Expression of recombinant nitrogenase synthetic pathway in non-leguminous plants

Abstract

The invention relates to expression of a recombinant nitrogenase synthetic pathway in non-leguminous plants. According to the invention, 11 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus polymyxa and 4 genes (nifU, nifS, nifF and nifJ) from Klebsiella oxytoca are introduced into non-leguminous plants such as rice, wheat, corn, arabidopsis thaliana or cotton, and the total 15 nitrogen fixation genes are introduced into the non-leguminous plants such as rice, wheat, corn, arabidopsis thaliana or cotton, so that the plants obtain the independent nitrogen fixation capability, the chemical nitrogen fertilizer consumption required by the growth of the non-leguminous plants is reduced, and the ecological environment is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology and synthetic biology, in particular, the application of a recombinant nitrogenase synthesis pathway in enhancing the nitrogen fixation ability of non-leguminous plants. BACKGROUND

[0002] Biological nitrogen fixation refers to the process by which a few prokaryotes reduce atmospheric nitrogen (N2) to ammonia in vivo at normal temperature and pressure. Biological nitrogen fixation can provide plants with a certain amount of nitrogen. Nitrogenase is a complex enzyme composed of two component proteins, ferredoxin (also known as NifH protein) and molybdenum-iron protein (also known as NifDK protein). The ferredoxin is about 64 kDa in size, and is a dimer (γ2) formed by two γ subunits (encoded by nifH gene), in which a [4Fe-4S] cluster is located at the interface of the two subunits and is connected by two cysteine residues. The molybdenum-iron protein is about 240 kDa in size, and is a tetramer (α2β2) composed of two α subunits (encoded by nifD gene) and two β subunits (encoded by nifK gene). The molybdenum-iron protein binds two metal clusters: FeMo-cofactor (Mo-7Fe-9S-C-homocitrate) is the site of N2 binding and reduction, and the P-cluster (8Fe-7S) is located at the interface of the α subunit and the β subunit. Electrons are transferred from the [4Fe-4S] cluster of the ferredoxin to the P-cluster, and then to the FeMo-cofactor to reduce N2 to ammonia. There are six essential genes for the synthesis of nitrogenase: nifH, nifD, nifK, nifB, nifE, and nifN. In addition to these essential genes, the number of nitrogen fixation genes (nif) required for the synthesis and maturation of nitrogenase varies greatly among nitrogen-fixing microorganisms. In Azotobacter vinelandii and Klebsiella oxytoca, the synthesis of active NifH protein requires the products of NifH, nifM, nifS, and nifU, while active NifDK requires the products of NifH, nifD, nifK, nifB, nifE, nifN, nifS, nifU, nifQ, nifX, nifV, nifW, nafY, and nifZ. Studies on A. vinelandii and K. oxytoca have shown that the synthesis of nitrogenase requires at least 16 genes.

[0003] In Paenibacillus polymyxa, a minimum nif gene cluster consisting of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV) enables E. coli to synthesize active nitrogenase. In addition to the nif gene cluster, the sufCDSUB operon, nifS-like gene and yutI gene on the P. polymyxa genome are also necessary for the synthesis of the Fe-S cluster of nitrogenase; groES and groEL play a role in protecting the stability of the Fe-S cluster. In Azotobacter vinelandii and Klebsiella oxytoca, nifS and nifU are responsible for the synthesis of the Fe-S cluster. Based on previous research results, the synthesis of nitrogenase requires at least 11 nitrogen fixation genes [nifH, nifD, nifK, nifB, nifE, nifN, nifV, nifX, nifQ (or hesA), nifU and nifS].

[0004] According to the different relationships between nitrogen-fixing bacteria and plants, they can be divided into: free-living nitrogen-fixing bacteria, symbiotic nitrogen-fixing bacteria and associative nitrogen-fixing bacteria. Free-living nitrogen-fixing bacteria live freely in the soil, water and other environments, and have a relatively loose relationship with plants; symbiotic nitrogen-fixing bacteria mainly include rhizobia, which form root nodules with leguminous plants, and perform nitrogen fixation in the root nodules, and the fixed ammonium is transported to other parts of the plant to promote plant growth. The nitrogen fixation efficiency of rhizobia is high, but they can only fix nitrogen symbiotically with leguminous plants; associative nitrogen-fixing bacteria mainly colonize on the root surface of non-leguminous plants such as corn, wheat and rice, and some enter the intercellular space of plants but do not form root nodules. The nitrogen fixation efficiency of associative nitrogen-fixing bacteria is easily inhibited by the concentration of ammonium and oxygen in the environment, and is lower than that of rhizobia. Regardless of which type of nitrogen-fixing bacteria, they all reduce nitrogen in the air to ammonium under the catalysis of the same nitrogenase.

[0005] Biological nitrogen fixation is a high-energy-consuming process, and it consumes 16 ATP to reduce 1 molecule of N2 to synthesize 2 molecules of ammonium (see the following reaction formula):

[0006] N2+8e - +10H + +16ATP→2NH4 + +H2+16[ADP+Pi]

[0007] Because the nitrogenase is very sensitive to oxygen, scientists believe that the mitochondria and chloroplasts of plants can be more suitable as the organelle for heterologous expression of nitrogenase in plants. The mitochondria have efficient oxygen-consuming respiratory enzymes and [Fe-S] cluster synthesis pathways, and can produce a large amount of ATP. Although the chloroplasts can perform photosynthesis to produce oxygen, the time separation mechanism similar to that in cyanobacteria can be used to make the chloroplasts perform photosynthesis during the day and nitrogen fixation at night.

[0008] However, food crops such as rice, wheat, corn and cotton lack the rhizobium symbiotic nitrogen fixation system, and their high yield and stable yield are highly dependent on chemical nitrogen fertilizer. Therefore, how to improve the nitrogen fixation performance of these non-legume plants is a problem to be solved. SUMMARY

[0009] In view of the defects in the prior art, the purpose of the present application is to provide expression of bacterial nitrogenase genes in non-legume plants such as rice, wheat, corn and cotton. The application uses synthetic biology to introduce the nitrogen fixation enzyme synthesis pathway (15 nitrogen fixation genes) of nitrogen-fixing microorganisms into the mitochondria of non-legume plants to obtain homozygous trans-nitrogen fixation gene plants, so that the non-legume plants can realize "autonomous nitrogen fixation", which will help to reduce the use of chemical nitrogen fertilizer during the growth of non-legume plants and improve the ecological environment.

[0010] To achieve the above purpose, the technical solution adopted by the present application is:

[0011] The expression of a recombinant nitrogenase synthesis pathway in non-legume plants is characterized in that the recombinant nitrogenase synthesis pathway enables the non-legume plants to have autonomous nitrogen fixation ability.

[0012] The recombinant nitrogenase synthesis pathway includes 15 nitrogen fixation genes, and the nucleotide sequences of the 15 nitrogen fixation genes are shown in SEQ ID NO. 1-15.

[0013] The sequences shown in SEQ ID NO. 1-15 are the optimized sequences of 11 nitrogen fixation genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES and groEL from Paenibacillus polymyxa (the sequences of these genes are registered in the NCBI database under accession number ALJV00000000) and 4 nitrogen fixation genes nifS, nifU, nifF and nifJ from Klebsiella oxytoca (formerly known as Klebsiella sp. M5al) (the sequences of these genes are registered in the NCBI database under accession number CP020657.1).

[0014] On the basis of the above scheme, the non-legume plants include rice, wheat, corn, cotton and the like.

[0015] A recombinant nitrogenase synthesis pathway is characterized in that the recombinant nitrogenase synthesis pathway is sequentially connected by a promoter, a mitochondrial signal peptide, the above-mentioned recombinant nitrogenase gene and a terminator; the mitochondrial signal peptide is connected with the coding region of the recombinant nitrogenase gene.

[0016] On the basis of the above scheme,

[0017] The promoter includes Cc1, Actin or Ubi; the nucleotide sequences of the Cc1, Actin and Ubi are shown in SEQ ID NO. 16-18;

[0018] The mitochondrial signal peptide includes SU9 or pFAγ; the nucleotide sequences of the SU9 and pFAγ are shown in SEQ ID NO. 19-20;

[0019] The terminator includes Nos, 35S or In21; the nucleotide sequences of the Nos, 35S and In21 are shown in SEQ ID NO. 21-23.

[0020] The application of the nitrogenase gene in the above-mentioned recombinant nitrogenase enzyme synthesis pathway in the cultivation of non-legume nitrogen-fixing plants is characterized in that the application is to introduce the nitrogenase gene into non-legume plants.

[0021] The expression of the recombinant nitrogenase enzyme synthesis pathway in non-legume plants has the beneficial effects that:

[0022] The active nitrogenase is expressed in non-legume plants, and the nitrogen in the air can be reduced to ammonium, which can be absorbed and utilized by plants, thereby reducing the application of chemical nitrogen fertilizer and improving the ecological environment. BRIEF DESCRIPTION OF DRAWINGS

[0023] The present application has the following drawings:

[0024] Figure 1 Two plant expression vectors used in Example 1 of the present application: pCAMBIA1300 and pCAMBIA2300.

[0025] Figure 2Four expression cassettes (nifK-nifX-hesA, nifH-nifD-nifF-nifJ, nifS-nifU-groES-groEL and nifB-nifE-nifN-nifV) were constructed for Example 1 of the present application, and each expression cassette was assembled into a plant expression vector (pCAMBIA1300 or pCAMBIA2300) to form four recombinant plant expression vectors: pCAM2301KXA, pCAM1301FJHD, pCAM2300USESEL and pCAM1301BENV.

[0026] Figure 3 Figure 1 is a photograph of the electrophoresis results of PCR detection of the nitrogen fixation genes (nif) in transgenic rice; WT is the product obtained by PCR using the wild-type Nipponbare rice genome as the template (negative control); M is a 1 kb plus DNA ladder.

[0027] Figure 4 Figure 2 is a photograph of the electrophoresis results of RT-PCR products; M is a 1 kb plus DNA ladder; Ml is a 100 bp DNA ladder; the label "-" is the band of the nif gene amplified using the wild-type rice cDNA as the template (negative control); the label "+" is the band of the nif gene amplified using the constructed nitrogen fixation gene plasmid DNA as the template (positive control); nifK, nifX, hesA, nifF, nifJ, nifH, nifD, nifU, nifS, groES, groEL, nifB, nifE, nifN and nifV represent the RT-PCR products from the positive transgenic nitrogen fixation gene rice. DETAILED DESCRIPTION

[0028] The application will be further described below in conjunction with the accompanying drawings.

[0029] The preferred embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be noted that the following examples are given only to illustrate the present application and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained commercially unless otherwise specified.

[0030] Example 1: Construction of plant expression vectors and transgenic rice

[0031] 1. Amino acid sequences encoded by 11 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES, groEL) of Paenibacillus polymyxa, codon-optimized according to eukaryotes (see SEQ ID NO. 1-11).

[0032] 2. Amino acid sequences encoded by 4 genes (nifS, nifU, nifF, nifJ) of Klebsiella oxytoca, codon-optimized according to eukaryotes (see SEQ ID NO. 12-15).

[0033] 3. The above 15 gene sequences (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES, groEL, nifS, nifU, nifF, nifJ) are added with a mitochondrial signal peptide (SU9 or pFAγ) in front of each gene sequence. Then, on the basis of the above addition of mitochondrial signal peptide, a promoter (Ccl or Actin or Ubi) and a terminator (Nos or 35S or In21) are added at the 5'-end and 3'-end respectively, to construct 15 expression kits.

[0034] 4. The 15 expression kits obtained in step 3 are divided into four groups, and assembled into a plant expression vector (such as pCAMBIA1300 or pCAMBIA2300 or other plant expression vectors) using Gibson assembly enzyme, to form four recombinant plant expression vectors. Specifically:

[0035] The coding region of each gene, mitochondrial signal peptide, promoter and terminator are amplified by PCR method respectively, and then the four DNA fragments amplified by PCR are assembled into the expression cassette of the gene using the assembly kit Gibson Assembly MasterMix of NEB company, to assemble 15 gene expression cassettes. Further, the nifK expression cassette, nifX expression cassette and hesA expression cassette are assembled with the vector pCambia2300 (with G418 resistance screening gene (NptII)) digested by BamH I enzyme, to form the recombinant plant expression vector pCAM2301KXA. The nifF expression cassette, nifJ expression cassette, nifH expression cassette and nifD expression cassette are assembled with the vector pCambia1300 (carrying hygromycin resistance screening gene (HygR)) digested by BamH I enzyme, to form the recombinant plant expression vector pCAM1301HDFJ. R) and groEL expression cassette were assembled with the BamH I digested vector pCambia 2300 (carrying G418 resistance selection gene (Npt II)) to form the recombinant plant expression vector pCAM2300USESEL. The nifB expression cassette, nifE expression cassette, nifN expression cassette and nifV expression cassette were assembled with the BamH I digested vector pCambia 1300 (carrying hygromycin resistance selection gene (Hyg R ) to form the recombinant plant expression vector pCAM1301BENV.

[0036] 5. Transform the plants with the recombinant expression vectors constructed in step 4, either individually or collectively. Specifically:

[0037] (1) Introduce the recombinant plant expression vector into Agrobacterium GV310

[0038] The four recombinant plant expression vectors pCAM2301KXA, pCAM1301FJHD, pCAM2300USESEL and pCAM1301BENV were transformed into Agrobacterium GV310 respectively to obtain four recombinant Agrobacterium carrying the recombinant plant expression vectors.

[0039] (2) Agrobacterium-mediated genetic transformation of rice

[0040] (i) Induction of rice callus: peel the mature embryo of rice and place it in a sterile 300 ml triangular flask, sterilize it with sterile water, 75% alcohol and sodium hypochlorite respectively. Then place the seeds on sterile filter paper and dry them for 30 minutes, inoculate them in N6 medium (formula see Table 1 below) to induce callus, 15-20 per dish, and place them in the light zone for culture.

[0041] Table 1 N6 medium formula (unit: mg / L)

[0042]

[0043]

[0044] (ii) Pre-culture: select uniform embryonic callus pieces and place them in pH = 5.6 pre-culture plates, 300-400 callus pieces per plate, in a dark incubation box at 25-28°C for three days. The day before pre-culture.

[0045] (iii) Co-cultivation: 300-400 pre-cultured embryogenic calli were placed in a sterile 250 ml flask, 50 ml of Agrobacterium carrying the recombinant plant expression vector pCAM2301KXA and 50 ml of Agrobacterium carrying the pCAM1301FJHD were added, mixed well and immersed in a shaker. The bacterial solution was discarded and the calli were placed on a sterile filter paper in a Petri dish and dried. A piece of filter paper was placed on a co-cultivation medium plate with pH 5.2 and the dried calli were placed on it. The Petri dish was sealed and incubated in the dark at 25-28°C for three days. The purpose was to co-transform the rice calli with the 7 nitrogen fixation genes (nifK, nifX, hesA, nifF, nifJ, nifH, nifD) carried by the two recombinant plant expression vectors.

[0046] Similarly, 300-400 pre-cultured embryogenic calli were placed in a sterile 250 ml flask, 50 ml of Agrobacterium carrying the recombinant plant expression vector pCAM2300USESEL and 50 ml of Agrobacterium carrying the pCAM1301BENV were added, mixed well and the same experimental operation was performed. The purpose was to co-transform the rice calli with the 8 nitrogen fixation genes (nifU, nifS, groES, groEL, nifB, nifE, nifN, nifV) carried by the two recombinant plant expression vectors.

[0047] (iv) Selection: As described above, the expression vector pCAM2301KXA (integrating the nifKXhesA expression cassette) and the expression vector pCAM1301FJHD (integrating the nifFJHD expression cassette) co-transformed rice; the expression vector pCAM1301BENV (integrating the nifBENV expression cassette) and the expression vector pCAM2300USESEL (integrating the nifUSgroUSUL expression cassette) co-transformed rice. Since the recombinant expression vector pCAM1301FJHD and the recombinant expression vector pCAM1301BENV both carry the hygromycin resistance selection gene (hyg R ), and the recombinant expression vector pCAM2301KXA and the recombinant expression vector pCAM2300USESEL both carry the G418 resistance selection gene (NptII). Therefore, both co-transformed groups were selected with two antibiotics, hygromycin and G418, to obtain rice calli containing 7 nitrogen fixation genes (nifK, nifX, hesA, nifF, nifJ, nifH, nifD) and rice calli containing 8 nitrogen fixation genes (nifB, nifE, nifN, nifV, nifU, nifS, groUS, groUL).

[0048] (v) Pre-differentiation: The yellow granular resistant calli were selected and transferred to pre-differentiation medium (15 calli per bottle) one by one, and cultured under illumination for about 10 days.

[0049] (vi) Differentiation: The fresh yellow and dense resistant calli were selected and transferred to differentiation medium (5 calli per bottle) one by one, and cultured under illumination for about 30 days.

[0050] (vii) Seedling rooting: The resistant calli with green seedlings of about 3-4 cm were selected according to the principle of one growth-advantageous resistant bud per callus, and then transferred to rooting medium to root. The seedlings were cultured under illumination for about 14 days.

[0051] (viii) Hardening-off: After the top membrane of the rooted and strong seedlings was removed, the water was added to adapt to the external environment, and the positive rate was detected. The hardening-off of the seedlings was observed daily, and the water was supplemented in time.

[0052] (3) Rice hybridization

[0053] The positive rice plants containing 7 nitrogen fixation genes (nifK, nifX, hesA, nifF, nifJ, nifH, and nifD) were obtained by screening the co-transformation expression vector pCAM2301KXA and the expression vector pCAM1301FJHD using antibiotics (hygromycin + kanamycin); the positive rice plants containing 8 nitrogen fixation genes (nifB, nifE, nifN, nifV, nifU, nifS, groUS, and groUL) were obtained by screening the co-transformation expression vector pCAM2300USESEL and the expression vector pCAM1301BENV using antibiotics (hygromycin + G418).

[0054] The positive rice plants containing 7 nitrogen fixation genes (nifK, nifX, hesA, nifF, nifJ, nifH, and nifD) were hybridized with the positive rice plants containing 8 nitrogen fixation genes (nifU, nifS, groES, groEL, nifB, nifE, nifN, and nifV). The hybridization of rice can be completed by emasculation and pollination. The positive rice containing 15 nitrogen fixation genes (nifK, nifX, hesA, nifF, nifJ, nifH, nifD, nifU, nifS, groES, groEL, nifB, nifE, nifN, and nifV) was screened from the hybrid offspring by PCR detection.

[0055] Example 2: Agrobacterium-mediated genetic transformation of cotton

[0056] Cotton is transformed by Agrobacterium-mediated cotton hypocotyl genetic transformation method, through callus, embryoid, transgenic tissue culture seedling each stage, finally grafted to stock seedling to obtain transgenic cotton plant. The specific operation steps are as follows:

[0057] (1) seed de-fuzzing: 100g cotton seeds are added with appropriate amount of concentrated H2SO4, and stirred quickly to remove the fiber on the cotton seeds, and then washed with plenty of tap water and dried for standby;

[0058] (2) seed disinfection and cleaning: select full cotton seeds and put them in 100mL triangular flask, 10 seeds per flask, add appropriate amount of 75% ethanol for surface disinfection, pour off, add appropriate amount of 10% sodium hypochlorite for disinfection for 20min, pour off sodium hypochlorite and add appropriate amount of sterile water, shake and clean, repeat for more than 3 times, finally soak the seeds in appropriate amount of sterile water in 28℃ incubator overnight;

[0059] (3) explant preparation: the next day, when the seeds are cracked and white, peel the seeds and plant them in 1 / 2MS seedling culture medium, and cultivate in 28℃ light incubator (16h light / 8h dark) for 6-7d;

[0060] (4) infection bacteria liquid preparation: pick a little bacteria liquid of Agrobacterium carrying nitrogen fixation gene and streak it on LB solid culture medium plate (containing 50mg / L Kan and 50mg / L Rif), invert and cultivate in 28℃ incubator for 2-3d, pick single colony and inoculate in 10mL LB liquid culture medium (containing 50mg / L Kan and 50mg / L Rif) for activation, cultivate overnight in 28℃ shaker at 200rpm, stop cultivation when the OD600 value reaches 0.6-0.8, take 5mL bacteria liquid and dilute it with dilution bacteria liquid medium to OD600 value of 0.3-0.4 for standby;

[0061] (5) infection and co-culture: take out the sterile seedlings cultivated in light incubator, cut off the root and top leaves with surgical blade, cut the remaining hypocotyl into small pieces of about 1cm, soak in the diluted infection bacteria liquid for 7min, take out and absorb the bacteria liquid on the surface of hypocotyl with sterile filter paper, and place evenly on co-culture medium with a layer of filter paper, cultivate in dark at 25℃ for 2d;

[0062] (6) resistant callus induction: after 2d, transfer the hypocotyls to resistant callus induction medium, continue to cultivate in 28℃ light incubator (16h light / 8h dark), observe the callus growth during 1-2d, subculture once after about 20d;

[0063] (7) Primary embryogenic callus induction: After 2-3 months of subculture, the callus was peeled off from the hypocotyl and transferred to differentiation medium (without any antibiotics and hormones) to continue the induction and differentiation, and subcultured once every 20 days until embryogenic callus appeared;

[0064] (8) Embryoid induction: After 2-3 months of subculture, the embryogenic callus formed by differentiation was transferred to embryoid induction medium (without any antibiotics and hormones) to continue the induction and differentiation, and subcultured once every 20 days until embryoid appeared, and continued to be cultured to differentiate into seedlings;

[0065] (9) Tissue culture seedling culture: The differentiated seedlings were inserted into seedling culture medium for continued culture, and subcultured once every 1 month until the stems of the tissue culture seedlings were lignified;

[0066] (10) Grafting: After the tissue culture seedlings were lignified, they were grafted onto rootstock seedlings (25-day-old sea island cotton) and cultured under greenhouse conditions to obtain transgenic cotton T0 generation.

[0067] Example 3: PCR detection of transgenic rice

[0068] CTAB method for extracting rice genomic DNA

[0069] (1) Preheat 2xCTAB buffer solution in a 65°C water bath;

[0070] (2) Weigh 0.1 g of rice leaves into a 2.0 mL centrifuge tube, add a 0.3 mm steel ball, freeze in liquid nitrogen, and grind on a sample grinder for 1-2 min to fully grind the leaves into powder;

[0071] (3) Add 500 μL of 2xCTAB buffer solution, mix well, and incubate at 65°C for 30-60 min, mixing several times during the incubation;

[0072] (4) Take out the incubated solution, cool to room temperature, add 500 μL of chloroform / isopentanol (24:1) mixture to the centrifuge tube, mix well, and centrifuge at 13400xg for 15 min; repeat once to completely remove proteins;

[0073] (5) Transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube, add 400 μL of isopropanol, mix gently, and let stand at room temperature for 10 min to precipitate the DNA, then centrifuge at 13400xg for 15 min at room temperature, and discard the supernatant;

[0074] (6) Wash the precipitate with 0.5 mL of 75% ethanol, centrifuge at 13400xg for 2 min, and discard the supernatant; repeat 2-3 times, and dry the precipitate at 55°C for 10 min;

[0075] ​

[0076] (7) Add 60 μL TE buffer to dissolve the DNA precipitate, and take an appropriate amount of sample to detect the DNA mass and concentration. Store in a refrigerator at -20°C for standby use.

[0077] The PCR primers for amplifying the nitrogen fixation gene (nif) in the transgenic rice are shown in Table 2. The PCR results are shown in Figure 3 .

[0078] Table 2 PCR primers

[0079]

[0080]

[0081] Example 4: RT-PCR detection of the expression of the nitrogen fixation gene in transgenic rice

[0082] On the basis of the above DNA extraction, further extract the rice RNA.

[0083] Extraction of total RNA from rice by Trizol method

[0084] (1) Take 0.1 g of rice leaves in a 2.0 mL centrifuge tube, add a 0.3 mm small steel ball, freeze in liquid nitrogen, and grind on a sample grinder for 1-2 min to fully grind the leaves into powder;

[0085] (2) Add 1 mL Trizol, and blow and suck several times with a gun, mix well, and stand at room temperature for 5 min to completely separate the nucleic acid protein complex;

[0086] (3) Further add 200 μL chloroform, cover the cap, and shake vigorously for 30 s, and stand at room temperature for 5 min to fully react;

[0087] (4) Centrifuge at 13400 x g at 4°C for 10 min, and the sample will be divided into three layers: the upper colorless water phase is the RNA liquid (about 60%), the middle layer is the phenol-chloroform phase, and the bottom layer is light red;

[0088] (5) Carefully aspirate 500 μL of the upper water phase and move to another centrifuge tube, add 500 μL of isopropanol, mix gently by inverting, and white flocculent material can be seen, and stand at room temperature for 10 min to completely precipitate the RNA;

[0089] (6) Centrifuge at 13400 x g at 4°C for 10 min, and the RNA precipitate is a layer of gel-like transparent small block attached to the bottom and wall of the tube;

[0090] (7) Discard the supernatant, add 1 mL of pre-cooled 75% ethanol, and shake by blowing with a gun to fully wash the precipitate;

[0091] (8) 4℃, 13400 x g centrifugation for 5 min, discard the supernatant, after short centrifugation, carefully suck the residual ethanol with the gun head, dry for 10 min in the clean bench (note not to open the fan), resuspend the precipitate with sterilized 1‰ DEPC water, blow and suck several times to dissolve the RNA;

[0092] (9) Use ultramicro UV-visible spectrophotometer (Bio-Sciences AB Healthcare nano) to determine the concentration and purity of RNA, and store it in -80℃ refrigerator for standby.

[0093] Use the reverse transcription kit Prime Script RT reagent Kit with gDNA Eraser of TaKaRa company to reverse transcribe RNA into cDNA, and operate according to the instruction manual.

[0094] Synthesis of cDNA

[0095] Prepare the following reaction mixture on ice:

[0096] RT Primer Mix 1 μL

[0097] 5 x PrimeScript Buffer 2 (for Real Time) 4 μL

[0098] RNase Free dH2O 4 μL

[0099] Dispense the reaction solution obtained in the above step into two PCR tubes with premix, add 1 μL Prime Script RT Enzyme Mix I to one tube, and add 1 μL RNase Free dH2O as a control to the other tube, centrifuge briefly, mix, reverse transcribe at 37℃ for 15 min, and at 85℃ for 5 s, then the product is cDNA. Then use cDNA as a template to perform PCR amplification to obtain RT-PCR product. RT-PCR primers are shown in Table 2. RT-PCR results are shown in Figure 4 . The research results show that 15 nitrogen fixation genes are expressed in rice.

[0100] The contents not described in detail in the specification belong to the prior art known to those skilled in the art.

Claims

1. Use of a recombinant nitrogenase synthesis pathway in a non-legume plant, characterized in that, The recombinant nitrogenase synthesis pathway enables non-legume plants to have the ability of independent nitrogen fixation; The recombinant nitrogenase synthesis pathway comprises 15 nitrogen fixation genes, and the nucleotide sequences of the 15 nitrogen fixation genes are shown as SEQ ID NO. 1-15.

2. Use according to claim 1, wherein The non-legume plants include rice, wheat, corn and cotton.

3. A recombinant nitrogenase synthesis pathway as claimed in claim 1, wherein, The recombinant nitrogenase synthesis pathway is sequentially connected by a promoter, a mitochondrial signal peptide, the recombinant nitrogenase gene in claim 1 and a terminator; and the mitochondrial signal peptide is connected with the coding region of the recombinant nitrogenase gene.

4. The recombinant nitrogenase synthesis pathway according to claim 3, wherein: The promoter comprises Cc1, Actin or Ubi, and the nucleotide sequences of the Cc1, Actin and Ubi are shown as SEQ ID NO. 16-18; The mitochondrial signal peptide comprises SU9 or pFAγ, and the nucleotide sequences of the SU9 and pFAγ are shown as SEQ ID NO. 19-20; The terminator comprises Nos, 35S or In21, and the nucleotide sequences of the Nos, 35S and In21 are shown as SEQ ID NO. 21-23.

5. Use of a nitrogen fixation gene in the recombinant nitrogenase enzyme synthetic pathway as claimed in claim 1 for breeding non-leguminous nitrogen fixation capable plants, characterized in that, The application is to introduce the nitrogen fixation gene into non-legume plants.

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