CAPE polypeptide and application thereof in symbiotic nitrogen fixation of leguminous plants

Abstract

The invention discloses a CAPE polypeptide released by a protein coded by a soybean disease course related gene GmPR1-6 and application of the CAPE polypeptide. The nucleotide sequence of the soybean disease course related gene is as shown in SEQ ID NO.1, the amino acid sequence of protein coded by the soybean disease course related gene is as shown in SEQ ID NO.2, and the sequence of CAPE polypeptide released at the C terminal of the protein is as shown in SEQ ID NO.9. The soybean disease course related gene GmPR1-6 provided by the invention is a gene coding a core protein participating in soybean immune defense reaction, the C tail end of the GmPR1-6 protein can be sheared to release CAPE polypeptide, the CAPE polypeptide is externally applied to weaken early immune defense reaction triggered by soybean during infection of rhizobium, the weight and size of a single root nodule are increased, and the disease resistance of the soybean is improved. The symbiotic nodulation and nitrogen fixation capabilities of the soybeans are improved, and the method has potential application value in planting of leguminous plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a CAPE polypeptide released by a pathogenesis-related protein and application thereof in symbiotic nitrogen fixation of leguminous plants. Background Art

[0002] Pathogenesis-related Protein 1 (PR1) is a core component of plant immune defense response. Most PR1s are induced to be secreted into the apoplast when attacked by pathogens, playing an important role in plant defense. The 11 C-terminal residues of PR1 contain the PxGNxxxxxPY motif, which is called CAPE. The CAPE motif of PR1 is conserved in different flowering plants, and the residue CNYx motif before the cleavage site is also conserved. CAPE polypeptide has biological activity and is induced to participate in the innate immune response of plants and defense against bacteria. Some PR1s of leguminous plants are induced to express in both early rhizobium infection and late nitrogen fixation, and may play a role in nitrogen fixation in root nodule symbiosis.

[0003] As the main way for legumes to obtain nitrogen from nature, the strength of the symbiotic nitrogen fixation process between legumes and rhizobia has the most direct impact on the yield of the former. From the perspective of bacterial-plant interaction, the early infection of rhizobia and the early recognition of legumes are the beginning of the entire symbiotic process, and their importance can be seen. Rhizobia in the soil gather to the roots of host plants after being induced by flavonoids, and promote the invasion of rhizobia into plant root cells by secreting various nodulation factors. However, the nodulation factors secreted by rhizobia are foreign antigens for host plants. Therefore, when legumes recognize these nodulation signals, they will also trigger an immune defense response, which will undoubtedly inhibit the transmission of nodulation signals and thus affect the entire symbiotic nodulation process. Therefore, if the intensity of the early immune defense triggered by the host plant after rhizobium infection can be weakened, it may enable rhizobia to better coexist with legumes, thereby achieving the purpose of increasing the latter's yield.

[0004] At present, the research direction is mainly focused on genes that improve crop resistance to diseases and pests, but the proteins encoded by these genes are essentially not involved in the legume-rhizobium symbiotic nitrogen fixation process, and therefore cannot fundamentally increase soybean yields. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide with symbiotic nitrogen-fixing effect, which has potential application value in the field of leguminous plant cultivation.

[0006] In order to achieve the above-mentioned object, the present invention provides a CAPE polypeptide, the amino acid sequence of which is shown in SEQ ID NO.9. The polypeptide is obtained by shearing and releasing the C segment of the protein encoded by the gene with the nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0007] The CAPE polypeptide provided by the present invention can be applied to the field of leguminous plant cultivation, including weakening the immune response triggered by soybean when infected by rhizobia; increasing the nodule weight and size of leguminous plant root nodules; and improving the symbiotic nodulation and nitrogen fixation capabilities of leguminous plants.

[0008] The present invention has the following advantages:

[0009] The CAPE polypeptide at the C-terminus of the protein encoded by the soybean pathogenesis-related gene GmPR1-6 provided by the present invention can be cleaved and released. Applying the CAPE polypeptide to the roots of legumes can inhibit the early immune defense response triggered by legumes when infected by rhizobia, increase the weight and size of single nodules, and improve the activity of nitrogenase, thereby improving the symbiotic nodulation and nitrogen fixation capabilities, providing a new method for efficient nitrogen fixation of legumes, and providing a favorable tool for the combined utilization and application of soybeans and rhizobia, thereby serving the development of environmentally friendly green sustainable agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the construction of the recombinant protein obtained in Experimental Example 1 of the present invention.

[0011] Figure 2 This is the electrophoresis result of the GmPR1-6 protein after in vivo shearing in Experimental Example 1 of the present invention.

[0012] Figure 3 Schematic diagram of the construction of the recombinant protein obtained in Experimental Example 2 of the present invention.

[0013] Figure 4 This is the in vitro shearing result of GmPR1-6 protein in Experimental Example 2 of the present invention.

[0014] Figure 5 The results of measuring the expression of immune-related genes in soybean lateral roots after applying CAPE.

[0015] Figure 6 This is a graph showing the H2O2 content in soybean lateral roots after applying CAPE.

[0016] Figure 7 This is the phenotype of soybean plants after applying CAPE.

[0017] Figure 8 The statistical results of soybean plant phenotype after applying CAPE.

[0018] Fig. 9 These are the results of measuring the expression levels of genes related to symbiotic nitrogen fixation in soybean nodules after application of CAPE. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0021] Experimental Example 1 Verification of protein cleavage of protein GmPR1-6 in vivo

[0022] A soybean disease course-related gene GmPR1-6 was obtained by cloning the primer pair PR1-6-F / R from the soybean line Williams82, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the GmPR1-6 protein encoded by the gene is shown in SEQ ID NO.2. The sequences of the primers PR1-6-F / R are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0023] The specific sequence is as follows:

[0024] Gene GmPR1-6 (SEQ ID NO.1):

[0025] atggggttgtgcaaggtttcatttcctgtattatgtgtgttggggttggtcatgattgtgagtcacgttgccaatgctcaagactcaccagcagattacgtcaatgcacacaacgctgcaagatcagaggtgggtgttcaaaacttggcttgggatgacacggttgctgcttttgcacagaactatgctaatcaacgtaaaggtgattgtcaactgatccactctggtggtggtggccaatacggggagaatcttgctatgagcactggtgacctaagtggcacagatgcagtgaaattgtgggttgatgagaagtctaactatgactacaattctaattcctgtgttggtggagagtgtctgcattacactcaggttgtttggagagactctgtgcgtcttggatgtgccaaagtagcatgtgataatggaggcactttcatcacttgcaactatgctcctcctggcaactatgttggccaaagaccctactag。

[0026] GmPR1-6 protein (SEQ ID NO.2):

[0027] MGLCKVSFPVLCVLGLVMIVSHVANAQDSPADYVNAHNAARSEVGVQNLAWDDTVAAFAQNYANQRKGDCQLIHSGGGGQYGENLAMSTGDLSGTDAVKLWVDEKSNYDYNSNSCVGGECLHYTQVVWRDSVRLGCAKVACDNGGTFITCNYAPPGNYVGQRPY。

[0028] PR1-6-F (SEQ ID NO.3): atggggttgtgcaaggtttca;

[0029] PR1-6-R (SEQ ID NO.4): ctagtagggtctttggccaacata。

[0030] Reference Figure 1 and Figure 2, by verifying the protein cleavage of soybean pathogenesis-related proteins in vivo, the full-length CDs of the GmPR1-6 gene cloned from the soybean line Williams82 were cloned into the vector p1301-GFP, so that it could be fused and expressed with GFP, and the GmPR1-6-GFP recombinant protein was transiently expressed in tobacco. The obtained tobacco apoplast fluid was subjected to SDS-PAGE gel electrophoresis and Western-Blot experiments. GmPR1-6-eGFP was cleaved in tobacco cells. Therefore, this method can be used to verify that soybean pathogenesis-related proteins are cleaved in tobacco and release CAPE polypeptides. The specific steps are as follows:

[0031] Step 1: Construction of tobacco transient expression vector. First, the genomic DNA of soybean line Williams82 was amplified using the primer pair (p1301-PR1-6-F / R) with nucleotide sequences as shown in SEQ ID NO. 5 and 6 to obtain a GmPR1-6 fragment with partial vector p1301-GFP homologous segments at both ends. Then, the fragment was connected to the vector p1301-GFP after double digestion with KpnI and XbaI using the homologous recombination method to obtain the tobacco transient expression vector p1301-GFP-GmPR1-6. The schematic diagram of the construction structure of the recombinant vector is shown in FIG. Figure 1 As shown, the CNYx motif is a conserved motif in the PR1-6 protein. The recombinant vector was introduced into Agrobacterium GV3101 by electroporation;

[0032] The sequences of the primer pair p1301-PR1-6-F / R are as follows:

[0033] p1301-PR1-6-F (SEQ ID NO.5):

[0034] gagaacacgggggactctagaatggggttgtgcaaggtttca;

[0035] p1301-PR1-6-R (SEQ ID NO.6):

[0036] gtccatcccgggagcggtaccgtagggtctttggccaacatagtt.

[0037] Step 2: Inoculate the successfully transformed Agrobacterium GV3101 into a PA bottle containing an appropriate amount of LB+Rif resistance+Kan resistance liquid culture medium, culture at 28°C in a shaking incubator for 1-2 days, centrifuge at 5000r / min for 5 minutes at room temperature, discard the supernatant, add 30mL Activation Buffer to resuspend, adjust the bacterial solution to OD = 0.3-0.4, and then stand in the dark for 2 hours;

[0038] Step 3: Use a 1mL syringe with the needle removed to absorb the bacterial solution, select a fresh and tender tobacco leaf at the top, and gently inject the bacterial solution into the leaf from the back so that the bacterial solution fills the entire leaf. Put the tobacco back to its original place and continue light culture for 2-3 days.

[0039] Step 4: Pick the injected tobacco leaves and put them into a 50 ml syringe, and fill the syringe with PBS. Create negative pressure in the syringe and hold it for a few seconds before releasing the plunger until the leaves are completely infiltrated with water; roll up the leaves with a sealing film and put them into a 10 ml syringe. Insert the syringe into a clean 50 ml centrifuge tube and centrifuge at 4 °C and 1000 g for 10 min; collect the apoplast wash fluid (AWF) and transfer it to a clean, sterilized 1.5 ml centrifuge tube.

[0040] Step 5: The extracted AWF was subjected to SDS-PAGE gel electrophoresis and Western-Blot experiments. While the Agrobacterium transformed with the GmPR1-6-GFP vector was used as the experimental group, an Agrobacterium transformed with an empty vector alone was also set up for a control experiment.

[0041] The electrophoresis results of GmPR1-6 protein after in vivo shearing were obtained as follows Figure 2 As shown, lane 1 is the AWF of the experimental group, lane 2 is the AWF of the control group, and lane 3 is the AWF sample obtained from the untreated leaves. The upper gel image is the WB result, and the lower gel image is the Coomassie Brilliant Blue (CBB) staining result. Figure 1 , 2 As shown, three bands were observed with molecular weights similar to those of the full-length GmPR1-6-eGFP protein (46.1 kDa), CAPE fused to eGFP (29.5 kDa), and empty eGFP (27 kDa), respectively, indicating that GmPR1-6-eGFP was cleaved in tobacco cells.

[0042] Experimental Example 2: Verification of protein cleavage of GmPR1-6 in vitro

[0043] refer to Figure 3 and Figure 4 , the protein shearing of soybean pathogenesis-related proteins in vitro was verified, and the specific steps are as follows:

[0044] Step 1: Construction of His-tagged protein expression vector. First, the genomic DNA of soybean line Williams82 was amplified using primer pair pET28a-PR1-6-F / R to obtain a GmPR1-6 fragment with partial vector pET28a homologous segments at both ends. Then, the fragment was connected to the pET28a vector using homologous recombination to obtain the His-tagged protein expression vector pET28a-GmPR1-6. The schematic diagram of the construction structure of the recombinant expression vector is shown in FIG. Figure 3 As shown; the His-tagged protein expression vector was transformed into Escherichia coli Rosetta, and the primer pair pET28a-F / R was used to screen the positive transformants by PCR;

[0045] The primer sequences are shown below;

[0046] pET28a-PR1-6-F (SEQ ID NO.7):

[0047] tgccgcgcggcagccatatgcaagactcaccagcagattacgtc;

[0048] pET28a-PR1-6-R (SEQ ID NO.8):

[0049] tggtggtggtggtgctcgaggtagggtctttggccaaca.

[0050] Step 2: Induce the target protein. Inoculate the successfully transformed Rosetta strain into a PA bottle containing 3 mL LB+Chl resistance+Amp resistance liquid medium and culture it in a shaking incubator at 37°C for 12-16 h. Transfer the bacterial liquid to 250 mL LB liquid medium at a rate of 1%-2%, and culture it in a shaking incubator at 37°C for 2-3 h until the OD value is between 0.4 and 0.5. Add 250 μL 0.5 mol / L IPTG to the LB liquid medium and induce it in a shaking incubator at 16-18°C and 160 r / min for 18-20 h.

[0051] Step 3: Purify the target protein. Centrifuge the bacterial solution at 4°C and 7000r / min for 10min; discard the supernatant, add 30mL Lysis Buffer, 30μL DTT solution and 30μL 1mol / LPMSF solution, oscillate with a vortexer and mix with a pipette, set the pressure to 1000bar, add the bacterial solution to crush 2-3 times until the bacterial solution becomes obviously clear and transparent, centrifuge at 4°C and 130r / min for 45min; purify the protein solution with an affinity chromatography gravity column.

[0052] Step 4: See Figure 4 40 μL of the purified His-tag fused GmPR1-6 recombinant protein was co-incubated with 40 μL of the soybean line Williams82 apoplast fluid at 28°C for 0 h (see lane 1), 4 h (see lane 2), 12 h (see lane 3), and 24 h (see lane 4), respectively. The soybean line Williams82 apoplast fluid was used as the empty control (see lane 5). SDS-PAGE gel electrophoresis and Western-Blot experiments were performed.

[0053] The electrophoresis results of GmPR1-6 protein after in vitro shearing were as follows: Figure 4 As shown, the upper gel image is the Coomassie Brilliant Blue (CBB) staining result, and the lower gel image is the WB result. It can be seen that His-GmPR1-6 can be cleaved and release CAPE small peptide in soybean AWF, and the molecular weight of His-GmPR1-6 changes from 16kDa to 14kDa. Figure 4 As shown, after His-GmPR1-6 was incubated with AWF, the protein band shifted downward and the molecular weight became smaller, indicating that it was cleaved in AWF.

[0054] Experimental Example 3: Application of CAPE peptide

[0055] This experimental example is to explore the application effect of CAPE peptide, as follows:

[0056] Step 1: Artificially synthesize the C-terminal CAPE polypeptide released by GmPR1-6 according to the protein sequence, and dilute it into four concentration gradients, namely 1nM, 10nM, 100nM, and 1μM. The synthesis of the CAPE polypeptide used in this experimental example was completed by Nanjing GenScript Biotechnology Co., Ltd., and CAPE was dissolved in ultrapure water;

[0057] The C-terminal CAPE polypeptide sequence released by GmPR1-6 is PPGNYVGQRPY, recorded as SEQ ID NO.9.

[0058] Step 2: Soybean seed germination treatment: first sterilize the soybean seeds with chlorine gas, then sow them in vermiculite completely soaked with CAPE polypeptide dilution, add 100 mL of polypeptide dilution to each pot for the first sowing, keep the vermiculite completely moist but not overflowing with excess water, add CAPE polypeptide dilution once every 2 to 3 days, 30 mL per pot, and inoculate rhizobium USDA6 one week after sowing;

[0059] Step 3: Identification and phenotypic analysis. Two days after inoculation, the expression levels of GmPR1-6 genes, some immune defense genes, and symbiotic nitrogen fixation-related genes in lateral roots were analyzed using the qRT-PCR method using primer pairs qPCR-PR1-6-F / R, qPCR-NIN1a-F / R, qPCR-MPK6-F / R, qPCR-MKK4-F / R, and qPCR-RBOHD-F / R. The sequence information of the primers involved in the identification of the relevant genes is as follows:

[0060] qPCR-PR1-6-F (SEQ ID NO. 10): gtgagtcacgttgccaatgc;

[0061] qPCR-PR1-6-R (SEQ ID NO. 11):ccacttaggtcaccagtgct;

[0062] qPCR-NIN1a-F (SEQ ID NO. 12): tgtgtctcggtgttgtcgag;

[0063] qPCR-NIN1a-R (SEQ ID NO. 13): acggctgcaggtggaataaa;

[0064] qPCR-MPK6-F (SEQ ID NO. 14): tcattgcatcaccgtcaccacc;

[0065] qPCR-MPK6-R (SEQ ID NO. 15): tcctcctccttccattgctctg;

[0066] qPCR-MKK4-F (SEQ ID NO. 16): gatctccgccgtggatatgatg;

[0067] qPCR-MKK4-R (SEQ ID NO. 17): atcggcctcatctcatgtctcg;

[0068] qPCR-RBOHD-F (SEQ ID NO. 18): acatgaaggccatggaagag;

[0069] qPCR-RBOHD-R (SEQ ID NO. 19): ccgaagctaactgaccaagc.

[0070] Step 4: Identification and phenotypic analysis: Analyze the H2O2 content in the lateral roots 2 days after inoculation.

[0071] Step 5: Identification and phenotypic analysis: After 28 days of inoculation, the symbiotic phenotype of the plants was investigated, and the expression levels of the GmPR1-6 gene and some symbiotic nitrogen fixation-related genes in the nodules were analyzed using the qRT-PCR method with primer pairs qPCR-PR1-6-F / R, qPCR-ENOD40A-F / R, qPCR-LB1-F / R and qPCR-RNPB-F / R. The identification primer sequences of the related genes are as follows:

[0072] qPCR-ENOD40A-F (SEQ ID NO. 20): gtcctcaccactcacactcctccactt;

[0073] qPCR-ENOD40A-R (SEQ ID NO. 21): gccttttcgtgacttgccggtttg;

[0074] qPCR-LB1-F (SEQ ID NO. 22): ctttttgcattggtgcgtgac;

[0075] qPCR-LB1-R (SEQ ID NO. 23): gctgccaattcatcgtaggc;

[0076] qPCR-RNPB-F (SEQ ID NO. 24): ccctattcggttttgctccc;

[0077] qPCR-RNPB-R (SEQ ID NO. 25): gctcccttgacatacggtg.

[0078] The expression changes of related genes in soybean lateral roots after 2 days of hydroponic treatment with 1 μmol / L CAPE (CAPE) and rhizobium USDA6 (U6) were obtained by RT-qPCR. Figure 5 Among them, the CK group was the control group treated with water only; the CAPE group was treated with 1 μmol / L CAPE peptide added to the water; the U6 group was treated with rhizobium USDA6 inoculated in the water, and the bacterial solution OD 600 The U6+CAPE group was inoculated with OD 600 =0.1 rhizobium USDA6 and 1 μmol / L CAPE peptide treatment.

[0079] Depend on Figure 5 It can be seen that compared with the rhizobia inoculated alone, the expression level of GmPR1-6 in the lateral roots of the plants inoculated with rhizobia and applied CAPE was significantly increased, and the expression level of marker genes related to immunity and prevention was significantly decreased.

[0080] The hydrogen peroxide (H2O2) content in soybean lateral roots after 2 days of hydroponic treatment with 1 μmol / L CAPE (CAPE) and rhizobium USDA6 (U6) was obtained. Hydrogen peroxide, as one of the most abundant reactive oxygen species (ROS) in cells, is a key signal molecule for plants to resist adverse stress. Figure 6 As shown, the CK group is the control group treated with water only; the 1nmol / L CAPE group is the water with 1nmol / L CAPE peptide added, representing the low-concentration peptide treatment; the 1μmol / L CAPE group is the water with 1μmol / L CAPE peptide added, representing the high-concentration peptide treatment; the U6 group is the water inoculated with rhizobium USDA6, and the bacterial solution OD 600The U6+1nmol / LCAPE group was inoculated with OD 600 =0.1 rhizobium USDA6 and 1 nmol / L CAPE peptide treatment; U6+1μmol / L CAPE group was inoculated with OD 600 =0.1 rhizobium USDA6 and 1 μmol / L CAPE peptide treatment.

[0081] As can be seen from the figure, compared with the plants inoculated with rhizobia alone, the accumulation of H2O2 in the lateral roots of the plants inoculated with rhizobia and applied with CAPE was reduced, and the CAPE polypeptide weakened the soybean immune response.

[0082] The differences between the symbiotic phenotypes of potted plants (USDA6+CAPE) treated with different concentrations of CAPE peptide and the phenotypes of the control plants (USDA6) are as follows Figure 7 At the same time, the phenotypes, related indicators and some symbiotic nitrogen fixation related genes of soybean plants in different groups were statistically analyzed and measured. The results are shown in Figure 8 and 9 As shown in the figure, it can be seen that the expression level of symbiotic nitrogen fixation related genes in the plants treated with CAPE was significantly increased, the weight and size of single nodules were significantly increased, and the nitrogenase activity was significantly improved.

[0083] In summary, the CAPE polypeptide provided by the present invention can weaken the early immune defense response triggered by soybean when infected by rhizobia, and the polypeptide can increase the weight and size of a single nodule, improve the symbiotic nodulation and nitrogen fixation ability of soybean, and has potential application value in the field of leguminous plant cultivation.

[0084] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A CAPE polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO.

9.

2. The CAPE polypeptide according to claim 1, characterized in that The polypeptide is obtained by shearing and releasing the C segment of the protein encoded by the gene with the nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. Use of the CAPE polypeptide as claimed in claim 1 in the field of leguminous plant cultivation.

4. The use according to claim 3, characterized in that: The application includes any of the following: Weaken the immune response triggered by soybeans when infected by rhizobia; Increase the weight and size of nodules in leguminous plants; Improve the symbiotic nodulation and nitrogen fixation ability of legumes.

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