CN115043917A

CN115043917A - Application of soybean GmNAC039 or GmNAC018 in regulation and control of nitrogen fixation and/or yield of plant nodules

Info

Publication number: CN115043917A
Application number: CN202210230086.1A
Authority: CN
Inventors: 曹扬荣; 肖爱芳; 朱辉; 余海翔
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2022-09-13
Anticipated expiration: 2042-03-10
Also published as: CN115043917B

Abstract

The invention belongs to the technical field of gene editing, and particularly relates to application of a soybean GmNAC039 or GmNAC018 regulation transcription unit in regulation and control of nitrogen fixation of plant nodules and/or regulation and control of yield; the amino acid sequence of the protein coded by the GmNAC039 is shown in SEQ ID NO. 1; the amino acid sequence of the protein coded by the GmNAC018 is shown as SEQ ID NO. 2. The GmNAC039(Glyma.06G157400) and the GmNAC018(Glyma.04G208300) are homologous genes, and the GmNAC039 and the GmNAC018 can regulate and control the senescence of plant nodules.

Description

Application of soybean GmNAC039 or GmNAC018 in regulation and control of nitrogen fixation and/or yield of plant nodules

Technical Field

The invention belongs to the technical field of gene editing, and particularly relates to application of soybean GmNAC039 or GmNAC018 in regulation and control of nitrogen fixation of plant nodules and/or regulation and control of yield.

Background

Legumes interact with rhizobia to form nodules for symbiotic nitrogen fixation, the host plant provides a carbon source for the rhizobia, and the rhizobia provides the nitrogen needed for plant growth. Legumes require strict control of the number, development and senescence of nodules to regulate symbiotic nitrogen fixation efficiency, ultimately maximizing host plant benefits. Soybeans are the main source of human vegetable protein and vegetable oil, and play an important role in the food structure of China, but the soybean yield of China is not enough to meet the requirements of people. Therefore, there is a need to improve the yield of soybeans to meet the increasing demand of people. The symbiosis of the soybean root nodules provides most of nitrogen for the whole growth period of plants, so that the contradiction between the increase of the nitrogen demand and the reduction of the nitrogen fixation efficiency in the later period of the soybean is solved, the senescence of the root nodules can be properly delayed, the nitrogen fixation time of the root nodules is prolonged to improve the total nitrogen fixation amount in the growth period of the soybean and guarantee the sufficient supply of the nitrogen in the reproductive growth period, the environmental problem caused by nitrogen fertilizers is effectively relieved, and the sustainable development of agricultural ecology is realized.

Root nodule senescence in legumes is a highly controlled process, and both developmental and environmental factors can contribute to root nodule senescence, such as drought and high nitrate levels. During the senescence process of nodules, the bacteroids lyse, forming a large number of vesicles in the cytoplasm of the invading cells, which subsequently invade cells to senesce. In the legume-mode plant alfalfa, MtNAC920 directly activates the transcription of the senescence-associated gene, cysteine protease MtCP2, promoting root nodule senescence (de zelicoort et al, 2012). Gene silencing of the alfalfa NAC potential target genes MtCP6 and MtVPE has been shown to delay root nodule senescence, prolong root nodule growth and nitrogen fixation time (Perez Guerra et al, 2010; Pierre et al, 2016). Similarly, there is a large upregulated expression of NAC transcription factor in the early senescence mutants of leguminous plants (Wang et al, 2016; Wang et al, 2019). There are no reports of GmNAC039 or GmNAC018 in regulating the senescence of plant nodules.

Disclosure of Invention

The invention aims to provide application of a soybean GmNAC039 or GmNAC018 transcription regulation unit in regulation and control of nitrogen fixation and/or yield of plant nodules. The GmNAC039 and GmNAC018 disclosed by the invention can regulate and control the senescence of plant nodules.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides application of soybean GmNAC039 or GmNAC018 in regulating and controlling nitrogen fixation of plant nodules, wherein the regulation and control of nitrogen fixation of the plant nodules comprise one or more of the following aspects 1) to 5):

1) regulating and controlling the plant root nodule aging;

2) regulating and controlling the growth of plant root nodules;

3) regulating and controlling the number of plant nodules;

4) regulating and controlling the activity of azotobacter in the plant root nodule;

5) regulating and controlling the nitrogen fixation efficiency of the plant root nodule;

the amino acid sequence of the protein coded by the GmNAC039 is shown in SEQ ID NO. 1; the amino acid sequence of the protein coded by the GmNAC018 is shown as SEQ ID NO. 2.

Preferably, the modulating senescence in plant nodules comprises delaying senescence in plant nodules by negatively modulating expression of GmNAC039 or GmNAC 018.

Preferably, said modulating plant nodule development comprises promoting plant nodule development by positively modulating expression of GmNAC039 or GmNAC 018.

Preferably, said modulating the number of plant nodules comprises increasing the number of plant nodules by positively modulating expression of GmNAC039 or GmNAC 018.

Preferably, said modulating yield comprises increasing yield by negatively modulating expression of GmNAC039 or GmNAC 018.

Preferably, said modulating azotase activity in plant nodules comprises enhancing azotase activity in plant nodules by negatively modulating expression of GmNAC039 or GmNAC 018.

Preferably, the modulating nitrogen fixation efficiency of plant nodules comprises increasing nitrogen fixation efficiency of plant nodules by negatively modulating expression of GmNAC039 or GmNAC 018.

The invention also provides application of soybean GmNAC039 or GmNAC018 in high-yield plant molecular breeding, wherein the amino acid sequence of the protein encoded by GmNAC039 is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by the GmNAC018 is shown as SEQ ID NO. 2.

Preferably, the plant comprises a leguminous plant.

Preferably, the leguminous plant comprises one or more of soybean, alfalfa and lotus japonicus.

Has the advantages that:

the invention provides application of soybean GmNAC039 or GmNAC018 in regulating and controlling nitrogen fixation of plant nodules, wherein the regulation and control of nitrogen fixation of the plant nodules comprise one or more of the following aspects 1) to 5): 1) Regulating and controlling the plant root nodule aging; 2) regulating and controlling the growth of plant root nodules; 3) regulating and controlling the number of plant nodules; 4) regulating and controlling the activity of azotobacter in the plant root nodule; 5) regulating and controlling the nitrogen fixation efficiency of the plant root nodule; the amino acid sequence of the protein coded by the GmNAC039 is shown in SEQ ID NO. 1; the amino acid sequence of the protein coded by the GmNAC018 is shown as SEQ ID NO. 2. The GmNAC039(Glyma.06G157400) and the GmNAC018(Glyma.04G208300) are homologous genes, are up-regulated to be expressed in aged nodules, and the functions of the genes in the process of the aging of the nodules are predicted. The application of the gene of the invention is not reported at home and abroad.

Moreover, the plant root nodule azotase activity of the over-expressed gene is obviously reduced and the root nodule is premature senility through over-expression in the soybean root nodule, compared with a contrast, so that the gene regulates the aging process of the root nodule; by using a CRISPR-Cas9 technology and knocking out GmNAC039 and GmNAC018 genes through a soybean root transformation method, the activity of the nodule azotobacter is obviously higher than that of a control group, the nodule senescence is delayed, the effective nitrogen-fixing time of the nodule is prolonged, and the nodule azotobacter has an application prospect in leguminous crops.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1 is a diagram of an alignment of GmNAC039 and GmNAC018 protein sequences;

FIG. 2 is an expression graph of GmNAC039 and GmNAC018 in fluorescent quantitative PCR detection, wherein A is an expression level of LbA, B is an expression level of LbC1, C is an expression level of GmNAC039, and D is an expression level of GmNAC 018;

FIG. 3 is a graph of the growth of a GmNAC039 overexpressing plant, wherein A is the plant phenotype, B is the control root nodule, and C is the GmNAC039 overexpressing nodule;

FIG. 4 is a graph of GmNAC018 overexpressing plant growth, wherein A is the plant phenotype, B is the control nodule, and C is the GmNAC018 overexpressing nodule;

FIG. 5 is a paraffin section view of nodules from GmNAC039 and GmNAC018 overexpressing plants, wherein A is a GmNAC039 overexpressing nodule and B is a GmNAC018 overexpressing nodule;

fig. 6 is gene editing for GmNAC039 and GmNAC018 CRISPR knockout plants;

wherein, in FIG. 3, FIG. 4 and FIG. 5, Control is transferred into empty vector plants, and NAC039-OE and NAC018-OE are over-expression plants; in FIG. 6, Control is a plant with an empty vector transferred, and CRISPR-NAC is a knockout plant;

indicates a direct comparison of differences between Control and over-expressed plants (t-test), wherein differences are significant when the significance level is 0.01 < p.ltoreq.0.05; differential significance was between significant and very significant when significance level 0.001 < p.ltoreq.0.01; when p is 0.001, the difference is extremely significant.

Detailed Description

The invention provides application of soybean GmNAC039 or GmNAC018 in regulating and controlling nitrogen fixation of plant nodules, wherein the regulation and control of nitrogen fixation of the plant nodules comprise one or more of the following aspects 1) to 5): 1) Regulating and controlling the plant root nodule aging; 2) regulating and controlling the growth of plant root nodules; 3) regulating and controlling the number of plant nodules; 4) regulating the activity of azotobacter in the plant root nodule; 5) regulating and controlling the nitrogen fixation efficiency of the plant root nodule;

the amino acid sequence of the protein coded by the GmNAC039 is shown in SEQ ID NO.1, and specifically comprises the following steps:

MKGELELPPGFRFHPTDEELVNHYLCRKCAGQPIAVPIIKEVDLYKFDPW QLPEIGYYGEKEWYFFSPRDRKYPNGSRPNRAAGSGYWKATGADKAIGKPK ALGIKKALVFYAGKAPKGVKTNWIMHEYRLANVDRSASKKNNNNLRLDDW VLCRIYNKKGKIEKYNTTAPKMNLEMIHSFEHENETKPEIHKLGNEQLLYTET SDSVPRLHTDSSSSEHVVSPDVRCEREVQSDPKWNNDDYDLGLQLENAFDFQ FNYLDDNNLSVDDDPFGTVQYQMGQLSPLQDMFMYLQKM*；

the amino acid sequence of the protein coded by GmNAC018 is shown in SEQ ID NO.2, and specifically comprises the following steps:

MKGELELPPGFRFHPTDEELVNHYLCRKCAGQPIAVPVIKEVDLYKFDP WQLPEIGFYGEKEWYFFSPRDRKYPNGSRPNRAAGSGYWKATGADKPIGKP KALGIKKALVFYAGKAPKGVKTNWIMHEYRLANVDRSASKKKNNNLRLDD WVLCRIYNKKGKIEKYNTGAAKMNVEMVHSFEHENETKPEIHKLGNEQLYM ETSDSVPRLNTDSSSSEHVVSPDVTCEREVQSDPKWNDDLDLKLENAFDFQF NYLDDNNLSVDDYLFGTVQYQMGQLSPLQDMFMYLQKM*。

in the present invention, the nucleotide sequence of GmNAC039 is preferably represented by SEQ ID No.29, and specifically:

ATGAAGGGAGAATTAGAGTTGCCACCAGGGTTCAGATTTCACCCCACT GATGAAGAATTGGTGAATCACTACTTGTGTAGGAAGTGCGCGGGTCAACC AATCGCGGTTCCCATCATCAAAGAGGTCGATTTGTACAAGTTTGATCCATG GCAGCTTCCAGAAATTGGCTACTACGGCGAGAAAGAATGGTACTTCTTTTC TCCTCGGGATCGGAAATACCCGAACGGTTCACGGCCGAACCGTGCCGCCG GAAGCGGCTATTGGAAAGCCACCGGCGCCGATAAGGCGATCGGAAAACCG AAAGCGCTAGGGATCAAGAAAGCTCTGGTTTTTTACGCCGGAAAAGCCCC CAAAGGAGTGAAAACCAATTGGATCATGCACGAATATCGCCTCGCCAATGT TGACCGATCTGCCTCCAAGAAAAACAACAACAACTTGAGGCTTGATGATT GGGTGTTGTGTCGAATCTACAACAAGAAAGGGAAGATTGAGAAATACAAC ACGACCGCACCGAAGATGAATCTTGAAATGATTCATAGTTTTGAGCACGAG AACGAGACGAAGCCTGAGATTCATAAGCTTGGAAATGAGCAATTGTTGTA CACGGAGACTTCAGATTCGGTGCCAAGGTTGCACACGGACTCGAGCAGCT CGGAGCACGTGGTTTCGCCCGATGTGAGGTGCGAGAGGGAAGTGCAGAG CGACCCCAAGTGGAATAACGATGATTATGATCTGGGCCTACAGCTAGAAAA CGCGTTTGATTTTCAGTTTAATTACTTGGACGATAATAACCTTTCCGTCGAC GATGACCCCTTTGGCACTGTTCAGTACCAAATGGGTCAGCTCTCGCCCTTG CAGGACATGTTCATGTACCTACAGAAGATGTGA。

in the invention, the nucleotide sequence of the GmNAC018 is preferably shown as SEQ ID NO.30, and specifically comprises:

ATGAAGGGAGAATTAGAGTTGCCACCTGGGTTCAGATTTCACCCCACT GATGAAGAATTGGTGAATCACTACTTGTGTAGGAAGTGCGCTGGTCAACC AATCGCGGTTCCCGTCATCAAAGAGGTCGATTTGTACAAGTTTGATCCATG GCAGCTTCCAGAAATTGGTTTTTACGGCGAGAAAGAATGGTACTTCTTTTC TCCTCGGGACCGGAAATACCCGAACGGTTCACGGCCGAACCGCGCCGCCG GAAGCGGCTATTGGAAAGCCACCGGCGCTGATAAACCGATCGGAAAACCG AAAGCGCTTGGGATCAAGAAAGCTCTGGTTTTTTACGCCGGAAAAGCCCC CAAAGGTGTGAAAACCAATTGGATTATGCACGAATATCGCCTCGCCAATGT TGACCGATCTGCCTCCAAGAAAAAAAACAACAACTTGAGGCTTGATGATT GGGTGTTGTGTCGAATCTACAACAAGAAAGGGAAGATTGAGAAATACAAC ACAGGCGCAGCGAAGATGAATGTTGAGATGGTTCATAGTTTTGAGCACGA GAACGAGACGAAGCCAGAGATTCATAAGCTAGGAAATGAGCAATTGTACA TGGAGACTTCGGATTCGGTGCCAAGGTTGAACACGGACTCGAGCAGTTCG GAGCACGTGGTTTCGCCCGATGTCACGTGCGAGAGGGAGGTGCAGAGCG ACCCCAAGTGGAACGATGATCTGGACCTAAAGCTAGAAAACGCGTTTGAT TTTCAGTTTAATTACTTGGACGATAATAACCTTTCCGTGGATGATTACCTTTT TGGCACTGTTCAGTATCAAATGGGCCAGCTCTCGCCCTTGCAGGACATGTT CATGTACCTACAGAAGATGTGA。

in the present invention, the regulating senescence of plant nodules preferably comprises delaying senescence of plant nodules by negatively regulating expression of GmNAC039 or GmNAC 018.

In the present invention, the modulation of plant root nodule development preferably comprises promoting plant root nodule development by positively modulating expression of GmNAC039 or GmNAC 018.

In the present invention, the regulation of the number of plant nodules preferably includes increasing the number of plant nodules by positively regulating the expression of GmNAC039 or GmNAC 018.

In the present invention, the modulating yield preferably comprises increasing yield by negatively modulating expression of GmNAC039 or GmNAC 018.

In the present invention, said modulating azotase activity in plant nodules preferably comprises enhancing azotase activity in plant nodules by negatively modulating expression of GmNAC039 or GmNAC 018.

In the present invention, the regulating nitrogen fixation efficiency of plant nodules preferably comprises increasing nitrogen fixation efficiency of plant nodules by negatively regulating expression of GmNAC039 or GmNAC 018.

In the present invention, the negative regulation means preferably includes CRISPR knockouts GmNAC039 and GmNAC 018.

In the present invention, the positive regulation mode preferably comprises overexpression of GmNAC039 or GmNAC 018.

The invention discovers NAC transcription factors (GmNAC039 and GmNAC018) derived from soybean and capable of regulating the senescence of nodules, wherein the senescence of the nodules can be promoted after the GmNAC039 or GmNAC01 is overexpressed, and the senescence of the nodules can be delayed after the GmNAC039 or GmNAC018 is knocked out by using a CRISPR-Cas9 technology.

According to the invention, a soybean hairy root transformation method is utilized to overexpress GmNAC039 or GmNAC018, so that the number of nodules is obviously increased; according to the invention, after GmNAC039 or GmNAC018 is knocked out by using a CRISPR-Cas9 technology, the root nodule aging can be delayed, more nitrogen is fixed by the root nodule and is provided for plants, and the effect of improving the yield is achieved.

The CRISPR knockout GmNAC039 or GmNAC018 disclosed by the invention can improve nitrogen fixation efficiency, improve the nitrogen content of a plant, and increase the biomass and yield of the plant, and has important significance for developing environment-friendly sustainable agriculture.

According to the invention, NAC transcription factors (GmNAC039 and GmNAC018) derived from soybean and capable of regulating the root nodule senescence are found, the activity of the root nodule azotase can be reduced after the GmNAC039 or GmNAC01 is overexpressed, and the effect of improving the activity of the root nodule azotase can be achieved after the GmNAC039 or GmNAC018 is knocked out by using a CRISPR-Cas9 technology.

According to the invention, after GmNAC039 or GmNAC018 is knocked out by using a CRISPR-Cas9 technology, the effective nitrogen fixation time of rhizobia can be prolonged, and the nitrogen fixation efficiency of plant rhizobia can be further ensured to be improved.

The invention also provides application of soybean GmNAC039 or GmNAC018 in high-yield plant molecule breeding, wherein the amino acid sequence of the protein encoded by GmNAC039 is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by the GmNAC018 is shown as SEQ ID NO. 2.

In the present invention, the plant includes leguminous plants, and more preferably includes one or more of soybean, alfalfa and lotus japonicus.

According to the invention, the GmNAC039 and GmNAC018 are knocked out by a soybean root transformation method by using a CRISPR-Cas9 technology, so that the activity of the root nodule enzyme of leguminous plants is improved, the senescence time of the root nodule and the effective nitrogen fixation time of the root nodule are prolonged, the nitrogen content of the plants is improved, and the biomass and the yield of the plants are increased.

For further illustration of the present invention, the following detailed description of the application of soybean GmNAC039 or GmNAC018 provided by the present invention in regulating nitrogen fixation and/or yield of plant nodules is provided in connection with the drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1

GmNAC039(Glyma.06G157400) and GmNAC018(Glyma.04G208300) protein sequences were obtained in soybean data Phytozome (https:// phytozome.jgi. doe.gov/pz/port. html # | infoalias. Org. Gmax) for protein sequences of GmNAC039(Glyma.06G157400) and GmNAC018(Glyma.04G208300), and the sequence comparison was performed online with Clustal Omega (https:// www.ebi.ac.uk/Tools/msa/clustalo). The aligned sequences were stained with ESPrip 3.0 (https:// ESPript. ibcp.fr/ESPrip/cgi-bin/ESPrip. cgi) on-line, and the results are shown in FIG. 1; it can be seen that GmNAC039 and GmNAC018 are homologous proteins.

Example 2

Fluorescent quantitative PCR detection of expression of GmNAC039 and GmNAC018 in nodules is carried out by inoculating (Bradyrhizobium diazoefficiens) USDA110(USDA-ARS culture collection, NRRLB-4361) on wild-type plant Williams 82(Wm82), wherein each inoculation amount is 2ml, OD600 is 0.1, respectively collecting rhizobia (Bradyrhizobium diazoefficiens) USDA110(USDA-ARS culture collection, NRRLB-4361) nodules 4 weeks (W), 8W and 12W after inoculation of the rhizobia, respectively extracting RNA of 4W, 8W and 12W nodules, and carrying out fluorescent quantitative PCR detection of expression levels of GmNAC039 and GmNAC 018.

The RNA extraction method comprises the following steps: total RNA from soybean nodules was extracted using RNA extraction kit (Aidlab, RN 3302). The specific method refers to a kit operation manual. After RNA elution, the concentration and purity of the RNA were determined using NanoDrop 2000 (Thermo), and the samples were stored at-70 ℃.

The reverse transcription method comprises the following steps: reverse transcription was performed using a reverse transcription kit (ABCloanl, RK 20403). The specific method refers to a kit operation manual.

The fluorescent quantitative PCR primer is as follows:

NAC039:GmNAC039-qRT-F/GmNAC039-qRT-R；

NAC018:GmNAC018-qRT-F/GmNAC018-qRT-R；

ACT11-qRT-F:ATCTTGACTGAGCGTGGTTATTCC，SEQ ID NO.3；

ACT11-qRT-R:GCTGGTCCTGGCTGTCTCC，SEQ ID NO.4；

GmNAC039-qRT-F:GAGCGACCCCAAGTGGAATA，SEQ ID NO.5；

GmNAC039-qRT-R:CTGAACAGTGCCAAAGGGGT，SEQ ID NO.6；

GmNAC018-qRT-F:AGCACGAGAACGAGACGAAG，SEQ ID NO.7；

GmNAC018-qRT-R:AGGTCCAGATCATCGTTCCAC，SEQ ID NO.8；

GmLbA-qRT-F：GAAGGCATAATTAGTATCTATTG，SEQ ID NO.9；

GmLbA-qRT-R：TATCAGGAACTTGTCTAATAG，SEQ ID NO.10；

GmLbC1-qRT-F:ACAATAAAGGAAGCTGTTGGCGG，SEQ ID NO.11；

GmLbC1-qRT-R:TTACACTTTACGGCAATGCAG，SEQ ID NO.12。

the fluorescent quantitative PCR system is (10 μ L): SYBR Select master mix (ABI, 2X) 5. mu.L, PCR Forward Primer (10. mu.M) 0.4. mu.L, PCR Reverse Primer (10. mu.M) 0.4. mu. L, cDNA template 0.4. mu.L and ddH ₂ O 3.8μL；

The fluorescent quantitative PCR program is: 2min at 50 ℃; 10min at 95 ℃; 95 ℃ for 15sec, 60 ℃ for 1min, 40 cycles.

Taking soybean housekeeping gene ACT11 as an internal reference gene as an internal reference to standardize the gene expression level, and adopting relative quantification method ^△△Ct The relative expression level is the ratio of the expression level of the target gene to the expression level of the reference gene, and the results are shown in FIG. 2. It is known that, in the process of root nodule senescence, the expression levels of the leghemoglobin genes LbA and LbC1 are gradually reduced, and the expression of GmNAC039 and GmNAC018 is significantly induced.

Example 3

Effect of GmNAC039 and GmNAC018 overexpression on plants.

1. Vector construction

Designing primers according to GmNAC039 and GmNAC018 full-length cDNAs, and respectively amplifying NAC039 and NAC018 fragments by using soybean nodule cDNA as a template and using the primers GmNAC039-F/R and GmNAC 018-F/R; and performing second round amplification by using NAC039 and NAC018 fragments as templates and using primers GmNAC039/018-XbaI (Gibson) -F/GmNAC039/018-StuI (Gibson) -R to obtain final GmNAC039 and GmNAC018 fragments.

The corresponding gene is amplified. The primers are as follows:

GmNAC039-F:ACAAAGAGAGGAAGAAACGCGA，SEQ ID NO.13；

GmNAC039-R:TCCTATCATCCATCATTCGGCT，SEQ ID NO.14；

GmNAC018-F:AAACCACCCAGTGTGTCCTC，SEQ ID NO.15；

GmNAC018-R:CTTGGCCTAGCCCACATCAC，SEQ ID NO.16；

GmNAC039/018-XbaI(Gibson)-F：TGATGTGATTACAGTCTAGAATGAAG GGAGAATTAGAGTTGC，SEQ ID NO.17；

GmNAC039/018-StuI(Gibson)-R：GGATCCACTAGTAGGCATCTTCTGT AGGTACATGAAC，SEQ ID NO.18；

use of PCR amplification

Max Super-Fidelity DNApolymerase (Vazyme), PCR amplification system (20. mu.L): 2X PhantaMax Buffer 10. mu. L, dNTP (10mM research) 0.5. mu. L, F-primer (10. mu.M) 0.5. mu. L, R-primer (10. mu.M) 0.5. mu.L, Phanta Max Super-Fidelity DNA Polymerase 0.5. mu.L, template DNA 1. mu.L and ddH ₂ O7 mu L; the PCR amplification procedure was:

the vector pUB-GFPC-3xFLAG is derived from (Yu, H.H., et al, Suppression of immunological mediated by the CDPK-Rboh complex is required for rhizobiul degradation in medical procedure. New Phytol,2018.220(2): p.425-434.), is digested with restriction enzymes Pst I and Xba I (Thermo, FD0684), and the original Ubiquitin promoter is replaced with the leghemoglobin promoter of alfalfa, thus finally obtaining the overexpression vector pLb 2-GFP-C' 3 xFLAG. The vector is subjected to double enzyme digestion by Xba I/Stu I and is respectively connected with GmNAC039 and GmNAC018 through a Ginson reaction; the two recombinant vectors obtained were transformed into Agrobacterium rhizogenes K599(NCPPB No.2659) to obtain Agrobacterium rhizogenes K599 containing GmNAC039 and Agrobacterium rhizogenes K599 containing GmNAC018, respectively.

The restriction enzymes for vector digestion are Xba I (Thermo, FD0684) and StuI (Thermo, ER0421), and the digestion system is 50. mu.L: 2 mu g of carrier,XbaI 1.5. mu. L, Stu I1.5. mu.L, 10 Xbuffer 5. mu.L and the balance ddH ₂ O；

The vectors were ligated to GmNAC039 and GmNAC018, respectively, using the Gibson reaction method (Yeasen, 10911ES20) with a reaction system of 5. mu.L: 2x Hieff

2.5 mu L of Enzyme Premix, 80-100 ng of gene fragment, 300ng of vector and the balance of ddH ₂ O；

Gibsion reaction conditions were: reacting at 50 ℃ for 20min

2 transformation of soybean hairy root

The formula of the FM liquid culture medium is as follows: 0.5mM MgSO ₄ ·H ₂ O、0.7mM KH ₂ PO ₄ 、0.8mM Na ₂ HPO ₄ ·2H ₂ O, 50. mu.M Fe-EDTA (with FeSO) ₄ Formulated with disodium EDTA), 0.1. mu.g MnSO ₄ 、0.1μg CuSO ₄ 、0.1μg ZnSO ₄ 、0.1μg H ₃ BO ₃ 、0.1μg Na ₂ MoO ₄ Add ddH ₂ Adjusting the volume of O to 1L, adjusting the pH value to 6.5, sterilizing, and storing at normal temperature for 5-6 months; the FM medium formulation reference (Toth, K., J.Batek, and G.Stacey, Generation of Soybean (Glycine max) Transgenic Roots.Current protocol Plant Biol,2016.1(1): p.1-13.) can be prepared as a stock solution and diluted for use, with 1% (w/v) Agar added to the solid medium.

The HM liquid culture medium formula is as follows: 0.125g Na ₂ HPO ₄ 、0.25g Na ₂ SO ₄ 、0.32g NH ₄ Cl、0.18g MgSO ₄ ·7H ₂ O、0.25g yeast extract、1g D-Arabinose、1g sodium gluconate、0.004g FeCl ₃ 、0.001g CaCl ₂ 1.30g HEPES, and 1.10g MES, plus ddH ₂ Adjusting the pH value of the solution to be 1L by using NaOH, and adjusting the pH value of the solution to be 6.6;

HM solid medium: 1.5% (w/v) agar was added on the basis of the liquid medium.

The LB culture medium formula is: 10g of Tryptone, 5g of Yeast extract, 10g of NaCl, ddH2O adjusted to 1L, pH adjusted to 7.2 with NaOH, and 1.5% (w/v) agar added to the solid medium.

The method comprises the following steps of: sterilizing soybean seeds with chlorine overnight, washing with sterile water for 6 times, soaking the seeds in sterile water for 3h to swell, rolling up the soybean seeds with sterilized filter paper, placing in a sterilized plastic beaker, adding 1/2 volume of FM (Fahraeus medium) liquid culture medium into the plastic beaker, and finally sealing the beaker with a plastic film. Culturing at 26 deg.C in dark for 2d, and culturing in light incubator (16h light, 8h dark) at 26 deg.C for 2 d.

The method comprises the following steps: activating a control vector pLb 2-GFP-C' 3xFlag and the agrobacterium rhizogenes K599 containing GmNAC039 and the agrobacterium rhizogenes K599 containing GmNAC018, which are obtained in the vector construction step, on a plate containing plasmid resistance (Kanamycin, 50 mu g/mL), after activation, picking a single colony in an LB liquid culture medium, and performing shaking culture at 28-30 ℃ for 24 h. And sucking 200 mu L of bacterial liquid, coating the bacterial liquid on an LB solid flat plate, and culturing the bacterial liquid in an incubator at 28-30 ℃ to form a bacterial membrane containing the soybean agrobacterium rhizogenes K599 of the control vectors pLb 2-GFP-C' 3xFlag and GmNAC039 and a bacterial membrane containing the soybean agrobacterium rhizogenes K599 of the GmNAC 018.

And c, infection and culture: scraping a mycoderm of the agrobacterium rhizogenes K599 containing the control and GmNAC039 and a mycoderm of the agrobacterium rhizogenes K599 containing the GmNAC018, beveling soybean seedlings obtained in the step (1) at alternate white and green positions of a hypocotyl to cut off the hypocotyl, dipping the hypocotyl-cut soybeans in the bacteria of the agrobacterium rhizogenes K599 containing the control and GmNAC039 and the bacteria of the agrobacterium rhizogenes K599 containing the GmNAC018 respectively, and placing the bacteria on an FM solid culture medium for co-culture; the co-culture steps are as follows: culturing in dark at 26 deg.C for 2 days, washing with sterile water to remove Agrobacterium, placing the seedling on new FM solid culture medium, and culturing in light incubator at 26 deg.C (16h light and 8h dark) for about 6 days.

Fourth, rooting: taking out the soybean seedlings, placing the soybean seedlings in a rooting box added with an FM liquid culture medium, and culturing for 8-10 days at 26 ℃ in an illumination incubator (16h for illumination and 8h for darkness).

Identification of positive transgenic roots: under a stereoscopic fluorescence microscope, positive seedlings are selected and non-positive roots are removed through GFP screening marks.

Sixthly, rhizobium inoculation: after the rhizobia (Bradyrhizobium disazoefficiens) USDA110 was activated on an HM (HEPES-MES) solid plate, the rhizobia was picked up and cultured in an HM liquid medium at 28 ℃ for 2 to 3 days with shaking. Centrifuging at 7000r/min for 5min, collecting thallus, resuspending thallus with FM culture medium, and adjusting concentration to OD ₆₀₀ Is approximately equal to 0.02 and is inoculated to the root of the soybean seedling to obtain a hairy root transformation plant. After 4 weeks, the phenotype was observed and the results are shown in fig. 3 and 4: compared to control (empty vector), GmNAC039 and GmNAC018 overexpressed plants yellow and nodules also whiten or turn green, indicating premature senescence, indicating that GmNAC039 and GmNAC018 overexpression promotes nodule and plant senescence.

Example 4

Effect of GmNAC039 and GmNAC018 overexpression on Azotoxin Activity and nodule Numbers

1 measurement of Activity of Soybean root nodule nitrogenase

After the rhizobium japonicum USDA 1104W was inoculated to the resultant hairy root transformed plant obtained in example 3, the underground part of the soybean was placed in a 40mL glass bottle, 3mL of air was evacuated from the glass bottle by a syringe, and 2mL of acetylene gas was injected into the glass bottle by a syringe. The glass bottle was placed in a plastic box containing water, reacted at 28 ℃ for 2 hours, and then taken out and detected by a gas chromatograph.

2 preparation of ethylene Standard Curve

A glass bottle was filled with a predetermined volume of ethylene standard gas by a microsyringe, 3 replicates were set for each concentration, and the ethylene peak was detected by a gas chromatograph. The standard curve of ethylene volume versus ethylene peak area was plotted on an excel table.

3 calculation of nitrogenase Activity

The Activity of the nodule nitrogenase is Acetylene Reduction Activity (ARA), which can be expressed as the number of moles of Acetylene reduced by a unit weight of nodule in a unit time, and the calculation formula is: acetylene reduction activity ═ moles of ethylene/fresh nodule weight × reaction time (unit of enzyme activity is usually:μmol/g.h).

The number of moles can be determined from the volume of ethylene, based on the relationship of the number of moles of gas to volume, temperature, pressure:

C ₂ H ₄ (μmol)＝C ₂ H ₄ volume (. mu.L). times.1/22.4X 273/(273+ t ℃ C.) times.P/760, wherein: t ℃ is as follows:

reaction temperature (centigrade temperature, 28), P: air pressure, typically 760 mm hg, 22.4: the volume of 1mol of gas in the standard state was 22.4 liters; 273: absolute temperature.

The number of the nodules of each plant is counted respectively, and the fresh weight of the nodules is weighed. The results are shown in fig. 5, table 1 and table 2: compared to the control, GmNAC039 and GmNAC018 overexpressed nitrogenase activity decreased and the number of nodules increased, indicating that GmNAC039 and GmNAC018 overexpressed promoted nodule senescence.

TABLE 1

TABLE 2

Example 5

Effect of GMNAC039 and GmNAC018 overexpression on nodule development

After soybean hairy root transformed seedling is inoculated with rhizobium USDA1104 weeks, underground part is taken for measuring enzyme activity, FAA stationary liquid (Sericebio, G1103-500mL) is fixed, after paraffin embedding and slicing, toluidine blue is used for staining, and then a stereoscopic microscope is used for observation, and the result is shown in figure 6: overexpression of GmNAC039 and GmNAC018 promoted the lysis of the symbiont, advancing nodulation senescence.

Example 6

Effect of GmNAC039 and GmNAC018 CRISPR knockouts on plant nitrogenase activity and nodule count

1. Vector construction

According to the needs of experiments, guide-RNA 1-guide-RNA 4 (the specific sequence is guide-RNA 1: TTTGATCCATGGCAGCTTCC, SEQ ID NO. 19; guide-RNA 2: TTTCCAATAGCCGCTTCCGG, SEQ ID NO. 20; guide-RNA 3: ATATTCGTGCATGATCCAAT, SEQ ID NO. 21; guide-RNA 4: ATGGTGCACAGCTTGATGAT, SEQ ID NO.22) are connected in series on the same vector to knock out the same gene. Primers F1/R1, F2/R3 and F3/R3 are designed, and the primers are amplified by taking a vector pGTR as a template to obtain 3 gRNA-tRNA sequences containing 4 guide-RNAs, and the sequences are called PTGs sequences.

BbsI digests the vector LjU6-tRNA-sgRNA, constructs the PTGs sequence to LjU6-tRNA-sgRNA using the Gibson method (same as example 3), and finally constructs the PTGs sequence to the vector pCAMBIA1300-sGFP-2x35S-Cas9 by double digestion with KpnI and XbaI (Yu, H., et al, prediction of innovative biological mediated by the CDPK-Rboh complex for rhizobiul degradation in medical trunular noduli. New Phytol,2018.220(2): p.425 + 434.).

The primers are as follows:

F1：CGATTCCCGGCTGGTGCATTTGATCCATGGCAGCTTCCGTTTTAGA GCTAGAAATAG，SEQ ID NO.23；

R1：CCGGAAGCGGCTATTGGAAATGCACCAGCCGGGAATC，SEQ ID NO.24；

F2：TTTCCAATAGCCGCTTCCGGGTTTTAGAGCTAGAAATAG，SEQ ID NO.25；

R2：ATTGGATCATGCACGAATATTGCACCAGCCGGGAATC，SEQ ID NO.26；

F3：ATATTCGTGCATGATCCAATGTTTTAGAGCTAGAAATAG，SEQ ID NO.27；

R3：CTATTTCTAGCTCTAAAACATCATCAAGCTGTGCACCATTGCACC AGCCGG GAATCG，SEQ ID NO.28。

2 Soybean root transformation As in example 3

3 Soybean root nodule nitrogenase activity assay the same as in example 3.

4 ethylene standard curve was made as in example 3.

5 nitrogenase activity was calculated as in example 3.

The number of the nodules of each plant is counted respectively, and the fresh weight of the nodules is weighed. The results are shown in Table 3: compared with the control, the azotase activity of the GmNAC039 and GmNAC018 CRISPR knockout plants is improved, the number of nodules is increased, and the GmNAC039 and GmNAC018 CRISPR knockout is proved to delay the senescence of the nodules.

TABLE 3

6. Genome extraction

After 6W of hairy root transformed plants obtained from 6 rhizobium inoculation in 2 soybean hairy root transformation in example 3 were inoculated, the rhizobia were harvested in a 1.5mL centrifuge tube. Freezing the tube in liquid nitrogen, grinding with a grinding rod, repeating several times until the plant is powdered, adding 300. mu.L of extraction buffer (genome extraction buffer, CTAB 4g, NaCl 16.364g, 1M Tris-HCl 20mL (pH8.0) and 0.5M EDTA 8mL, adding 70mL ddH ₂ Dissolving O, diluting to 200mL, sterilizing), fully reversing and mixing, incubating in a water bath at 65 ℃ for 30min, reversing and mixing for 1 time every 5min, adding 200 μ L of a mixture of phenol, chloroform and isoamyl alcohol (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), fully reversing and mixing, standing at room temperature for 2min, and centrifuging at 12000r/min for 10 min.

Sucking 250 mu L of supernatant, adding isovolumetric isopropanol and 10 wt.% of 3M NaAc, fully and uniformly mixing, precipitating at-20 ℃ for 30min, centrifuging at 12000r/min for 10min, discarding the supernatant, adding 1mL of precooled ethanol with the volume percentage content of 75%, centrifuging at 12000r/min for 5min, and discarding the supernatant; drying the precipitate with a dryer, adding about 40 μ L ddH ₂ Dissolving O to obtain the plant genome DNA.

7. Gene editing assay

The genomic DNA of the plant obtained above was subjected to PCR detection.

The PCR primers were as follows:

GmNAC039-F：ACAAAGAGAGGAAGAAACGCGA，SEQ ID NO.13；

GmNAC039-R：TCCTATCATCCATCATTCGGCT，SEQ ID NO.14；

GmNAC018-F：AAACCACCCAGTGTGTCCTC，SEQ ID NO.15；

GmNAC018-R：CTTGGCCTAGCCCACATCAC，SEQ ID NO.16。

the PCR procedure and reaction are as in example 3.

The gene editing result of PCR detection shows that large fragment deletion occurs in both GmNAC039 and GmNAC 018.

Taken together, NAC transcription factors GmNAC039 and GmNAC018 have important functions in regulating senescence in soybean nodules. The GmNAC039 and GmNAC018 induce expression in aging nodules, and the overexpression of the GmNAC039 and GmNAC018 obviously reduces the activity of nitrogen-fixing enzyme, obviously increases the number of nodules, and obviously increases the activity of nitrogen-fixing enzyme and the number of nodules after the GMNAC039 and GmNAC018 are knocked out, which shows that the GmNAC039 and GmNAC018 have important meanings in the aspects of improving the nitrogen-fixing efficiency and yield of soybeans.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are within the scope of the present invention.

Sequence listing

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atggagactt cggattcggt gccaaggttg aacacggact cgagcagttc ggagcacgtg 660

gtttcgcccg atgtcacgtg cgagagggag gtgcagagcg accccaagtg gaacgatgat 720

ctggacctaa agctagaaaa cgcgtttgat tttcagttta attacttgga cgataataac 780

ctttccgtgg atgattacct ttttggcact gttcagtatc aaatgggcca gctctcgccc 840

ttgcaggaca tgttcatgta cctacagaag atgtga 876

Claims

1. The application of soybean GmNAC039 or GmNAC018 in regulating and controlling nitrogen fixation of plant nodules and/or regulating and controlling yield, wherein the regulation and control of nitrogen fixation of plant nodules comprises one or more of the following aspects 1) to 5):

1) regulating and controlling the plant root nodule aging;

2) regulating and controlling the growth of plant root nodules;

3) regulating and controlling the number of plant nodules;

2. The use of claim 1, wherein modulating senescence in plant nodules comprises delaying senescence in plant nodules by negatively modulating expression of GmNAC039 or GmNAC 018.

3. The use of claim 1, wherein modulating plant nodule development comprises promoting plant nodule development by positively modulating expression of GmNAC039 or GmNAC 018.

4. The use of claim 1, wherein modulating the number of plant nodules comprises increasing the number of plant nodules by positively modulating expression of GmNAC039 or GmNAC 018.

5. The use of claim 1, wherein said modulating yield comprises increasing yield by negatively modulating expression of GmNAC039 or GmNAC 018.

6. The use of claim 1, wherein modulating azotase activity in plant nodules comprises enhancing azotase activity in plant nodules by negatively modulating expression of GmNAC039 or GmNAC 018.

7. The use as claimed in claim 1, wherein said modulating nitrogen fixation efficiency of plant nodules comprises increasing nitrogen fixation efficiency of plant nodules by negatively modulating expression of GmNAC039 or GmNAC 018.

8. The application of soybean GmNAC039 or GmNAC018 in high-yield plant molecular breeding is characterized in that the amino acid sequence of a protein encoded by GmNAC039 is shown as SEQ ID No. 1; the amino acid sequence of the protein coded by the GmNAC018 is shown as SEQ ID NO. 2.

9. Use according to any one of claims 1 to 8, wherein the plant comprises a leguminous plant.

10. The use according to claim 9, wherein the leguminous plants comprise one or more of soybean, alfalfa and lotus japonicus.