopecuroide (Sophora is had been reported that before It alopecuroides) can be with the rhizobium symbiosis from 6 11 kinds of categories.2015, hardship of the Zelanian scholar from local Ginseng platymiscium is separated to 48 bacterial strains and belongs to Autoinducer, while possessing unique nodulation gene nodC development status. These all show that kuh-seng platymiscium is the promiscuity host of symbiosis.

Leguminous plant and rhizobium symbiotic nitrogen fixation are important the approach of nitrogen cycle.Rhizobium can be planted by infecting pulse family Object forms root nodule, and the nitrogen in fixed air becomes the ammonia that plant can be absorbed.Increase with people to kuh-seng demand, is adopted The demand that wild kuh-seng is unable to the growth of meet demand amount is dug, therefore, the artificial cultivation section of kuh-seng is gradually set up, main to be distributed On Shanxi, Gansu, Ningxia, Inner Mongol and other places.Kuh-seng is can to pass through dross with a kind of leguminous plant of rhizobium symbiotic nitrogen fixation Factor receptor proteins NFP goes identification rhizobium, enters to form root nodule so that rhizobium infect plant root, allows rhizobium in root nodule The inside symbiotic nitrogen fixation.

Kuh-seng (Sophora flavescens) is distributed across the medicinal perennial leguminous plant of Asia northeast.In history The root of kuh-seng is taken as traditional Chinese medicine to treat disease.The leguminous plant feature of kuh-seng can be by exploitation nitragin at present Develop sustainable agriculture.Although people recognize this plant of kuh-seng very early, but for the nod factor receptor egg of kuh-seng Known to white NFP information has no, this albumen is most important for identification kuh-seng rhizobium.It, cannot after this protein mutation With rhizobium symbiotic nitrogen fixation, so that the ability of symbiotic nitrogen fixation cannot be played.

During perceiving NF, two members of LysM family are considered playing a significant role: first is that being located at LysM I The LYK3 of NFR1 and clover in branch including crowtoe；Second is that LysM II branch includes NFR5 and the clover of crowtoe NFP.Protein model has predicted receptor protein and the binding site of NF, while two receptors go to tie with nanomolar range concentration Close NF.This affinity is consistent with the concentration of NF needed for activation symbiosis signal especially calcium ion oscillations.Experiments have shown that NFR1 It is of equal importance with intrusion of the NFR5 for rhizobium, and interaction is combined together.But the intracellular kinase of NFR5 seemingly nonfunctional , and also indicate that this structural domain is not needed for infecting after mutation analysis.On the contrary, NFR1 has one extremely to close symbiosis signal Important kinase domain, and have self-phosphorylation ability.The kinases area for showing dross receptor protein is in the form of polymer It functions, the function of this phosphorylation transfer is most important for the activation of downstream signal.

In conclusion the information of nodulation factor receptor proteins NFP is not studied in kuh-seng at present, knot is excavated from kuh-seng The gene order of tumor factor receptor proteins NFP and its coding, and the protein is applied in other leguminous plants, improve pulse family The symbiotic nitrogen fixation rates of plant reduces the use of chemical fertilizer, cultivates leguminous plant new varieties, has important biological significance and economy Value.

Summary of the invention

For this purpose, the embodiment of the present invention provides a kind of protein from kuh-seng and its encoding gene and application.

To achieve the goals above, the embodiment of the present invention provides the following technical solutions:

A kind of protein, which is characterized in that the protein is selected from following (a1) or (a2):

(a1): protein of the amino acid sequence as described in SEQ ID NO.1；

(a2): by amino acid sequence shown in SEQ ID NO.1 by one or several amino acid residues substitution and/or It is deleted and/or added and is nod factor receptor protein as derived from (a1).

The encoding gene of the protein provided in an embodiment of the present invention.

The encoding gene is selected from following (b1) or (b2):

(b1) nucleotide sequence encoding gene as shown in SEQ ID NO.2；

(b2) there is the coding for the nucleotide sequence that can hybridize under strict conditions with DNA sequence dna shown in SEQ ID NO.2 Gene.

Protein provided in an embodiment of the present invention improves the application in leguminous plant nitrogen fixing capacity in preparation.

Application provided in an embodiment of the present invention, including into recipient plant, obtaining the channel genes of code for said proteins The nitrogen fixing capacity plant higher than recipient plant the step of.

The recipient plant is dicotyledon or monocotyledon.

The gene of code for said proteins is the expression cassette by the nucleic acid molecules containing code for said proteins and contains Have in recipient plant described in the vector introduction of the nucleic acid molecules of code for said proteins.

In the embodiment of the present invention, NFP: nod factor acceptor gene.

In the embodiment of the present invention, MtNFP: M. truncatula nod factor acceptor gene.

In the embodiment of the present invention, SfNFP: kuh-seng nod factor acceptor gene.

In the embodiment of the present invention, Nfp: nodulation factor receptor proteins.

In the embodiment of the present invention, pNFP:Mt-NFP: the plasmid of M. truncatula nod factor receptor covering nfp mutant.

In the embodiment of the present invention, pNFP:Sf-NFP: the plasmid of kuh-seng nod factor receptor covering nfp mutant.

The embodiment of the present invention has the advantages that

Description of test, the embodiment of the present invention by the kuh-seng NFP gene that is obtained in the transcript profile of kuh-seng root, by the gene By being connected to carrier, and clover nfp mutant can be covered, so that the nfp clover mutant of covering has Noduling ability, Kuh-seng NFP albumen can be used as nod factor receptor, be applied in recipient plant, to improve the nitrogen fixing capacity of recipient plant.Kuh-seng NFP gene is improving legume symbiosis nitrogen-fixing efficiency, reduces the use aspect of chemical fertilizer with good application prospect.

Detailed description of the invention

It, below will be to embodiment party in order to illustrate more clearly of embodiments of the present invention or technical solution in the prior art Formula or attached drawing needed to be used in the description of the prior art are briefly described.It should be evident that the accompanying drawings in the following description is only It is merely exemplary, it for those of ordinary skill in the art, without creative efforts, can also basis The attached drawing of offer, which is extended, obtains other implementation attached drawings.

Fig. 1 is that the kuh-seng albumen NFP of ML phylogenetic tree provided in an embodiment of the present invention building and other related pulse families are planted Object homologous protein phylogenetic tree, wherein five-pointed star indicates two different systematic growth branches；Scale, 10% amino acid difference It is anisotropic；

Fig. 2 is the Weblogo figure that three LysM domain conservations provided in an embodiment of the present invention compare；

Fig. 3 is that kuh-seng NFP gene provided in an embodiment of the present invention successfully covers clover nfp mutant electron microscope, wherein S.meliloti 2011-gfp is inoculating strain；From left to right covering plasmid is emptyvector (A, D, G), clover respectively NFP covers the kuh-seng mutant SfNFP (C, F, I) of clover mutant MtNFP (B, E, H), kuh-seng NFP covering；Promoter sequence It is the original promoter of NFP of M.truncatula；D, E, F, which are shown, utilizes RFP fluorescent screening transgenosis root；G, H, I are shown S.meliloti 2011-gfp infects the GFP fluorescence issued after dross；Scale, 1mm；

It is formed after Fig. 4 optical microphotograph sem observation SfNFP and MtNFP covering clover nfp provided in an embodiment of the present invention The slice map of root nodule, wherein A, C refer to the root nodule slice formed after MtNFP covering nfp；B, D are formed after referring to SfNFP covering nfp Root nodule slice；Red arrow, which identifies, infects line.Scale: A, B25uM；C,D 7.5uM.

Fig. 5 be different carriers provided in an embodiment of the present invention cover clover nfp after with 2011 interaction dross of S.meliloti Situation compares histogram, wherein Empty vector refers to the negative control of empty plasmid covering clover mutant nfp；PNFP refers to lucerne The promoter of Mu NFP；PNFP:Mt-NFP is the positive control of clover NFP gene covering clover mutant nfp；pNFP:Sf-NFP Refer to and covers clover mutant nfp with kuh-seng NFP.Experimental plants statistics numbers are labeled in above histogram, and data collection is from three times Biology repeats to test.The dross number difference of MtNFP and SfNFP covering experiment is not significant (P > 0.05, t are examined).

Fig. 6 is the map of the P1 carrier of the embodiment of the present invention.

Specific embodiment

Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation Content disclosed by book is understood other advantages and efficacy of the present invention easily, it is clear that described embodiment is the present invention one Section Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not doing Every other embodiment obtained under the premise of creative work out, shall fall within the protection scope of the present invention.

1 kuh-seng NFP gene of embodiment is transferred

1, culture medium and reagent prepare:

TY culture medium (1L): tryptone 5g, yeast powder 3g, CaCl₂0.6g, solid medium add agar powder 15g.It is low Nitrogen nutrition liquid (1L): ironic citrate 0.075g, Ca (NO₃)₂, 0.03g, KCl 0.075g, MgSO₄·7H₂O 0.06g, K₂HPO₄ 0.136g,CaSO₄0.46g, microelement 1mL.Microelement (1L): H₃BO₃2.86g MnSO₄1.81g CuSO₄·5H₂0 0.8g, ZnSO₄0.22g, H₂MoO₄0.02g.Root nodule fixer (2.5% glutaraldehyde) (Van de Velde et al.2006)。

The preparation of 0.05M cacodylate buffer (pH 7.2): Solution A (1L): Sodium cacodylate trihydrate 42.8g.603mL is added in the concentrated hydrochloric acid 10mL of Solution B:0.2M HCl, 36-38% In water.Add in 4.2mL solution B to 50mL solution A, adds water polishing to 200mL.

The preparation of root nodule fixer: by 25% glutaraldehyde solution (Sigma) and the 0.05M cacodylate prepared Buffer is mixed by 1:9, as root nodule fixer.

Toluidine blue dye liquor (100mL): 1g Toluidine bule O is dissolved in 100mL ddH₂In O, 0.22 μm is used It dispenses and uses after bacterial filter filtering.

Bacteroid Extraction buffer: Extraction buffer (DTT containing 10mM, the 300mM sucrose, 10mM of 0.33g PVP Phosphate buffer pH 7.0,2mM MgCl₂) total RNA extraction reagent box RNAiso Plus be purchased from TaKaRA company；It is high Salt precipitation solution leads to company purchased from Beijing six directions；DEPC is purchased from Beijing Baeyer enlightening biotech firm；Quantitative PCR reagent StarScript II First-strand cDNA Synthesis Mix, reverse transcription try II First-strand cDNA of box StarScript Synthesis Mix is purchased from GenStar；PCR primer synthesis and sequencing, high-flux sequence work all by Beijing Huada gene company It completes.

2, transcript profile sample sequencing quality in kuh-seng root is assessed

It is respectively as follows: R.yanglingense CCBAU 01603 and S.fredii CCBAU using 2 plants of different bacterial strains 45436 are inoculated with kuh-seng root, and the corresponding position in kuh-seng root was collected at 15 days or so.The selection of materials time is according to hardship It is found after ginseng process of nodulation observation, compare other leguminous plants, and kuh-seng is that cotyledon stays soil type, and the dross time is later.Respectively 6 kuh-seng roots are taken, transcript profile sample sequencing is carried out to it, 6 kuh-seng root transcript profile sample sequencing quality assessment results are shown in Table 1.

Table 1

By local blast, c51657_g1 gene order number and clover and the homologous base of crowtoe NFP in database are found Because of similitude height.Kuh-seng NFP gene is obtained from transcript profile data, gene order is as shown in SEQ ID NO.2.

3, the building of kuh-seng phylogenetic tree and protein sequence alignment

As shown in Figure 1, the kuh-seng albumen NFP of ML phylogenetic tree building and other related leguminous plant homologous protein systems Development tree, five-pointed star indicate two different systematic growth branches；Scale, 10% amino acid difference are anisotropic.Pass through phylogenetic tree Building and protein sequence relatively show that this latent gene and other known pulse family nod factor receptor similitudes are high.Kuh-seng The amino acid sequence of NFP albumen is as shown in SEQ ID NO.1.

As shown in Fig. 2, kuh-seng NFP albumen passes through Weblogo, website http://weblogo.threeplusone.com/ Three LysM domain conservations of the protein being calculated online compare figure, wherein different amino acid big minispread generation Table conservative differences, such as different letters represent different aminoacids, and different alphabetical sizes on each position, represent the ammonia in figure The conservative of base acid, letter is bigger, and conservative is stronger.

Implement the clone of 2 kuh-seng NFP genes

1, the extraction of kuh-seng plant tissue RNA

(1) prepare liquid nitrogen, with Liquid nitrogen precooler 1.5mL centrifuge tube, 1mLRNAiso Plus is added in the pipe of pre-cooling and extracts Liquid；

(2) dehydrated alcohol is added in advance and burns for mortar, goes RNA enzyme, and it is (steady to liquid nitrogen that Liquid nitrogen precooler mortar is then added It is fixed)；

(3) liquid nitrogen is used, the root nodule of about 0.1g or the rhizobium of pure body is ground in the cooling mortar of N2, is thoroughly ground to It is powdered；

(4) about 0.1g tissue is added in the centrifuge tube of the RNAiso Plus equipped with 1mL, is shaken with oscillator uniform；

(5) it is stored at room temperature 5~10min；

(6) 12000rmp, 4 DEG C of centrifugation 15min lysates, to remove sundries, by supernatant be transferred to a new 1.5mL from In heart pipe, 2mL chloroform is added；

(7) acutely oscillation is reversed test tube and is mixed well several times；

(8) it is incubated for 5~10min at room temperature；

(9) water phase (about 400 μ L) is then transferred in new 1.5mL pipe, is sure not by 12000rmp, 4 DEG C of centrifugation 20min White middle layer is sucked out；

(10) isopropanol of 1 times of volume and the precipitation solution with high salt of 1 times of volume is added in the corresponding water phase being sucked out；

(11) it is gently mixed by inversion, is incubated for 10 minutes, is then incubated overnight at -20 DEG C at room temperature；

(12) 12000rmp, 4 DEG C of centrifugation 10min remove supernatant, are sure not to touch precipitating, remain a small amount of isopropanol and do not close System；

(13) ethyl alcohol of 1mL 70% is added, 12000rmp, 4 DEG C of centrifugation 10min remove supernatant, obtain kuh-seng plant Organize RNA；

(14) kuh-seng plant tissue RNA addition 1mL dehydrated alcohol is used for preservation and transport into each pipe.

2, the clone of kuh-seng NFP gene

According to the nucleotide sequence of kuh-seng NFP gene as shown in SEQ ID NO.2, specific primer pair is designed, forward direction is drawn Object is (SEQID NO.3):

ggggacagct ttcttgtaca aagtggaaat gtctgccttc tttcttcct；

Reverse primer is (SEQID NO.4):

ggggacaact ttgtataata aagttgctta acgagctatt acagaagt。

Kuh-seng the plant tissue RNA, reverse transcription cDNA that embodiment 1 is extracted；Using cDNA as template, with above-mentioned primer pair PCR amplification is carried out, pcr amplification product is recycled.PCR reaction system and amplification program are as shown in table 2:

Table 2

3, the detection of pcr amplification product

1% Ago-Gel is prepared using 0.5 × TBE electrophoretic buffer, micro-wave oven Gao Huozhi is completely dissolved, is cooled to not It scalds one's hand, ethidium bromide (0.5 μ gmL of final concentration is added^-1), it rocks to ethidium bromide and agarose solution and is mixed thoroughly, Enter plastic tank, insertion glue comb after agarose is completely dissolved, extracts glue comb, the glue made is put into electrophoresis tank, pours into 0.5 × TBE electrophoretic buffer was not to having offset plate just, and each sample point sample 6 μ L, 100V electrophoresis 30 minutes.Offset plate is taken out, is made With glue instrument UV scanning development is swept, the sample for the single band that becomes clear is taken to carry out digestion.Sample is stored in -20 DEG C.

By the carrier pDONR201 carrier of the PCR product of above-mentioned acquisition insertion Gateway system, then carry out BP reaction and LR reaction.The marker gene of the recombinant vector of acquisition is red fluorescent protein RFP.This carrier is named as P, in existing starting On the basis of subcarrier, in conjunction with P1 carrier, the map of P1 carrier is as shown in fig. 6, the kuh-seng NFP that building clover NFP promoter drives The P2 carrier of expression.

2 kuh-seng NFP gene of embodiment covers mutant experiment

1, P2 carrier transfects clover mutant root hair:

The M. truncatula seed that will have been sprouted, root growth about 1cm are being cut at the tip of a root about 3-4mm with scalpel It is disconnected, top half seedling is then stained with the Agrobacterium being ready for and is placed on FM nitrogen-free agar, is set with sealed membrane closing It is co-cultured in 22 DEG C of illumination boxs.By the root system complete resection of seedling new life after 7 days, and it is transferred to containing suitable antibiotic HRE (Hairy Root Emergence) culture medium on cultivate 10 days, after transgenosis root growth come out after, it is glimmering by RFP Light screening.

2, kuh-seng mutant root hair is transfected using the carrier containing clover NFP gene:

Method equally based on the transfection of above-mentioned clover root hair, obtains the clover mutant plants of covering, the root system of transgenosis Pass through RFP fluorescent screening.

3, blank control group transfects clover mutant root hair using empty plasmid vector.

In the embodiment of the present invention, the carrier and primer sequence used in transfection process are as shown in table 3.

The covering experiment of 3 kuh-seng NFP receptor of table

It is blue that main background carrier used in the work of transgene receptor part (empty plasmid for carrying RFP) comes Holland naturally The universal plasmid that the laboratory Wageningen University Ton has had been built up.Carrier details see document (Xiao et al., 2014)。Xiao,T.T.,Schilderink,S.,Moling,S.,Deinum,E.E.,Kondorosi,E.,Franssen, H.,Kulikova,O.,Niebel,A.,and Bisseling,T.(2014).Fate map of Medicago truncatula root nodules.Development.141,3517-3528。

The present embodiment 2 can get, the clover that clover mutant, clover NFP gene containing the covering of kuh-seng NFP gene cover Mutant and the clover mutant of empty plasmid covering.

The clover mutant dross energy that clover mutant, the clover NFP gene of 3 kuh-seng NFP gene of embodiment covering cover Force estimation

The clover for the kuh-seng NFP gene covering that the representative strain S.meliloti 2011 filtered out needs tieback to cover is prominent The clover mutant that variant, clover NFP gene cover, the clover mutant of empty plasmid covering, and detect its Noduling ability.

1, strain culturing: all representative strain S.meliloti 2011 are from -80 DEG C of activation to YMA plate, 28 DEG C of inversion trainings 2-5d is supported, picking single colonie is seeded to TY fluid nutrient medium, 180rpm, 28 DEG C of culture 2-3d to logarithmic phase；

2, the preparation of the double-deck alms bowl: deionized water is added in the can of 500mL capacity, every bottle of about 250mL uses sealing Film seals bottleneck, 121 DEG C of sterilizing 30min.Vermiculite is mixed to holding is agglomerating using 1 × Poor nitrogen nutrition liquid and is not dripped, black is used Polybag (two layers) installs the vermiculite mixed thoroughly, 121 DEG C of sterilizing 90min.By the disposal plastic cup bear of 350mL and wear A piece gauze, black plastic bag load, 121 DEG C of sterilizing 30min.In superclean bench, by sterile vermiculite dispense to In 350mL aseptic plastic cup, aseptic plastic cup is put into sterile can, guarantees that gauze can absorb water from can, supplies To needed for the plant growth in vermiculite.

3, germination seed: clover mutant, the clover NFP gene for respectively covering the kuh-seng NFP gene of covering cover The seed of clover mutant and the clover mutant of empty plasmid covering is outwelled the concentrated sulfuric acid with concentrated sulfuric acid skin breaking 5min, It is washed to and does not lose color.It turns next in superclean bench and is operated, the seed after skin breaking is put into sterile small burning In cup, 95% ethyl alcohol is added, stands 30s, outwells ethyl alcohol, and blot net remaining ethyl alcohol with sterile pipette tips, hypochlorous acid is added Sodium thimerosal handles 5min, and 1min rocks once, outwells javelle water, pipette tips clean remaining thimerosal, then use nothing Bacterium is washed 7 times.Sterilized seed is uniformly put on 0.5% water agar plate, 28 DEG C are protected from light germination.

4, seedling: this process operates in superclean bench, by the seed kind of the long 1-2cm of bud to the vermiculite in the double-deck alms bowl In, inoculation 1mL bacterium solution to seed root, sealed membrane sealing.35d is cultivated in 16h illumination, the illumination greenhouse of 8h dark, is harvested Count dross situation.

Experimental result, as shown in figure 3, compared with the control group, containing under the promoter driving of clover NFP (MtNFP) MtNF carrier can cover clover nfp mutation type surface by the transfection of root hair, and knot can be infected by S.meliloti 2011-gfp Tumor.Equally, the P2 carrier of kuh-seng NFP (SfNFP) has successfully covered clover nfp mutant after the starting of clover NFP promoter Phenotype.The SfNFP gene of the embodiment of the present invention has the function similar to other leguminous plant NFP homologous genes identification rhizobium, Kuh-seng but can be with S.meliloti 2011-gfp symbiosis dross (Jiao et al., 2015).

The embodiment of the present invention is observed after the root nodule tied after covering being done slice and Toluidine blue staining, as shown in figure 4, Inside the root nodule formed after SfNFP covering, the line that infects of S.meliloti 2011 extends normally, can observe after simultaneously amplifying Developmental condition to the arrangement of bacteroid and inside root nodule also (Xiao et similar with bacteroid in the root nodule of MtNFP covering al.,2014).The SfNFP of the embodiment of the present invention can restore the phenotype of clover nfp to wild-type levels completely.But in ammonia Base acid sequence compares display, SfNFP not consistent to the vital amino acid L and SfNFP albumen of the function of MtNFP albumen Different protein steric structurals may be taken when identifying S.meliloti 2011 from MtNFP.

As shown in figure 5, the present invention is real after different carriers covering nfp compared with 2011 interaction dross situation of S.meliloti It applies example and has counted nodule number on different disposal red fluorescence transgenosis root, cover clover mutant dross situation class with MtNFP Seemingly, SfNFP is fine with 2011 dross situation of S.meliloti after covering clover nfp, and nodule number is averagely 3, without significant Difference.This illustrates that SfNFP and MtNFP do not have notable difference in identification 2011 nod factor efficiency of S.meliloti.

Although above having used general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.

Sequence table

<110>Jiao Yinshan Chen Wenfeng

<120>a kind of from the protein and its encoding gene of kuh-seng and application

<130> GG19443618A

<160> 6

<170> SIPOSequenceListing 1.0

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<212> PRT

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Met Ala Val Phe Phe Leu Pro Ser Arg Ser Val Ser Leu Leu Ile Ala

1 5 10 15

Phe Met Leu Phe Ser Thr Asn Ile Ala Ala Gln Ala Gln His Thr Asn

20 25 30

Gly Thr Asp Phe Ser Cys Pro Val Asp Ser Pro Pro Ser Cys Glu Thr

35 40 45

Tyr Val Thr Tyr Ile Ala Gln Ser Pro Asn Phe Val Ser Leu Phe Asn

50 55 60

Ile Ser Asn Leu Phe Asp Thr Ser Pro Leu Ser Ile Ala Ser Ala Ser

65 70 75 80

Asn Leu Lys Ala Glu Asp Asn Lys Leu Val Pro Gly Gln Val Leu Leu

85 90 95

Ile Pro Val Thr Cys Gly Cys Ile Gly Asn Arg Ser Phe Ala Asn Met

100 105 110

Ser Tyr Glu Ile Lys Lys Gly Asp Thr Tyr Asp Phe Val Ala Thr Thr

115 120 125

Ser Tyr Glu Asn Leu Thr Ile Trp Gln Val Val Val Asp Phe Asn Pro

130 135 140

Gly Leu Thr Pro Ala Val Leu Pro Val Arg Val Lys Val Val Phe Pro

145 150 155 160

Leu Phe Cys Arg Cys Pro Ser Lys Asn Gln Leu Asn Lys Gly Ile Lys

165 170 175

His Leu Ile Thr Tyr Val Trp Gln Pro Asn Asp Thr Val Ser Ile Val

180 185 190

Gly Ala Lys Phe Gly Ala Ser Pro Val Asp Leu Leu Thr Glu Asn Asn

195 200 205

Tyr Gly Lys Asn Phe Thr Ala Ala Thr Tyr Leu Pro Val Leu Ile Pro

210 215 220

Val Arg Gln Leu Pro Thr Leu Asn Gln Ser Gln Pro Asn Pro Ser Asn

225 230 235 240

Gly Arg Arg Ser Ser Ser His Leu Met Val Ile Ile Gly Thr Thr Leu

245 250 255

Gly Cys Thr Phe Leu Val Ala Val Leu Ala Val Leu Leu Val Tyr Val

260 265 270

Tyr Tyr Leu Lys Lys Lys Ile Leu Lys Arg Asn Ala Ser Ser Val Glu

275 280 285

Thr Ala Asp Lys Leu Leu Ser Gly Val Ser Gly Tyr Val Ser Lys Pro

290 295 300

Thr Met Tyr Glu Ile Asp Ala Ile Met Glu Ala Thr Met Asn Leu Gly

305 310 315 320

Glu Gln Cys Lys Ile Gly Glu Ser Val Tyr Lys Ala Lys Met Glu Gly

325 330 335

Arg Val Leu Ala Val Lys Arg Ile Lys Glu Asp Val Ser Glu Glu Leu

340 345 350

Lys Ile Leu Gln Lys Val Asn His Gly Asn Leu Val Lys Leu Ile Gly

355 360 365

Val Ser Ser Asp Asn Asp Gly Asn Phe Phe Leu Val Tyr Glu Tyr Ala

370 375 380

Glu Asn Gly Ser Leu Asp Asp Trp Met Phe Ser Lys Ser Thr Ser Asn

385 390 395 400

Ser Met Val Ser Leu Thr Trp Ser Gln Arg Leu Ser Ile Ala Val Asp

405 410 415

Val Ala Met Gly Leu Gln Tyr Met His Glu His Thr His Pro Arg Ile

420 425 430

Val His Arg Asp Ile Thr Thr Ser Asn Ile Leu Leu Asp Ser Asn Phe

435 440 445

Lys Ala Lys Ile Ala Asn Phe Ser Met Ala Arg Thr Ser Thr Asn Pro

450 455 460

Met Met Pro Lys Ile Asp Val Phe Ala Phe Gly Val Val Leu Ile Glu

465 470 475 480

Leu Leu Thr Gly Arg Lys Ala Ile Thr Thr Lys Ala Asn Gly Glu Val

485 490 495

Val Met Leu Trp Lys Asp Val Arg Lys Ile Phe Asp Leu Glu Glu Asn

500 505 510

Arg Glu Glu Ser Leu Gln Arg Trp Met Asp Pro Lys Leu Glu Asn Phe

515 520 525

Tyr Pro Val Asp Tyr Ala Leu Ser Leu Ala Ser Leu Ala Val Asn Cys

530 535 540

Thr Ala Asp Lys Pro Leu Ser Arg Pro Thr Met Ala Glu Ile Val Leu

545 550 555 560

Cys Leu Ser Leu Leu Ala Gln Pro Ser Pro Pro Ile Leu Glu Arg Ser

565 570 575

Leu Thr Ser Gly Leu Asp Val Glu Val Thr Gln Thr Met Ala Pro Ile

580 585 590

Ala Ala Arg

595

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atggctgtct tcttcctccc ctcacgttct gtgagtcttc ttattgcatt catgttgttt 60

tctactaaca tagcagctca agcacaacac accaatggaa cagacttttc atgccctgtg 120

gattcacctc cttcctgtga aacttatgtg acatacattg cacagtctcc aaattttgtg 180

agcctgttca acatatctaa tttatttgat acaagtcctt tatccattgc aagtgccagt 240

aacttaaagg ccgaggacaa caagctggtt ccaggccaag tcctactgat acctgtaact 300

tgtggttgca ttggaaatcg ctcttttgcc aatatgtcct atgagatcaa gaaaggtgat 360

acctacgact ttgttgctac aacttcatac gagaatctca caatctggca ggtagtggta 420

gatttcaacc ctggtctaac tccagcggtg ttgccagtgc gcgtcaaagt tgtattccct 480

ttattctgca ggtgcccttc aaagaaccag ttgaacaaag ggataaagca tctgattact 540

tatgtgtggc agcctaatga cactgtttcc attgtaggtg ccaagtttgg tgcatcccca 600

gtggacttat tgactgaaaa caactatggt aaaaacttca ctgctgcaac ctacctgcca 660

gtgttgatcc cagtgagaca gttgccaact cttaatcaaa gtcaacctaa tccttcaaat 720

ggaagaagga gcagcagtca tctcatggtt ataatcggca ctaccctggg atgcacattt 780

ctagttgcag ttttagcggt attactggtg tatgtttatt atctgaaaaa gaagatattg 840

aaaaggaatg cttcttctgt tgagactgca gataagctac tttctggcgt ttcaggctat 900

gtaagtaagc caacaatgta tgaaattgat gcaattatgg aagctaccat gaacctcggt 960

gaacagtgca agattgggga atcagtatac aaggccaaaa tggaaggtcg agttttagca 1020

gttaaaagaa tcaaggaaga tgtctcggag gagcttaaaa ttctacagaa ggtgaaccat 1080

ggaaatctgg taaaattaat tggcgtctct tcagacaatg acgggaattt tttcctggtt 1140

tatgaatatg ctgaaaacgg gtctcttgat gactggatgt tctccaagtc cacctcaaac 1200

tcaatggtct cactcacatg gagtcagagg ttaagcatag cagtggatgt tgccatgggt 1260

ctgcaataca tgcatgaaca tactcatcca agaatagtcc acagggacat cacaacaagt 1320

aatatccttc ttgactcaaa ctttaaggcc aagatagcaa atttctccat ggccagaact 1380

tctaccaacc ctatgatgcc aaaaatagat gtctttgctt ttggggtggt tctgatagag 1440

ttgcttactg gcaggaaagc cataacaacc aaagcaaatg gtgaggtggt tatgctgtgg 1500

aaggatgtta ggaagatctt tgatctagaa gagaatagag aggagagtct ccaaagatgg 1560

atggatccta agctagagaa cttttatcct gtagattatg ctctcagctt ggcctcctta 1620

gcagtgaatt gcactgcaga taagccttta tccagaccaa ccatggcaga aattgttctt 1680

tgcctctccc ttctagctca gccatctccc ccaatattag agagatcctt gacttctggg 1740

ctagatgtgg aagttactca aacaatggct cccatagcag ctcgttga 1788

<210> 3

<211> 49

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 3

ggggacagct ttcttgtaca aagtggaaat gtctgccttc tttcttcct 49

<210> 4

<211> 48

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 4

ggggacaact ttgtataata aagttgctta acgagctatt acagaagt 48

<210> 5

<211> 48

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 5

ggggacagct ttcttgtaca aagtggaaat ggctgtcttc ttcctccc 48

<210> 6

<211> 47

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 6

ggggacaact ttgtataata aagttgctaa gcttcatgca tccagtg 47

Claims (7)

1. a kind of protein, which is characterized in that the protein is selected from following (a1) or (a2):

(a1): protein of the amino acid sequence as described in SEQ ID NO.1；

(a2): amino acid sequence shown in SEQ ID NO.1 is passed through to the substitution and/or missing of one or several amino acid residues And/or it adds and is nod factor receptor protein as derived from (a1).

2. the encoding gene of protein described in claim 1.

3. the encoding gene of protein as claimed in claim 2, which is characterized in that the encoding gene be selected from following (b1) or (b2):

(b1) nucleotide sequence encoding gene as shown in SEQ ID NO.2；

(b2) there is the encoding gene for the nucleotide sequence that can hybridize under strict conditions with DNA sequence dna shown in SEQ ID NO.2.

4. protein described in claim 1 improves the application in leguminous plant nitrogen fixing capacity in preparation.

5. application as claimed in claim 4, which is characterized in that including planting the channel genes of code for said proteins to receptor In object, the step of obtaining the nitrogen fixing capacity plant higher than recipient plant.

6. application as claimed in claim 5, which is characterized in that

The recipient plant is dicotyledon or monocotyledon.

7. application as claimed in claim 5, which is characterized in that

The gene of code for said proteins is by the expression cassette of the nucleic acid molecules containing code for said proteins and containing volume In recipient plant described in the vector introduction of the nucleic acid molecules of the code protein.