CN116555329A - Application of LjAP3M protein in regulation and control of nitrogen fixation efficiency of plant root nodule - Google Patents
Application of LjAP3M protein in regulation and control of nitrogen fixation efficiency of plant root nodule Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/06—Roots
Abstract
The invention relates to the technical field of genetic engineering, in particular to application of LjAP3M protein in regulating and controlling nitrogen fixation efficiency of plant root nodule. The invention uses CRISPR-Cas9 technology to knock out LjAP3M gene, obtains transgenic plants rapidly through agrobacterium rhizogenes mediated hundred-pulse root hair root transformation technology, and performs root nodule count after rhizobium inoculation 14d and 21d, and results show that LjAP3M can obviously improve the root nodule count after knocking out and promote the formation of root nodules; in addition, the LjoP 3M gene is over-expressed by using the root transformation technology, and the result shows that the number of root nodules of the over-expressed plant is obviously lower than that of a wild control group, which indicates that the LjoP 3M gene regulates the number of root nodules. The LjAP3M protein has great significance in reducing the use amount of nitrogen fertilizer and improving the utilization rate of plant nitrogen fertilizer, and has great potential in crop genetic improvement.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of LjAP3M protein in regulating and controlling nitrogen fixation efficiency of plant root nodule.
Background
Nitrogen is one of the key factors for determining the high and stable yield of crops, and although nitrogen is the element with the highest content in the atmosphere, plants cannot directly absorb and utilize the nitrogen. In order to meet the nitrogen demand of crops, agricultural production mainly provides absorbable nitrogen sources for crops by applying industrial nitrogen fertilizer. The overuse of industrial nitrogenous fertilizers creates a series of ecological and economic problems. On one hand, the production of industrial nitrogenous fertilizer consumes mineral energy and can discharge a large amount of greenhouse gases, on the other hand, the utilization efficiency of the nitrogenous fertilizer by plants is low, the use of the nitrogenous fertilizer in agriculture is excessive, the nitrogenous fertilizer which is not absorbed and utilized can flow into rivers, lakes and seas, water eutrophication is caused, the soil structure is changed, and the diversity of soil microorganisms can be influenced. Therefore, how to reduce the use of nitrogenous fertilizer while ensuring high and stable yield of crops and to make environmental protection work is a great problem in agricultural production. In nature, nitrogen-fixing microorganism can utilize its own nitrogen-fixing enzyme to make N in atmosphere by means of autogenous or symbiotic mode with plant 2 The direct catalytic reduction to ammonia, a process known as biosolidation, provides 60% to 70% of the absorbable nitrogen source by means of biosolidation. In view of the characteristics of high nitrogen fixation efficiency and environmental friendliness, the application of the biological nitrogen fixation to agriculture has become a great measure for promoting the green sustainable development of agriculture, and is also a target and direction in the research field of biological nitrogen fixation.
The symbiotic nitrogen fixation system formed by the reciprocal symbiosis of leguminous plants and rhizobia is the nitrogen reduction system with highest nitrogen fixation efficiency in nature. Leguminous plants interact with rhizobia to form a new organ, namely a rhizobium, which is a place for nitrogen fixation by the rhizobia, and the number of the rhizobium directly influences the nitrogen fixation efficiency of the rhizobium. The Baimai root is a model leguminous plant, and has important significance for improving the number of root nodules and the nitrogen fixation efficiency of the leguminous plant by identifying functional genes in the Baimai root symbiotic nodulation and nitrogen fixation, and also provides possibility for reducing the use of nitrogenous fertilizer, increasing the yield of leguminous crops and improving the quality of the leguminous crops in agricultural production.
There are many reports on the aspect of regulating the pore closure of arabidopsis thaliana about the AP3M gene at present, but no report on the aspect of regulating the nitrogen fixation of plant root nodule about LjoP 3M protein in the Baimai root exists at present.
Disclosure of Invention
In order to solve the problems, the invention provides application of LjAP3M protein in regulating and controlling nitrogen fixation efficiency of plant root nodule. The LjAP3M protein can regulate and control the nitrogen fixation efficiency of plant nodules, particularly can regulate and control the number of the nodules, and has wide application prospect on leguminous crops.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of LjAP3M protein in regulating nitrogen fixation efficiency of plant root nodule, wherein the amino acid sequence of the LjAP3M protein is shown as SEQ ID NO. 1.
Preferably, the regulating and controlling the nitrogen fixation efficiency of the plant root nodule comprises: regulating and controlling the number of plant root nodules.
Preferably, the regulating and controlling the nitrogen fixation efficiency of the plant root nodule comprises: the number of plant nodules is increased by negatively regulating the expression of the LjAP3M gene or decreased by positively regulating the expression of the LjAP3M gene.
Preferably, the gene encoding the LjoP 3M protein is the LjoP 3M gene; the nucleotide sequence of the LjAP3M gene is shown as SEQ ID NO. 2.
The invention also provides application of the LjAP3M protein in cultivating transgenic plants with improved nitrogen fixation efficiency, wherein the amino acid sequence of the LjAP3M protein is shown as SEQ ID NO. 1.
Preferably, the nitrogen fixation efficiency improvement includes: the number of plant root nodules is increased.
Preferably, the plant comprises a leguminous plant.
Preferably, the leguminous plants include one or more of soybean, alfalfa and centella asiatica.
The beneficial effects are that:
the invention provides application of LjAP3M protein in regulating nitrogen fixation efficiency of plant root nodule, wherein the amino acid sequence of the LjAP3M protein is shown as SEQ ID NO. 1. The invention uses CRISPR-Cas9 technology to knock out LjAP3M gene, obtains transgenic plants rapidly through agrobacterium rhizogenes mediated hundred-pulse root hair root transformation technology, and performs root nodule count after rhizobium inoculation 14d and 21d, and results show that LjAP3M can obviously improve the root nodule count after knocking out and promote the formation of root nodules; in addition, the LjoP 3M gene is over-expressed by using the root transformation technology, and the result shows that the number of root nodules of the over-expressed plant is obviously lower than that of a wild control group, which indicates that the LjoP 3M gene regulates the number of root nodules. The LjAP3M protein has great significance in reducing the use amount of nitrogen fertilizer and improving the utilization rate of plant nitrogen fertilizer, and has great potential in crop genetic improvement.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a graph of the number of nodules of LjoP 3M knockout mutant plants and control plants after inoculation with rhizobia 14 d;
FIG. 2 is a graph of the number of nodules of LjoP 3M knockout mutant plants and control plants after inoculation with rhizobia 21 d;
FIG. 3 is a schematic representation of LjoP 3M knockout mutant plants and control plants after inoculation with rhizobia 21d and corresponding schematic representation of rhizomes;
FIG. 4 is a graph of nodule numbers for LjoAP 3M overexpressing plants and control plants after 14d inoculation with rhizobia;
FIG. 5 is a graph of nodule numbers for LjoAP 3M overexpressing plants and control plants after 21d inoculation with rhizobia;
FIG. 6 is a schematic representation of LjoAP 3M overexpressing plants and control plants after inoculation with rhizobia 21d and corresponding schematic representation of nodules;
FIG. 7 is a target sequencing result of 3 sgRNAs in LjAP3M knockout mutant plants after 14d inoculation with rhizobia;
FIG. 8 is a graph showing the transcription level of LjAP3M in LjAP3M-KO transgenic hair roots;
FIG. 9 is the transcript levels of LjoP 3M in LjoP 3M-OX transgenic roots;
wherein, in fig. 1, 2 and 3, ljop 3M-KO is a knockout mutant plant; in FIGS. 4, 5 and 6, ljoP 3M-OX is an overexpressing plant; the scale bar in fig. 3 and 6 is 1cm;
* When the significant level is 0.01 < p is less than or equal to 0.05, the difference is significant; * When representing the significance level of 0.001 < p.ltoreq.0.01, the difference significance was between significant and very significant; * When p is less than or equal to 0.001, the difference is very significant.
Detailed Description
The invention provides application of LjAP3M protein in regulating nitrogen fixation efficiency of plant root nodule, wherein the amino acid sequence of the LjAP3M protein is shown as SEQ ID NO.1, and the application is as follows: MLQCIFLLSDSGEVMLEKQLTGHRVDRSICAWFWDHAISQPDSFKQQPVIASPTHYLFQVFRDGITFLACTQVEMPPLMAIEFLCRVADVLKDYLGGLNEDVIKDNFVIVYELLDEMIDNGFPLTTELNVLQEMIAPPNIVSKVLSVVTGRSSNVSDTLPGATASVVPWRTADPKYASNEVYVDLVEEMDATINRDGVLVKCEISGEVQVNSQITGLPDLTLSFANPSVLDDVRFHPCVRFRPWESNQILSFVPPDGQFKLMSYRVRKLKSTPIYVKPQLTSDGGTCRLSVLVGIRNDPGKTIDSVIVQFQLPSCILSADLTSNHGTVNILANKTCIWTIGRIPKDKAPSMSGTLVLETGLERLHVFPTFQVDFRIMGVALSGLQIDKLDLQTVPYRFYKGFRALTRAGQFEVRS.
In the present invention, the gene encoding the LjAP3M protein is preferably an LjAP3M gene; the nucleotide sequence of the LjoP 3M gene is preferably shown in SEQ ID NO.2, and is specifically as follows: 5'-ATGTTGCAGTGCATATTTCTTCTCTCCGATTCCGGAGAGGTAATGCTAGA GAAACAGCTCACTGGCCACCGCGTAGATCGCTCCATATGTGCCTGGTTCTGGGATCACGCTATTTCTCAACCTGATTCCTTCAAGCAACAACCAGTGATTGCTTCTCCAACTCATTATCTTTTCCAAGTTTTTCGTGATGGAATCACTTTTTTGGCTTGCACTCAAGTTGAAATGCCACCATTGATGGCCATTGAGTTCCTTTGTAGGGTAGCTGATGTCCTCAAAGATTATCTTGGTGGATTGAATGAAGACGTAATCAAAGATAACTTTGTCATTGTATATGAGCTGCTGGATGAGATGATAGACAATGGCTTCCCGCTAACTACGGAACTTAATGTCCTGCAAGAGATGATAGCTCCGCCAAATATCGTTAGCAAAGTCTTGAGTGTTGTGACTGGCAGAAGCTCCAATGTGAGTGACACGCTTCCGGGTGCCACTGCATCTGTTGTTCCCTGGAGAACAGCAGACCCGAAGTATGCTAGCAATGAAGTTTATGTAGATCTTGTTGAAGAAATGGACGCAACAATAAATAGGGATGGAGTTCTGGTGAAATGTGAGATCAGCGGTGAGGTTCAAGTAAATTCCCAGATCACAGGTCTTCCTGATTTGACCCTTTCATTTGCAAATCCTTCAGTCCTAGATGATGTGAGATTCCATCCCTGTGTTAGATTTCGGCCTTGGGAATCCAATCAAATTCTTTCCTTTGTGCCTCCTGATGGACAATTTAAGCTTATGAGTTACAGAGTTAGAAAATTGAAGAGCACCCCAATATATGTAAAGCCACAGTTGACTTCAGATGGTGGGACATGCCGTCTTAGTGTATTGGTTGGCATAAGAAATGATCCTGGAAAGACAATAGATTCAGTTATTGTTCAGTTTCAGCTTCCCTCTTGCATCTTATCAGCTGATCTGACTTCGAATCATGGAACAGTAAACATCCTTGCTAACAAGACATGCATTTGGACCATTGGTCGGATCCCTAAGGACAAAGCCCCATCAATGTCTGGCACATTGGTACTTGAGACTGGATTGGAGCGCCTTCATGTCTTTCCCACATTTCAAGTGGATTTTAGGATTATGGGTGTCGCCCTTTCTGGTCTGCAAATAGATAAACTGGATCTGCAGACTGTACCCTACCGTTTCTACAAAGGTTTTCGAGCTCTTACTCGGGCAGGCCAATTTGAAGTCAGGTCATAA-3'.
In the present invention, the regulation of nitrogen fixation efficiency of plant nodules preferably comprises: regulating and controlling the number of plant root nodules; more preferably includes increasing the number of plant nodules by negatively regulating expression of the LjAP3M gene or decreasing the number of plant nodules by positively regulating expression of the LjAP3M gene.
The invention proves that the LjAP3M gene and the LjAP3M protein expressed by the LjAP3M gene regulate nitrogen fixation efficiency of the root nodule by knocking out the coding gene of the LjAP3M protein and over-expressing the coding gene of the LjAP3M protein, and can improve the nitrogen fixation efficiency of leguminous plants, in particular to improve the root nodule number by inhibiting the expression of the LjAP3M gene.
In the present invention, the negative regulation means preferably comprises knockout of LjoP 3M gene; the sgRAN for knocking out the ljop 3M gene preferably comprises sgRAN1, sgRAN2 and sgRAN3, and the specific sequence is preferably as follows:
sgRAN1：5’-ATATGGAGCGATCTACGCGG-3’，SEQ ID NO.4；
sgRNA2：5’-TGAGTTGGAGAAGCAATCAC-3’，SEQ ID NO.5；
sgRNA3：5’-ACTTTGCTAACGATATTTGG-3’，SEQ ID NO.6。
in the present invention, the means of upregulation preferably includes overexpression of the LjoP 3M gene.
The invention also provides application of the LjAP3M protein in cultivating transgenic plants with improved nitrogen fixation efficiency, wherein the amino acid sequence of the LjAP3M protein is shown as SEQ ID NO. 1.
In the present invention, the nitrogen fixation efficiency improvement preferably includes: the number of plant root nodules is increased. The invention can increase the number of plant root nodules by negatively regulating the expression of LjAP3M genes.
In the present invention, the plant preferably includes a leguminous plant; the leguminous plants preferably include one or more of soybean, alfalfa and centella asiatica.
For further explanation of the present invention, the application of the LjoP 3M protein provided by the present invention to regulation of nitrogen fixation efficiency of plant nodules is described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Effect of the knockout of the Baimai root LjAP3M Gene on the number of nodules
1. selection of sgrnas
The sgrnas were designed based on the genomic sequence of ljop 3M, and were selected as follows: the target efficiency is higher than 50% and is as close as possible to the ATG downstream of the coding region of the gene, preferably on the first or second exon, 3 sgRNAs were selected, wherein the genomic sequence of LjAP3M is shown in SEQ ID NO.3, specifically:
5’-GGGAGTAATACCTTAGTTTTGTCGTTTGGGTGTGGTCTCAAGGAGG AAGAAAGCAGTGTAGCTTGTGAAACGATGAGAACTCCATCTCCTTCATCTTCATCATAAACCTACCTTGTTAGCTTCTTCTTCTTCTTCTTCGGATTTCATACAGAGAGATGTTGCAGTGCATATTTCTTCTCTCCGATTCCGGGTAAGATTGATTGTTGCAAACCAAAATCTCAAATTCAATTTCTATATTTTTTGAGTTGAATCTCTGAATTTTCGATTCGTTTAGAGAGGTAATGCTAGAGAAACAGCTCACTGGCCACCGCGTAGATCGCTCCATATGTGCCTGGTTCTGGGATCACGCTATTTCTCAACCTGATTCCTTCAAGGTTTGTTTTCCTCATTCTTCTCTTTTTCTCACATTTTTTGCCCATTCTTCTAATTTTATTTAGATTATTTCATTCTCTGTGTGTGTATAGCAACAACCAGTGATTGCTTCTCCAACTCATTATCTTTTCCAAGTTTTTCGTGATGGAATCACTTTTTTGGCTTGCACTCAAGTTGAAATGCCACCATTGATGGCCATTGAGGTATATTTCCAGTTACTTATACCTTATTCGCTTTGCAACTCGTAA
CAAACTGTATTTCTTTGCTTTTTAATTTTTAACCATATGCTTTTAGTTCCTTT
GTAGGGTAGCTGATGTCCTCAAAGATTATCTTGGTGGATTGAATGAAGACG
TAATCAAAGATAACTTTGTCATTGTATATGAGGTATTCATTCTGTGACTCATT
TGCCCAAATGTTATTAGTTTTTTGCCGTCCTCGTTTGATTTGCGGTTTAGAAT
TTAGATGTAAAAGTGATTGTTTTGTTTTTGTTATTGTAGCTGCTGGATGAGA
TGATAGACAATGGCTTCCCGCTAACTACGGAACTTAATGTCCTGCAAGAGA
TGATAGCTCCGCCAAATATCGTTAGCAAAGTCTTGAGTGTTGTGACTGGCA
GAAGCTCCAATGTGAGTGACACGCTTCCGGGTGCCACTGCATCTGTTGTTC
CCTGGAGAACAGCAGACCCGAAGTATGCTAGCAATGAAGTTTATGTAGATC
TTGTTGAAGAAATGGACGCAACAATAAATAGGTCTTTATGCTCTGACATGC
TTGTTCTTATTACGCATTTTGCAAGCTTAGTGAACAGGATATAACTGATTCT
TGGAAGTTTGAACTTTTGCAACCTAATATGGCTGAACTTTATAAGAAATCAT
CTTCAGACTCCTCCTTGGTAGTTTGTATCCTAGGAACCATTGATTTCAACTT
CTTTCTTATATACTCGTTGCTAAATAATACTGTTGATTCAGGGATGGAGTTCT
GGTGAAATGTGAGATCAGCGGTGAGGTTCAAGTAAATTCCCAGATCACAG
GTCTTCCTGATTTGACCCTTTCATTTGCAAATCCTTCAGTCCTAGATGATGT
GAGATTCCATCCCTGTGTTAGATTTCGGCCTTGGGAATCCAATCAAATTCTT
TCCTTTGTGCCTCCTGATGGACAATTTAAGCTTATGAGTTACAGGTATTACA
TCCACCTTTATGTTGATGTCTGAATTAGGATTTGAATTTTGAAACCTTTGTTT
GAGAAGTCAGTTTGGATATAGATATCATTGTGAATGTTGAAATTCATTCCAT
GATATTCCCAAATTTTGCTTTTTCTAAAAAAATTCTGGCATTCCACCTTTTTT
CCAGAGTTAGAAAATTGAAGAGCACCCCAATATATGTAAAGCCACAGTTGA
CTTCAGATGGTGGGACATGCCGTCTTAGTGTATTGGTTGGCATAAGAAATG
ATCCTGGAAAGACAATAGATTCAGTTATTGTTCAGTTTCAGCTTCCCTCTTG
CATCTTATCAGCTGATCTGACTTCGAATCATGGAACAGTAAACATCCTTGCT
AACAAGGTAGTTAATAATCTCTGCTTACTCTTTATCTATTGTAGAAACTAGG
AAGTCAAAATAGTCAGATGAGAACTTTGCTCTATTTGTGCAATAAAATTTG
AAGCTTTCTTCATATCTCATTCTATCAGACATGCATTTGGACCATTGGTCGG
ATCCCTAAGGACAAAGCCCCATCAATGTCTGGCACATTGGTACTTGAGACT
GGATTGGAGCGCCTTCATGTCTTTCCCACATTTCAAGTGGATTTTAGGATTA
TGGGTGTCGCCCTTTCTGGTCTGCAAATAGATAAACTGGATCTGCAGACTG
TACCCTACCGTTTCTACAAAGGTTTTCGAGCTCTTACTCGGGCAGGCCAAT
TTGAAGTCAGGTCATAATTTTGTTTTTGCCATTAACTCTTCTGAGACTTGTC
ATGATCCTGGACATGAAATTTCGTTGTGTGTACATGTCTGGTTTCATTTGCA
AAGTAAGTTGGTATTTTTTTTTTCTGGTTTCAAATTATGTCCATAGTGAACCA
TATCCTGGTTATCTGAATTATCCTGGTTTTGTGAGAGTCAAACCCTTGTCTT
TTGTGTCTTTTTTCCTTTCAAAAATTGGTATCAGTGAGAATGAATGATTTAG
ATGGATAGGTGATCTCCACTACTTCATCTTATCTGTATGCAGCCTAAATTTTA
GATGTGAAACACTACCATTTCAGTATCAATTGATTTATGGAGCAAGGAACCTTCA-3’；
the 3 sgRNA sequences selected were as follows:
sgRAN1：5’-ATATGGAGCGATCTACGCGG-3’，SEQ ID NO.4；
sgRNA2：5’-TGAGTTGGAGAAGCAATCAC-3’，SEQ ID NO.5；
sgRNA3：5’-ACTTTGCTAACGATATTTGG-3’，SEQ ID NO.6。
2. vector construction
(1) Amplification of fragments 1 and 2: amplifying different primers by using pGTR recombinant plasmid as a template and 2 respectively; the reaction system for amplifying the fragment 1 and the fragment 2 is as follows: powerpol 2x PCRMix with Dye 25. Mu.L, forward primer 1.5. Mu.L, reverse primer 1.5. Mu.L, template 1. Mu.L and sterile water 21. Mu.L; the concentration of the forward primer and the reverse primer is 100 mu M; the reaction procedure for amplifying both fragment 1 and fragment 2 was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 15s, for 32 cycles;
the pGTR recombinant plasmid takes pGTR plasmid as a template, gRNA-tRNA fusion fragments are inserted into the pGTR plasmid, and the construction method and the pGTR recombinant plasmid are disclosed in the literature [ FengY, wuP, liu C, et al, suppresion of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotusjaponicus [ J ]. Molecular plants: english version, 2021,14 (11): 16 ];
the nucleotide sequence of the gRNA-tRNA fusion fragment is shown in SEQ ID NO.7, and specifically comprises the following steps: 5'-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA CTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTG GTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGT GCA-3';
the 2 pairs of different primer sequences are as follows:
CRISPR-LjAP3M-sgRNA-F1：5’-TCGATTCCCGGCTGGTGCAATATGGAGC GATCTACGCGGGTTTTAGAGCTAGAAATAG-3’，SEQ ID NO.8；
CRISPR-LjAP3M-sgRNA-R1：5’-GTGATTGCTTCTCCAACTCATGCACCAG CCGGGAATC-3’，SEQ ID NO.9；
CRISPR-LjAP3M-sgRNA-F2：5’-TGAGTTGGAGAAGCAATCACGTTTTAG AGCTAGAAATAG-3’，SEQ ID NO.10；
CRISPR-LjAP3M-sgRNA-R2：5’-GCTATTTCTAGCTCTAAAACCCAAATAT CGTTAGCAAAGTTGCACCAGCCGGGAATC-3’，SEQ ID NO.11；
(2) And (3) carrying out enzyme cutting and connection of a carrier: intermediate vector pBluescript-SK with restriction endonuclease Bbs I + -LjU single cleavage to obtain pBluescript-SK + -LjU6 linearization carrier; the pBluescript-SK was reacted by Gibson + -LjU linearization vector is linked to fragment 1 and fragment 2 to obtain recombinant intermediate vector; recombinant intermediate vector pBluescript-SK + LjU6 is disclosed in [ FengY, wuP, liu C, peng L, wang T, wang C, tan Q, li B, ouY, zhu H, yuan S, huang R, starey G, zhang Z, cao Y.support of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonica. Molecular Plant,2021,14:1935-1950 ]
The single enzyme digestion system is as follows: 10X FastDigest Green buffer. Mu.L, intermediate vector pBluescript-SK + -LjU mu L, bbs I endonuclease 1.5. Mu.L and sterilized water 23.5. Mu.L; intermediate vector pBluescript-SK + LjU6 having a concentration of 350ngMu L; the single cleavage reagents were all purchased from Thermo Scientific company; the single enzyme cutting condition is 37 ℃ for 3 hours;
the Gibson reaction system is as follows: 2X MultiF Seamless Assembly Mix mu L, pBluescript-SK + LjU6 linearization of 0.5. Mu.L of vector, 11. Mu.L of fragment, 21. Mu.L of fragment and 0.5. Mu.L of sterile water; the pBluescript-SK + -LjU linearization of the carrier at a concentration of 98 ng/. Mu.L; the concentration of the fragment 1 and the fragment 2 is 50 ng/. Mu.L; the reagents for the Gibson reaction were all purchased from ABclonal corporation; the Gibson reaction condition is 50 ℃ for 1h;
(3) Cleavage of the recombinant intermediate vector and the final vector: double enzyme digestion is carried out on the recombinant intermediate vector and the final vector p1300-sGFP-2x35S-Cas9 by KpnI and XbaI; the final vector P1300-sGFP-2x35S-Cas9 is disclosed in [ Feng Y, wu P, liu C, peng L, wang T, wang C, tan Q, li B, ouY, zhu H, yuan S, huang R, starey G, zhang Z, caoY.support of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonica. Molecular plant,2021,14:1935-1950 ].
The double enzyme digestion system comprises: 10X FastDigest Green buffer. Mu.L, final vector p1300-sGFP-2X35S-Cas9 or recombinant intermediate vector 20. Mu. L, kpn I endonuclease 1.5. Mu. L, xba I endonuclease 1.5. Mu.L and sterilized water 22. Mu.L; the concentration of the final vector p1300-sGFP-2x35S-Cas9 is 180 ng/. Mu.L; the concentration of the recombinant intermediate vector is 200 ng/. Mu.L; the double digestion reagents were all purchased from Thermo Scientific company; the conditions of the double enzyme digestion are 37 ℃ and 3 hours;
(4) Recovery and ligation of cleavage products: recovering relatively smaller fragments after enzyme digestion of the recombinant intermediate vector, and recovering after enzyme digestion of the final vector p1300-sGFP-2x35S-Cas9 to obtain a p1300-sGFP-2x35S-Cas9 linearization vector; connecting the p1300-sGFP-2x35S-Cas9 linearization vector and the small fragment recovered after the recombination intermediate vector is subjected to enzyme digestion by using T4 ligase, so as to obtain a final knockout vector;
the connected reaction system is as follows: 10×T4 buffer 0.5.mu. L, T4 DNA library 0.5.mu. L, p1300-sGFP-2x35S-Cas9 linearized vector 1. Mu.L, small fragment recovered after cleavage of recombinant intermediate vector 2. Mu.L and sterilized water 1. Mu.L; the concentration of the p1300-sGFP-2x35S-Cas9 linearization vector is 30 ng/. Mu.L; the concentration of the small fragments recovered after the enzyme digestion of the recombinant intermediate vector is 15 ng/. Mu.L; the linked reagents were all purchased from Thermo Scientific company; the reaction conditions for the ligation were 22℃for 1h.
3. Transformation of Agrobacterium
Transforming agrobacterium rhizogenes Agrobacterium Rhizogenes LBA1334 from the obtained knockout vector to obtain knockout engineering bacteria; agrobacterium rhizogenes Agrobacterium Rhizogenes LBA1334 are disclosed in [ FengY, wu P, liu C, peng L, wang T, wang C, tan Q, li B, ouY, zhu H, yuan S, huang R, stacey G, zhang Z, cao Y.support of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus java. Molecular Plant,2021,14:1935-1950 ].
4. Root-to-root conversion of Baimai root
(1) Seed germination: adding 20mL of concentrated sulfuric acid into a 50mL sterile triangular flask, pouring wild seed of Baimaigen, gently shaking to make the seed fully contact with concentrated sulfuric acid, treating for 8min to make the seed color slightly lighter, and concentrating to obtain H 2 SO 4 The color turns yellow. Carefully sucking concentrated sulfuric acid into glassware containing a small amount of water, adding a large amount of ultrapure water into a triangular flask to quickly rinse seeds, and discarding waste liquid. Rinsing was repeated 4 times with a small amount of ultrapure water. Adding 2wt.% sodium hypochlorite solution to cover the seeds, gently shaking, and treating for 8min, wherein the transparent small ring at the umbilicus of the seeds falls, individual seeds expand, and the liquid is slightly pale yellow. The waste liquid was discarded, and the seeds were rinsed with ultrapure water and the rinsing was repeated 4 times. The seeds were immersed in 20mL of ultrapure water and vernalized at 4℃for 1d. The imbibed seeds were transferred to MS medium, dark cultured at 23℃in an illumination incubator for 2d, and light cultured for 3d.
The MS culture medium is prepared by the following steps: 4.43g MS salt (Sigma, M5519-1L), 20g sucrose, pH adjusted to 6.0, miliQ-H 2 O was set to 1L, 1.5wt.% agar powder was added to the solid, and sterilized at 115℃for 20min.
The wild type seed of Baimaigen is disclosed in [ FengY, wuP, liu C, peng L, wang T, wang C, tan Q, li B, ouY, zhu H, yuan S, huang R, stacey G, zhang Z, cao Y.support of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonica molecular plant,2021,14:1935-1950 ].
(2) Infection and co-cultivation: inoculating the knockout engineering bacteria obtained in the step 3 into LB liquid culture medium when seedling cotyledon grows up and true cotyledon does not grow out, culturing at 28 ℃ to make OD 600 =0.7. The cells were collected by centrifugation at 5000r/m for 8min, resuspended in sterile water containing acetosyringone at a final concentration of 40. Mu.g/mL, and the OD was adjusted 600 =0.8, transfer the bacterial solution into a 50mL sterile triangle; the sterile water with the final concentration of 40 mug/mL of acetosyringone is obtained by mixing 100mmol/L of acetosyringone with the sterile water; the acetosyringone is purchased from Shanghai, under the product number A601111. Dividing the germinated seedlings evenly, cutting 150 seedlings from the hypocotyl of each sample by scissors on a flat plate, and cutting down the enlarged part between the green stems and the white roots; and transferring the sheared seedlings to the resuspended bacterial liquid, and infecting for 30min. The seedlings are moved to MS culture medium, 30 seedlings are placed on each plate, a sealing film seals the flat plate, the illumination incubator is placed in dark for 3d, the illumination incubator is placed in light for 2d, whether root wounds cling to the culture medium or not is checked every day, and the tilted stems are pressed to cling to the culture medium. Seedlings were transferred to HRE medium containing final concentration of 300. Mu.g/mL cefotaxime (from Yeasan under the designation 60226ES 08) and grown for 12 days with two rows of 12 seedlings per plate and with the same orientation in a 23℃illuminated incubator with stems at 70-90℃positions.
The LB culture medium is prepared by the following steps: 5g of yeast extract, 10g of tryptone, 10g of NaCl and 0.45g of agar powder, adjusting the pH to 7.0, adjusting the volume of ultrapure water to 1L, and sterilizing for 30min at 121 ℃.
The HRE rooting culture medium is prepared by the following method: 50mL of 20 XSH-Asalt, 50mL of 20 XSM-Cvitamin, 10.0g Sucrose,3mL 1mol/L MES, 0.8wt.% final concentration of agar powder, ultrapure water to 1L, KOH adjusted pH to 5.8, and sterilization at 115℃for 30min.
The 20 XSH-A salt is prepared by the following method: 8.0g MgSO 4 ·7H 2 O，6.0g NH 4 H 2 PO 4 ，4.0gCaCl 2 ·2H 2 O，0.2g MnSO 4 ·4H 2 O，0.02g ZnSO 4 ·7H 2 O，0.004g CuSO 4 ·5H 2 O，0.002gNaMoO 4 ·2H 2 O，0.002g CoCl·6H 2 O，0.02g KI，0.1g H 3 BO 3 ，50.0g KNO 3 ，0.3gFeSO 4 ·7H 2 O，0.4g Na 2 EDTA, ultrapure water to 1L, and preserving at-20 ℃ for standby.
The 20 XUM-C vitamins are prepared by the following method: 2.0g Myoinositol,0.1g Nicotinic acid,0.2g Vitamin B6,0.2g Thiamine HCl,0.04g Glycine the ultrapure water is kept for standby after the volume is fixed to 1L and the temperature is minus 20 ℃.
1mol/L MES is prepared by the following steps: 4.88g MES, ultrapure water to 25mL, pH to 5.8, filtration sterilization and preservation at-20 ℃ for standby.
100mmol/L acetosyringone is prepared by the following method: 196.2mg acetosyringone, dimethyl sulfoxide to 10mL, filtering, sterilizing, and preserving at-20deg.C.
(3) And (3) identifying hair roots: under Nikon SMZ18 stereoscopic fluorescence microscope, the rooting plate was placed under a black bottom plate, positive seedlings (denoted LjoP 3M-KO) were selected through a fluorescence baffle, and non-positive roots were removed, wherein positive roots showed green fluorescence.
5. Transplanting seedlings and inoculating rhizobia
Wetting vermiculite, sterilizing at 121deg.C for 30min, cooling to room temperature, transferring the identified positive seedlings into soil, covering with transparent plastic cover, and culturing under illumination in a greenhouse. After 7d of culture, rhizobia was inoculated. Shake culturing rhizobia Mesorhizobium lotiMAFF303099/pHC60 with TY liquid medium at 28deg.C until OD 600 The cells were collected by centrifugation at 5000r/m for 5min and resuspended in sterile water to an OD 600 =0.04, inoculated on roots of the Baimaigen seedlings, each at an inoculum size of 10mL. Meanwhile, a wild type control group is set, and the difference is that no engineering bacteria are inoculated.
The TY culture medium is prepared by the following method: 3g yeast extract, 5g tryptone, 0.69g CaCl 2 pH was adjusted to 7.0, miliQ-H 2 And (3) sterilizing for 30min at 121 ℃ with constant volume of O to 1L.
Mesorhizobium lotiMAFF303099/pHC60 is disclosed in [ FengY, wuP, liu C, peng L, wang T, wang C, tan Q, li B, ou Y, zhu H, yuan S, huang R, stacey G, zhang Z, cao Y.support of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonica molecular plant,2021,14:1935-1950 ].
6. Counting the number of root nodules
After inoculation of rhizobia 14d and 21d, plants were excavated from vermiculite, roots of the plants were washed, numbers of nodules on wild type (EV) and positive roots were counted under Nikon SMZ18 stereoscopic fluorescent microscope, respectively, and the plants and nodules after inoculation of rhizobia 21d were photographed. The results are shown in fig. 1, fig. 2, fig. 3, table 1 and table 2.
Table 1 number of nodules on positive roots inoculated with rhizobia 14d (units: one)
Table 2 number of nodules on positive roots inoculated with rhizobia 21d (units: one)
As can be seen from fig. 1, 2, table 1 and 2, the number of nodules of the ljop 3M knockout mutant plants was significantly increased after inoculation with rhizobia 14d and 21d, compared to the control.
Example 2:
effect of Baimai root LjAP3M overexpression on root nodule number
1. Seed germination is the same as in step 4 of example 1;
2. obtaining the root cDNA of Baimai root
Collecting a sample of root and rhizomes inoculated with rhizobia Mesorhizobium loti MAFF303099/pHC6015d, extracting RNA, and reverse transcribing into cDNA; the inoculation method is the same as in step 4 of the example.
The RNA extraction method comprises the following steps: total RNA from the roots and nodules of Baimai was extracted using an RNA extraction kit (available from Aidlab under the accession number RN 3302). Specific methods refer to the kit operating manual. After elution of RNA, the concentration and purity of RNA was checked using NanoDrop 2000 (available from Thermo) and samples were stored at-80 ℃.
The reverse transcription method comprises the following steps: RNA was reverse transcribed into cDNA using a reverse transcription kit (available from ABcloanl under the trade designation RK 20403). Specific methods refer to the kit operating manual.
3. Vector construction
(1) Fragment amplification: designing a primer according to LjAP3M full-length cDNA, and amplifying by taking the cDNA as a template to obtain an LjAP3M fragment, wherein the primer is as follows:
LjAP3M-cDNA-GF：5’-TGATGTGATTACAGTCTAGAATGTTGCAGTGCAT ATTTCTT-3’，SEQ ID NO.12；
LjAP3M-cDNA-GR：5’-TGGTCCTTATAGTCGGTACCTGACCTGACTTCAA ATTGGCC-3’，SEQ ID NO.13；
the reaction system of the amplification chip is as follows: 2X Phanta Max buffer. Mu.L, dNTP Mix 1.5. Mu.L, phanta Max SUper-Fidelity DNA polymerase 1.5.5. Mu.L, forward primer 1.5. Mu.L, reverse primer 1.5. Mu.L, template cDNA 1. Mu.L and sterile water 18. Mu.L; the concentration of dNTP Mix is 10mM each; the concentration of the forward primer and the reverse primer is 100 mu M; the reaction procedure for the amplification was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 90s for 30 cycles;
(2) And (3) carrying out enzyme cutting and connection of a carrier: double-enzyme digestion is carried out on the pUB-GFP C-3xFLAG vector by restriction enzymes KpnI and XbaI to obtain a large fragment vector; connecting the large fragment vector with the LjoP 3M fragment through Gibson reaction to obtain a recombinant vector; the double cleavage and Gibson reactions are described in reference to example 1;
the pUB-GFP C-3xFLAG vector is disclosed in [ Yu, H., et al, suppression ofinnate immunity mediated by the CDPK-Rboh complex is required for rhizobial colonization in Medicago truncatula nodules.New Phytol,2018.220 (2): p.425-434 ]).
4. Transforming agrobacterium: and transforming the obtained recombinant vector into agrobacterium rhizogenes Agrobacterium Rhizogenes LBA1334 to obtain the over-expression engineering bacteria.
5. The process for conversion of the root of Baimai root hair is described in example 1. Wherein positive seedlings were noted as LjAP3M-OX.
6. The method of transplanting seedlings and inoculating rhizobia is described in example 1. Meanwhile, a wild type control group is arranged, and the difference is that the super-expression engineering bacteria are not inoculated.
7. Counting the number of root nodules.
After inoculation of rhizobia 14d and 21d, plants were excavated from vermiculite, roots of the plants were washed, the numbers of nodules on wild type (EV) and positive roots were counted under Nikon SMZ18 stereoscopic fluorescent microscope, and the plants and nodules after inoculation of rhizobia 21d were photographed. The results are shown in fig. 4, 5, 6, table 3 and table 4.
Table 3 number of nodules on positive roots inoculated with rhizobia 14d (units: one)
Table 4 number of nodules on positive roots inoculated with rhizobia 21d (units: one)
As can be seen from fig. 4, 5, table 3 and table 4, the number of ljop 3M overexpressing plant nodules was significantly reduced after inoculation with rhizobia 14d and 21d compared to the control.
Example 3
The genome of the transgenic positive roots inoculated with rhizobia 14d in example 1 was extracted and subjected to PCR amplification with the following primer sequences:
F：5’-ATGTTGCAGTGCATATTTCTTCTC-3’，SEQ ID NO.14；
R：5’-CATAAACTTCATTGCTAGCATACT-3’，SEQ ID NO.15；
the PCR products were sequenced and compared to the sequencing results of wild type plants to identify changes in the DNA sequence at the target position, as shown in FIG. 7.
From FIG. 7, 3 sgRNAs were able to target the LjoP 3M gene for knockout.
Example 4
After the positive seedlings in the example 1 and the example 2 are inoculated with rhizobia for 21d, respectively taking the underground part tissues of the plants to extract RNA, and reversely transcribing the RNA into cDNA, wherein the RNA extraction method comprises the following steps: extracting total RNA of the roots and nodules of the Baimai roots by using an RNA extraction kit (purchased from Aidlab and with the product number RN 3302), wherein the specific method refers to a kit operation manual; after elution of RNA, the concentration and purity of RNA was checked using NanoDrop 2000 (available from Thermo) and samples were stored at-80 ℃. The reverse transcription method comprises the following steps: RNA was reverse transcribed into cDNA using a reverse transcription kit (available from ABcloanl under the trade designation RK 20403), see the kit operating manual for specific procedures.
After reverse transcription into cDNA, the transcription level of LjoP 3M was detected by fluorescent quantitative PCR using the 2X Universal SYBR Green Fast qPCR Mix kit from ABclonal corporation. PCR reaction procedure using a Applied Biosystems ViiATM Real-Time PCR System instrument from ABI, USA: 50 ℃ for 2min;95 ℃ for 10min; 15s at 95℃and 1min at 60℃for 40 cycles. The relative quantification ΔΔct method was used to normalize gene expression levels with the use of the hundred vein root ATPase (Genbank ID: AW 719841) as an internal reference;
the primers for amplifying the LjoP 3M gene and the ATPase in the fluorescent quantitative PCR are respectively as follows:
LjAP3M-F：5’-TGTTGTGACTGGCAGAAGCT-3’，SEQ ID NO.16；
LjAP3M-R：5’-AACCTCACCGCTGATCTCAC-3’，SEQ ID NO.17；
ATPase-F：5’-CAATGTCGCCAAGGCCCATGGTG-3’，SEQ ID NO.18；
ATPase-R：5’-AACACCACTCTCGATCATTTCTCTG-3’，SEQ ID NO.19。
the results are shown in fig. 8 and 9, and the result shows that the expression amount of LjAP3M in the LjAP3M-KO plant is significantly different from that of the wild type; the expression level of LjAP3M in LjAP3M-OX plants was significantly different from that of the wild type, and was up-regulated 50-fold. This result shows that both knockout and overexpressing plants significantly affected the expression of gene LjoP 3M.
In conclusion, the Baimaigen LjoP 3M gene has important functions in regulating and controlling nitrogen fixation efficiency. The LjAP3M gene is knocked out to obviously increase the number of root nodules, and the overexpression of the LjAP3M gene obviously reduces the number of root nodules, so that the LjAP3M gene has important significance in the aspect of improving the nitrogen fixation efficiency of the hundred-vein roots.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
- The application of LjAP3M protein in regulating nitrogen fixation efficiency of plant root nodule is disclosed, wherein the amino acid sequence of the LjAP3M protein is shown as SEQ ID NO. 1.
- 2. The use of claim 1, wherein said modulating plant nodule nitrogen fixation efficiency comprises: regulating and controlling the number of plant root nodules.
- 3. The use according to claim 1 or 2, wherein said modulating plant nodule nitrogen fixation efficiency comprises: the number of plant nodules is increased by negatively regulating the expression of the LjAP3M gene or decreased by positively regulating the expression of the LjAP3M gene.
- 4. The use according to claim 1 or 2, characterized in that the gene encoding the ljop 3M protein is the ljop 3M gene; the nucleotide sequence of the LjAP3M gene is shown as SEQ ID NO. 2.
- Application of LjAP3M protein in culturing transgenic plants with improved nitrogen fixation efficiency, wherein the amino acid sequence of the LjAP3M protein is shown as SEQ ID NO. 1.
- 6. The use of claim 5, wherein the nitrogen fixation efficiency improvement comprises: the number of plant root nodules is increased.
- 7. The use according to claim 5, wherein the plant comprises a leguminous plant.
- 8. The use according to claim 7, wherein the leguminous plants comprise one or more of soybean, alfalfa and centella asiatica.
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