CN116606361A - LjPRP1 protein for regulating and controlling nitrogen fixation efficiency of plant root nodule and/or regulating and controlling plant yield and application thereof - Google Patents
LjPRP1 protein for regulating and controlling nitrogen fixation efficiency of plant root nodule and/or regulating and controlling plant yield and application thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- 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
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Abstract
The invention relates to the technical field of genetic engineering, in particular to LjPRP1 protein for regulating and controlling nitrogen fixation efficiency of plant root nodules and/or regulating and controlling plant yield and application thereof. Compared with a wild type, the activity of the LjPRP1 gene mutant plant nodule nitrogen fixation enzyme is obviously improved through experiments, which shows that the LjPRP1 protein coded by the LjPRP1 gene regulates the nitrogen fixation efficiency of the nodule; on the basis, the invention uses stable transformation technology to overexpress LjPRP1 gene, and obtains the overexpressed material, compared with wild type, the activity of the overexpressed material root nodule nitrogen fixation enzyme is obviously reduced, which indicates that the LjPRP1 gene and the LjPRP1 protein expressed by the gene regulate the nitrogen fixation efficiency of the root nodule, inhibit the expression of the gene, and can improve the nitrogen fixation efficiency of leguminous plants, thereby increasing the biomass and yield of plants, and having application prospect in the research of nitrogen fixation mechanism of leguminous crops and agricultural production.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to LjPRP1 protein for regulating and controlling nitrogen fixation efficiency of plant root nodules and/or regulating and controlling plant yield and application thereof.
Background
Nitrogen is one of the essential elements for plant growth and development. In conventional agriculture, the yield of plants can be effectively increased by applying industrial nitrogenous fertilizer. However, industrial nitrogen fixation effectively solves the limitation of nitrogen fertilizer in agricultural production, promotes the first agricultural green revolution, but causes a series of environmental pollution problems. The plant has low utilization efficiency of nitrogen fertilizer, and excessive applied nitrogen fertilizer can cause a large amount of unutilized nitrogen fertilizer to flow into rivers, lakes and seas along with the flushing of rainwater, so that serious nitrogen pollution is caused. Meanwhile, a large amount of industrial pollutants are generated in the process of industrially producing the nitrogen fertilizer, so that the earth ecological environment is seriously polluted. Therefore, how to reduce the application of chemical fertilizers, and not affect or even improve the yield of crops is an important direction of genetic improvement of crops.
In nature, some soil microorganisms are capable of forming nodules with legumes, converting nitrogen in the air to ammonia at normal temperature and pressure, a process known as biological nitrogen fixation. Symbiotic nitrogen fixation between leguminous plants and rhizobium is a high-efficiency biological nitrogen fixation system, provides sufficient nitrogen sources for leguminous plants, and is the most important green nitrogen source in natural soil. The lotus root (lotus japonica) is one of leguminous mode plants, improves nitrogen fixation efficiency of the lotus root, and is beneficial to genetic improvement of leguminous crops suitable for cultivation in various places throughout the country, such as grains, oil crop soybeans, peanuts, red beans, mung beans and the like; grass crops such as purple flower head Beibei, inclined stem yellow stripe and the like; green manure such as milk vetch, moss and the like; herb of licorice, yellow stripe, etc.; radix Saposhnikoviae and sand fixation plant and Leguminosae tree. Therefore, the nitrogen fixation efficiency of the crop root nodule is improved, the method has important scientific significance, and important guarantee can be provided for human improvement of ecological environment and realization of agricultural sustainable development.
There are many reports about the PRP1 gene in improving the salt tolerance of rice, but there are no reports about the LjPRP1 protein in regulating nitrogen fixation and/or regulating yield of plant root nodule in Baimai root.
Disclosure of Invention
In order to solve the problems, the invention provides an LjPRP1 protein for regulating and controlling nitrogen fixation efficiency of plant root nodule and/or regulating and controlling plant yield and application thereof. The LjPRP1 protein can regulate and control the nitrogen fixation efficiency of plant root nodules and/or regulate and control the plant yield.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides LjPRP1 protein for regulating and controlling nitrogen fixation efficiency of plant root nodule and/or regulating and controlling plant yield, wherein the amino acid sequence of the LjPRP1 protein is shown as SEQ ID NO. 1.
The invention also provides an LjPRP1 gene for encoding the LjPRP1 protein in the technical scheme, and the nucleotide sequence of the LjPRP1 gene is shown as SEQ ID NO. 2.
The invention also provides the LjPRP1 protein of the technical scheme or the application of the LjPRP1 gene of the technical scheme in regulating and controlling the nitrogen fixation efficiency of plant root nodules and/or regulating and controlling the plant yield.
Preferably, the regulating and controlling the nitrogen fixation efficiency of the plant root nodule comprises: regulating plant nodule development and/or regulating nitrogen fixation enzyme activity in plant nodule.
Preferably, the regulating and controlling the nitrogen fixation efficiency of the plant root nodule comprises: the nitrogen fixation efficiency of plant root nodules is improved by negatively regulating the expression of the LjPRP1 gene, or the nitrogen fixation efficiency of plant root nodules is reduced by positively regulating the expression of the LjPRP1 gene.
Preferably, the regulating plant yield comprises: plant yield is increased by negatively regulating expression of the LjPRP1 gene, or plant yield is decreased by positively regulating expression of the LjPRP1 gene.
The invention also provides application of the LjPRP1 protein in the technical scheme or the LjPRP1 gene in cultivation of transgenic plants with improved nitrogen fixation efficiency and/or yield.
Preferably, the nitrogen fixation efficiency improvement includes: promoting plant nodule development and/or increasing the activity of nitrogen fixation enzymes in plant nodules.
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 LjPRP1 protein for regulating and controlling nitrogen fixation efficiency of plant root nodule and/or regulating and controlling plant yield, wherein the amino acid sequence of the LjPRP1 protein is shown as SEQ ID NO. 1. The invention proves that the expression of the LjPRP1 gene is hardly detected in the plant of the mutant of the Baimaigen LjPRP1 gene, compared with a wild type, the activity of the nitrogen fixation enzyme of the plant root nodule of the LjPRP1 gene mutant is obviously improved, which proves that the LjPRP1 protein coded by the LjPRP1 gene regulates the nitrogen fixation efficiency of the root nodule, and indicates that the LjPRP1 protein has an important function in regulating the nitrogen fixation efficiency of the plant root nodule; on the basis, the invention uses stable transformation technology to overexpress LjPRP1 gene, and obtains the overexpressed material, compared with wild type, the activity of the overexpressed material root nodule nitrogen fixation enzyme is obviously reduced, which indicates that the LjPRP1 gene and the LjPRP1 protein expressed by the gene regulate the nitrogen fixation efficiency of the root nodule, inhibit the expression of the gene, and can improve the nitrogen fixation efficiency of leguminous plants, thereby increasing the biomass and yield of plants, and having application prospect in the research of nitrogen fixation mechanism of leguminous crops and agricultural production.
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 shows the relative expression level results of LjPRP1 mutant plants and wild type LjPRP1 genes;
FIG. 2 is a graph of the number of lines of infection for LjPRP1 mutant plants and wild type;
FIG. 3 is a graph of ethylene standard curve;
FIG. 4 is a graph showing the activity of LjPRP1 mutant plant individual seedling nitrogen fixation enzyme;
FIG. 5 is a graph showing the number of lines of infection of LjPRP1 overexpressing plants and wild-type;
FIG. 6 is a graph showing the activity of LjPRP1 overexpressing plant individual seedling azotase;
FIG. 7 is a paraffin section of plant nodule wherein A is a section of wild type and mutant nodules and B is a section of wild type and overexpressed plant nodules;
wherein Ljprp1-1 is a mutant plant; ljPRP1-OX-7 is an over-expression plant;
* Represents a direct differential comparison (t-test) between WT and mutant or overexpressing plants, where x represents significant levels 0.01 < p.ltoreq.0.05; * At significance levels 0.001 < p.ltoreq.0.01, the difference significance was between significant and very significant.
Detailed Description
The invention provides an LjPRP1 protein for regulating and controlling nitrogen fixation efficiency of plant root nodule and/or regulating and controlling plant yield, wherein the amino acid sequence of the LjPRP1 protein is shown as SEQ ID NO.1, and the LjPRP1 protein is specifically as follows: MASQVASVEEVAGDKYRSFIHEEADTTHWRHGGPPTYDVVNHLFEQGRTKEWPKGSLEETVQNAIKSWEMEVSHKTRLQDFRTINPEKFKLFVNGREGLSAEETLSIGSYNALLKSSLPEEFKYYKSEEETFESSHEAFRSAFPRGFAWEVIKVYTGPPEIAYKFRHWGFFEGPFKGHAPTGKMVEFYGLGTLKVDNARKGEEVEIYYDPEEWRGDLLPASGTATEDPTKTTPTSQACPFSK.
The invention proves that the LjPRP1 gene and the LjPRP1 protein expressed by the LjPRP1 gene regulate and control the nitrogen fixation efficiency of root nodules by inhibiting the expression of the LjPRP1 protein coding gene and promoting the expression of the LjPRP1 protein coding gene, and can improve the nitrogen fixation efficiency of leguminous plants so as to increase the biomass and the yield of the plants.
The invention also provides an LjPRP1 gene for encoding the LjPRP1 protein in the technical scheme, wherein the nucleotide sequence of the LjPRP1 gene is shown as SEQ ID NO.2, and the LjPRP1 gene is specifically as follows: 5'-ATGGCCAGCCAAGTAGCCTCCGTTGAAGAAGTTGCAGGAGACAAATAC AGATCTTTCATTCATGAAGAGGCTGACACAACCCACTGGAGACATGGTGGACCACCCACCTATGATGTTGTGAACCACCTTTTTGAACAAGGTCGAACCAAGGAATGGCCTAAAGGTTCATTAGAGGAGACAGTGCAAAACGCCATAAAATCATGGGAGATGGAGGTTTCTCACAAAACCCGCTTGCAGGATTTCAGAACCATAAATCCTGAAAAGTTCAAGCTCTTTGTTAATGGGAGGGAGGGGTTATCTGCAGAGGAAACTCTGAGCATAGGAAGTTATAATGCTTTGCTAAAAAGCTCTCTACCAGAAGAATTCAAGTATTACAAATCTGAAGAAGAGACTTTTGAATCATCTCATGAAGCTTTCAGATCAGCTTTTCCTCGTGGATTTGCATGGGAAGTGATCAAAGTTTATACCGGACCCCCTGAAATTGCTTACAAGTTTAGGCACTGGGGATTCTTTGAAGGTCCTTTCAAGGGACATGCCCCTACTGGGAAGATGGTTGAGTTCTATGGCTTGGGAACTCTCAAGGTTGACAACGCGCGGAAAGGGGAGGAGGTGGAGATCTACTACGACCCAGAAGAGTGGCGGGGTGATCTTCTACCTGCGAGTGGGACCGCCACAGAGGACCCCACTAAAACTACACCAACTTCTCAAGCATGTCCTTTCTCCAAATAA-3'.
The invention also provides the LjPRP1 protein of the technical scheme or the application of the LjPRP1 gene of the technical scheme in regulating and controlling the nitrogen fixation efficiency of plant root nodules and/or regulating and controlling the plant yield.
In the present invention, the regulation of nitrogen fixation efficiency of plant nodules preferably comprises: regulating plant nodule development and/or regulating nitrogen fixation enzyme activity in plant nodule; the modulating plant nodule development preferably comprises modulating the formation of an infection line and/or the formation of an infection cell to modulate plant nodule development.
In the present invention, the regulation of nitrogen fixation efficiency of plant nodules preferably comprises: the nitrogen fixation efficiency of plant root nodules is improved by negatively regulating the expression of the LjPRP1 gene, or the nitrogen fixation efficiency of the plant root nodules is reduced by positively regulating the expression of the LjPRP1 gene; more preferably comprises promoting plant nodule development and/or enhancing nitrogen fixation enzyme activity in plant nodules by negatively regulating expression of the LjPRP1 gene, or inhibiting plant nodule development and/or inhibiting nitrogen fixation enzyme activity in plant nodules by positively regulating expression of the LjPRP1 gene.
In the present invention, the controlling plant yield preferably comprises: plant yield is increased by negatively regulating expression of the LjPRP1 gene, or plant yield is decreased by positively regulating expression of the LjPRP1 gene.
In the present invention, the means of negative regulation preferably includes a mutation of the LjPRP1 gene.
In the present invention, the means of upregulation preferably includes overexpression of the LjPRP1 gene.
The invention also provides application of the LjPRP1 protein in the technical scheme or the LjPRP1 gene in cultivation of transgenic plants with improved nitrogen fixation efficiency and/or yield.
In the present invention, the nitrogen fixation efficiency improvement preferably includes: promoting plant nodule development and/or increasing the activity of nitrogen fixation enzymes in plant nodules. According to the invention, through negative regulation of the expression of the LjPRP1 gene, the plant nodule growth can be promoted, and the activity of the nitrogen fixation enzyme in the plant nodule can be improved, so that the nitrogen fixation efficiency of the nodule can be improved, more nitrogen can be fixed by the nodule to the plant, and the effect of improving the yield can be achieved.
In the present invention, the yield improvement preferably includes increasing the biomass and yield of the plant by increasing the nitrogen fixation efficiency of the plant.
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 following describes in detail an LjPRP1 protein for regulating nitrogen fixation efficiency of plant nodule and/or regulating plant yield and its application, with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Expression level detection of mutant of Baimaigen LjPRP1 gene
1. Seed germination
The mutant seed Ljprp1-1 of the Baimaigen LjPRP1 gene was purchased from Lotus Base (https:// lotus.au. Dk/lore 1/search), plantID:30037066 the insertion site of LORE1 of this mutant seed is on the first exon of LjPRP1, 168bp after the initiation codon ATG, and the LjPRP1 gene is numbered Lj0g3v0096089.1 in the Bai Mai Gen genome database miyakogusa. After purchase of seeds according to the invention, heterozygous and homozygous mutants were identified by PCR, and subsequent experiments were performed using the homozygous mutants, and wild type seeds of Baimaigen were disclosed in [ FengY, wuP, liuC, pengL, wangT, wangC, tanQ, liB, ouY, zhuH, yuanS, huangR, staceyG, zhangZ, caoY.SuppressionofLjBAK1-media imformation by SymRKpro-tes rhizobial fectionlotus plant,2021,14:1935-1950 ]. Germination is carried out on the mutant seeds LjPRP1-1 of the Baimaigen and the wild seeds of the Baimaigen respectively, and the specific method is as follows:
adding 20mL of concentrated sulfuric acid into a 100mL sterile triangular flask, pouring the seeds, gently shaking to enable the seeds to be fully contacted with the concentrated sulfuric acid, treating for 10min, enabling the color of the seeds to be slightly light, and concentrating H 2 SO 4 The color turns yellow. Carefully draw concentrated sulfuric acid into glassware with a small amount of water, add a large amount of MiliQ-H to a triangular flask 2 O quickly rinses the seeds and discards the waste liquid. Further using a small amount of MiliQ-H 2 O was repeatedly rinsed 4 times. Adding 2% sodium hypochlorite solution to cover the seeds, slightly shaking, and treating for 10min, wherein the transparent small ring at the umbilicus of the seeds is dropped, individual seeds are expanded, and the liquid is slightly pale yellow. Discard waste solution, use MiliQ-H 2 O rinsing the seeds, and repeating the rinsing for 4 times. With 20mL MiliQ-H 2 O submerges the seeds and vernalizes at 4℃for 1d. Transferring the imbibed seeds to an MS culture medium, and culturing in a dark condition at 23 ℃ for 2d in an illumination incubator for 4d to obtain germinated seedlings.
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.
2. Transplanting seedlings and inoculating rhizobia
Wetting vermiculite, sterilizing at 121 ℃ for 30min, cooling to room temperature, moving the germinated seedlings in the step 1 into soil, covering a transparent plastic cover, and culturing in a greenhouse under illumination.
After 7d of incubation, the cotyledons were fully expanded. Shake culturing rhizobia Mesorhizobium loti MAFF303099 with TY liquid culture medium at 28deg.C until OD 600 Centrifuging at 4000r/min for 5min at a value of 0.8 to collect bacterial cells, and collecting bacterial cells with B&D Nitrogen-free Medium resuspended and diluted to OD 600 =0.01, inoculated on roots of Baimaigen seedlings, each inoculum size being 3ml.
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.
B&D Nitrogen-free liquid culture Medium consists of the following group concentrationsThe method comprises the following steps: 1mmol/L CaCl 2 、0.5mmol/L KH 2 PO 4 、10μmol/L Fe(C 6 H 5 O 7 )、0.25mmol/L MgSO 4 、0.25mmol/L K 2 SO 4 、1.0μmol/L MnSO 4 、2.0μmol/L H 3 BO 3 、0.5μmol/L ZnSO 4 、0.2μmol/L CuSO 4 、0.1μmol/L CoSO 4 And 0.1. Mu. Mol/L Na 2 MoO 4 The method comprises the steps of carrying out a first treatment on the surface of the Sterilizing at 121deg.C for 30min.
Mesorhizobium loti MAFF303099 are disclosed in [ Feng Y, wu P, 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 ].
After 21d inoculation of rhizobia, extracting RNA from the underground part tissue of the plant, and carrying out reverse transcription to obtain cDNA, wherein the RNA extraction method comprises the following steps: extracting total RNA of the hundred vein root nodules by using an RNA extraction kit (Aidlab, RN 3302), wherein the specific method is referred to a kit operation manual; after RNA elution, the concentration and purity of RNA was checked using NanoDrop2000 (Thermo), and the samples were stored at-80 ℃. The reverse transcription method comprises the following steps: RNA was reverse transcribed into cDNA using a reverse transcription kit (ABcloanl, RK 20403), see the kit operating manual for specific methods.
After reverse transcription to cDNA, the transcript level of LjPRP1 was detected by fluorescent quantitative PCR using the SYBR Green qPCR Mix kit of monatin organism. PCR reaction procedure using a Applied Biosystems ViiATM Real-Time PCR System instrument from ABI, USA: 50 ℃ for 2min;95 ℃ for 10min; 15sec 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 dipyridyl ubaquitin (Genbank ID: AW 720576) and ATPase (Genbank ID: AW 719841) as internal controls;
the primers for amplifying the LjPRP1 gene, the Ubiquitin and the ATPase in the fluorescent quantitative PCR are respectively as follows:
LjPRP1-F：5’-GCCAGCCAAGTAGCCTCCGTTG-3’，SEQ ID NO.3；
LjPRP1-R：5’-TGCACTGTCTCCTCTAATGAACC-3’，SEQ ID NO.4；
Ubiquitin-F：5’-TTCACCTTGTGCTCCGTCTTC-3’，SEQ ID NO.5；
Ubiquitin-R：5’-AACAACCAGCACACACAGACAATC-3’，SEQ ID NO.6；
ATPase-F：5’-CAATGTCGCCAAGGCCCATGGTG-3’，SEQ ID NO.7；
ATPase-R：5’-AACACCACTCTCGATCATTTCTCTG-3’，SEQ ID NO.8。
as a result, it was found that expression of LjPRP1 was hardly detected in the LjPRP1-1 homozygous mutant plant relative to the wild type plant (FIG. 1). This result indicates that insertion of the LORE1 in the mutant seeds of the Baicalein LjPRP1 gene caused deletion mutation of the LjPRP1 gene, resulting in incapacity of expression of the LjPRP1 gene.
Example 2
Effect of mutation of Baimaigen LjPRP1 Gene on the number of infection lines
1. Seed germination was as in example 1.
2. Transplanting seedlings and inoculating rhizobia
Wetting vermiculite, sterilizing at 121 ℃ for 30min, cooling to room temperature, moving the germinated seedlings in the step 1 into soil, covering a transparent plastic cover, and culturing in a greenhouse under illumination.
After 7d of incubation, the cotyledons were fully expanded. Shake-culturing rhizobium mesorhizobium strain MAFF303099/pHC60 with TY liquid medium at 28deg.C until OD 600 Centrifuging at 4000r/min for 5min at a value of 0.8 to collect bacterial cells, and collecting bacterial cells with B&D Nitrogen-free Medium resuspended and diluted to OD 600 =0.01, inoculated on roots of Baimaigen seedlings, each inoculum size being 3ml.
TY medium: 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.
B&The nitrogen-free liquid culture medium consists of the following components in concentration: 1mmol/LCaCl 2 ，0.5mmol/LKH 2 PO 4 ，10μmol/LFe(C 6 H 5 O 7 )，0.25mmol/LMgSO 4 ，0.25mmol/LK 2 SO 4 ，1.0μmol/LMnSO 4 ，2.0μmol/LH 3 BO 3 ，0.5μmol/LZnSO 4 ，0.2μmol/LCuSO 4 ，0.1μmol/LCoSO 4 And 0.1. Mu. Mol/LNa 2 MoO 4 Sterilizing at 121deg.C for 30min.
Mesorhizobium longitumaff 303099/pHC60 is derived from the literature [ FengY, wuP, liuC, pengL, wangT, wangC, tanQ, liB, ouY, zhuH, yuanS, huangR, staceyG, zhangZ, caoY.SuppressionofLjBAK1-mediaedImmunity symRKprotein rhizobiaalfectionlotus japonica, molecular plant,2021,14:1935-1950 ], namely "M.lotitumaff303099 expressingGFP" in the literature.
3. Counting the number of infection lines
After rhizobium inoculation for 5d, digging out plants from vermiculite, cleaning roots of the plants, and counting the numbers of infection lines of Wild Type (WT) and LjPRP1 mutant plants respectively under a NikonSMZ18 fluorescent microscope, wherein the numbers of the wild type and the LjPRP1 mutant plants are 15. The results are shown in FIG. 2 and Table 1.
TABLE 1 number of lines of infection for wild-type and LjPRP1 mutant plants
From fig. 2 and table 1, it can be seen that the line of infection of LjPRP1 mutant plants is significantly increased compared to the wild type, and that negative regulation of LjPRP1 gene expression will promote the formation of the line of infection.
Example 3
Effect of mutation of the Baimaigen LjPRP1 Gene on the Activity of Nitrogen fixing enzyme
1. Seed germination was as in example 1.
2. Transplanting seedlings and inoculating rhizobia were as in example 1.
3. Determination of the activity of the Baimai root nodule azotase.
After 3 weeks of rhizobia inoculation, plants are dug out from vermiculite, roots of the plants are cleaned, the underground parts of the Baimai roots are placed into a 40mL glass bottle, 3mL of air is firstly pumped out of the glass bottle by using a syringe, and then 2mL of acetylene gas is injected into the glass bottle by using the syringe. The glass vials were placed in a plastic box containing water, and after 2 hours of reaction at 28℃were removed and examined by gas chromatography, wherein the number of wild-type and LjPRP1 mutant plants was 24.
4. Ethylene standard curve production
A glass bottle was filled with a volume of ethylene standard gas using a microsyringe, 3 replicates were set for each concentration, and the ethylene peak was detected using a gas chromatograph. The standard curve of ethylene volume versus ethylene peak area is plotted on an excel table. The specific method comprises the following steps:
10 volume gradients of ethylene gas (4. Mu.L, 6. Mu.L, 8. Mu.L, 10. Mu.L, 20. Mu.L, 40. Mu.L, 50. Mu.L, 100. Mu.L, 200. Mu.L, 500. Mu.L) were selected and injected into 10 equally sized rubber-capped glass vials, respectively, using a microsyringe. After the mixture is placed for a period of time and completely mixed, 100 mu L of gas is respectively extracted from 10 glass bottles by a microsyringe and is injected into a meteorological chromatograph, and the ethylene chromatographic peak area value under each volume gradient is recorded, wherein the selection of the ethylene volume gradient is determined according to the ethylene reduction capability of the root nodule sample. And drawing a standard curve by taking the volume gradient of ethylene as an abscissa and taking the recorded chromatographic peak area value of ethylene as an ordinate. If the volumetric differences between the sample glass vials and the operational errors are ignored, the ethylene volume in the vial should be linearly related to the measured ethylene chromatographic peak area over a range of conditions. The ethylene standard curve determined according to the invention is y=2362.5x+5600 (R 2 =0.9993), see fig. 3. Wherein R is 2 Representing the linear regression determinant, reflects the correlation between the two variables. It is generally considered that R 2 The closer the value is to 1, the stronger the correlation and the more accurate the analysis of the results. R of the invention 2 The value reaches 0.9993, which completely accords with the description of linear regression, so that the following determination of the activity of the root nodule azotase can be performed by referring to the ethylene standard curve.
5. Calculation of the activity of the Nitrogen fixing enzyme
The activity of the root nodule nitrogen fixation enzyme is acetylene reduction activity (Acetylene Reduction Activity, ARA), which can be expressed as the mole number of the root nodule reducing acetylene in unit time, and the calculation formula is as follows: acetylene reduction activity = moles of ethylene/reaction time (unit of enzyme activity is typically nmol c 2 H 4 /h/plant)。
According to the relation between the mole number of the gas and the volume, temperature and pressure, the mole number can be obtained by the volume of ethylene:
C 2 H 4 (μmol)＝C 2 H 4 volume (. Mu.L). Times.1/22.4X1273/(273+t). Times.P/760;
wherein: t DEG C: reaction temperature (temperature of celsius, 28), P: the air pressure, typically 760 mmHg, 22.4: the volume of 1mol of gas in the standard state is 22.4 liters; 273: absolute temperature.
The results are shown in FIG. 4 and Table 2.
TABLE 2 Azadirachta Activity of wild-type and LjPRP1 mutant plants
From fig. 4 and table 2, compared with the wild type, the activity of the azotase of the single seedling of the LjPRP1 mutant is significantly increased, which indicates that the mutation of the LjPRP1 (i.e., the negative regulation of the expression of the LjPRP1 gene) improves the nitrogen fixation efficiency of the plant.
Example 4
Effect of LjPRP1 overexpression on the number of lines of infection
1. Obtaining Baimai root nodule cDNA
In example 3, samples of nodules were harvested 3030993 weeks after inoculation with rhizobium MAFF, RNA was extracted and reverse transcribed into cDNA.
The RNA extraction method and the reverse transcription method were the same as in example 1.
2. Vector construction
Designing a primer according to the full-length cDNA of LjPRP1, and respectively amplifying a full-length cDNA fragment of LjPRP1 by using the primers LjPRP1-cDNA-F and LjPRP1-cDNA-R by using the hundred-vein root nodule cDNA as a template; and then using the full-length cDNA fragment of LjPRP1 as a template, and carrying out second round amplification by using the primers LjPRP1-pub-F and LjPRP1-pub-R to obtain the final LjPRP1 fragment.
Amplifying the corresponding gene. The primers were as follows:
LjPRP1-cDNA-F:5’-ATGGCCAGCCAAGTAGCCTCCGTTG-3’，SEQ ID NO.9；
LjPRP1-cDNA-R:5’-TTTGGAGAAAGGACATGCTTGAGAA-3’，SEQ ID NO.10；
LjPRP1-pub-F:5’-TCTAGACTGTAATCACATCAAATGGCCAGCCAAGTAG CCTCCGTTGA-3’，SEQ ID NO.11；
LjPRP1-pub-R:5’-ACCGGATCCACTAGTAGGCCTTTTGGAGAAAGGACA TGCTTGAGAAG-3’，SEQ ID NO.12；
both PCR amplifications were usedMax Super-Fidelity DNA Polymerase (Vazyme), both PCR amplification systems were (20. Mu.L): 2X Phanta Max Buffer mu L, dNTP (10 mM 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 MiliQ-H 2 O 7μL；
The two PCR amplification procedures were: denaturation at 95℃for 2min; denaturation at 95℃for 5s, annealing at 57℃for 30s, extension at 72℃for 50s,35 cycles; 72 ℃ for 3min;18℃for 5s.
The vector pUB-GFP C-3xFLAG was derived from (Yu, H., et al, suppression of innate immunity mediated by the CDPK-Rboh complex is required for rhizobial colonization in Medicago truncatula non-dules. New Phytol,2018.220 (2): p.425-434), digested with the restriction enzymes StuI (Thermo, ER 0421) and XbaI (Thermo, FD 0684), and ligated to the LjPRP1 fragment, respectively, by the Ginson reaction; the obtained recombinant vector was transformed into Agrobacterium tumefaciens Agrobacterium Rhizogenes EHA.
The cleavage system was 50. Mu.L: carrier 2. Mu.g, xbaI 1.5. Mu. L, stu I1.5. Mu.L, 10 Xbuffer 0.5. Mu.L and the balance MiliQ-H 2 O；
The carrier was ligated with LjPRP1, and the reaction system was 5. Mu.L by Gibson reaction method (Yeasen, 10911ES 20): 2X HieffEnzyme Premix 2.5. Mu.L, gene fragment 0.2. Mu.L, vector 0.5. Mu.L and MiliQ-H 2 O 1.8μL；
The Gibbing reaction conditions were: the reaction was carried out at 50℃for 20min.
3. Stable transformation of Baimaigen
(1) Plant material: adding 20mL of concentrated sulfuric acid into a 100mL sterile triangular flask, pouring the seeds, gently shaking to enable the seeds to be fully contacted with the concentrated sulfuric acid, treating for 10min, enabling the color of the seeds to be slightly light, and concentrating H 2 SO 4 The color turns yellow. Carefully draw concentrated sulfuric acid into glassware with a small amount of water, add 60mL MiliQ-H to a triangular flask 2 O quickly rinses the seeds and discards the waste liquid. Then 40mL MiliQ-H was used 2 O was repeatedly rinsed 4 times to MiliQ-H 2 The O color does not change. Adding 2% sodium hypochlorite solution to cover the seeds, slightly shaking, and treating for 10min, wherein the transparent small ring at the umbilicus of the seeds is dropped, individual seeds are expanded, and the liquid is slightly pale yellow. Discard waste solution, use MiliQ-H 2 O rinsing the seeds until the sterile water color does not change. With 20mL MiliQ-H 2 O submerges the seeds and vernalizes 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 4d.
(2) Strains: inoculating Agrobacterium Agrobacterium tumefaciensEHA105 into LB when seedling cotyledon grows and true leaf does not grow, culturing at 28deg.C to make OD 600 =0.6. The cells were collected by centrifugation at 4000r/m for 10min, resuspended in co-culture medium, and the bacterial solution was transferred to a 100mL sterile Erlenmeyer flask and incubated at 28℃for 30min.
(3) Infection and cultivation: acetosyringone (Shanghai, A601111) with a final concentration of 20 mug/mL is added into the bacterial liquid, cotyledons of seedlings are cut off, green stems are cut into small sections of 0.3cm, and the cut stem sections are placed into the bacterial liquid for infection for 30min. Plates containing 10 layers of sterile filter paper were wetted with 10mL of co-culture medium, explants were fished out and placed on plates and incubated at 23℃for 7d in dark. After co-cultivation for 7d, the explants were transferred to regeneration medium for 7d. Then placing the plant on a screening culture medium for screening, and carrying out secondary culture for 7 days until green calli grow out. The green calli were transferred to shoot induction medium and subcultured for 7d once until shoots developed. And then transferred to a bud growth medium for growth, and the bud growth medium is subjected to 7d secondary culture for 14d growth. The shoots were then elongated by transferring to shoot elongation medium and grown for 14d once for 7d. The shoots were excised and transferred to root induction medium for 7d subculture until basal enlargement. And transferring the buds to a root elongation culture medium, and repeating for 7 days until roots grow out to obtain LjPRP1 over-expression plants, which are marked as LjPRP1-OX-7.
Co-culture medium: 0.387g B5 salt, miliQ-H 2 O is fixed to volume of 1L, pH is adjusted to 5.5, and sterilization is carried out for 20min at 115 ℃.5mL of 1mol/LMES (pH 5.2), 1mL of 0.5mg/mL of 6-BA,0.1mL of 0.5mg/mLNAA and 0.1mL of 1000 XB 5 vitamin were added before use.
Regeneration medium: 3.87g B5 salt, 20g sucrose, 4g plant gel, miliQ-H 2 O is fixed to volume of 1L, pH is adjusted to 5.5, and the mixture is quenched at 115 ℃ for 20min. 1mL of 0.5mg/mL6-BA,0.1mL of 0.5mg/mL NAA,1mL of 1000 XB 5 vitamin and 1mL of 300mg/mL cefotaxime were added before use.
Screening the culture medium: 3.87g B5 salt, 20g sucrose, 4g plant gel, miliQ-H 2 O is fixed to volume of 1L, pH is adjusted to 5.5, and the mixture is quenched at 115 ℃ for 20min. 1mL of 0.5mg/mL6-BA,0.1mL of 0.5mg/mL NAA,1mL of 1000 XB 5 vitamin, 1mL of 300mg/mL cefotaxime and 0.3mL of 50mg/mL hygromycin were added before use.
Bud induction medium: 3.87g B5 salt, 20g sucrose, 4g plant gel, miliQ-H 2 O is fixed to volume of 1L, pH is adjusted to 5.5, and the mixture is quenched at 115 ℃ for 20min. 1mL of 0.5mg/mL6-BA,2.5mL of 2mol/L (NH) was added before use 4 ) 2 SO 4 0.1mL of 0.5mg/mL LNAA,1mL of 1000 XB 5 vitamin, 1mL of 300mg/mL cefotaxime and 0.15mL of 50mg/mL hygromycin.
Bud growth medium: 3.87g B5 salt, 20g sucrose, 4g plant gel, miliQ-H 2 O is fixed to volume of 1L, pH is adjusted to 5.5, and the mixture is quenched at 115 ℃ for 20min. Before use, 0.4mL of 0.5mg/mL6-BA,1mL of 1000 XB 5 vitamin, 1mL of 300mg/mL cefotaxime and 0.15mL of 50mg/mL hygromycin were added.
Bud elongation medium: 3.87g B5 salt, 20g sucrose, 4g plant gel, miliQ-H 2 O is fixed to volume of 1L, pH is adjusted to 5.5, and the mixture is quenched at 115 ℃ for 20min. 80 μL of 0.5mg/mL6-BA,1mL of 1000 XB 5 vitamin, 1mL of 300mg/mL cefotaxime and 0.15mL of 50mg/mL hygromycin were added prior to use.
Root induction medium: 1.94g B5 salt, 10g sucrose, 4g plant gel, miliQ-H 2 Constant volume of O to 1L, pH to 5.5, and sterilizing at 115deg.C to 20And (5) min. 1mL of 0.5mg/mLNAA,0.5mL of 1000 XB 5 vitamin, 1mL of 300mg/mL cefotaxime and 0.1mL of 50mg/mL hygromycin were added prior to use.
Root elongation medium: 1.94g B5 salt, 10g sucrose, 4g plant gel, miliQ-H 2 O is fixed to volume of 1L, pH is adjusted to 5.5, and the mixture is quenched at 115 ℃ for 20min. Before use, 0.5mL1000 XB 5 vitamin, 1mL300mg/mL cefotaxime and 0.1mL50mg/mL hygromycin were added.
LB medium: 5g of yeast extract, 10g of tryptone, 10g of NaCl, pH adjusted to 7.0, miliQ-H 2 O is fixed to volume to 1L, 1.8% of agar powder is added into the solid, and sterilization is carried out for 30min at 121 ℃.
MS medium: 4.43g MS salt (Sigma, M5519-1L), 20g sucrose, pH adjusted to 6.0, miliQ-H 2 O is fixed to volume to 1L, 1.5% of agar powder is added into the solid, and sterilization is carried out for 20min at 115 ℃.
Plant gel (Thermo, VG 0100B), B5 salt (Phytotech, G768-50L), B5 vitamin (MKbio, MS 0620-500G), cefotaxime (Yeasan, 60226ES 08), hygromycin (Yeasan, 60225ES 10), 6-BA (Sigma, B3408-25G), NAA (Sigma, N0640-25G).
4. Seed germination was the same as in example 1 using the LjPRP1 overexpressing plant seed obtained above and the wild-type seed of example 1.
5. Transplanting seedlings and inoculating rhizobia were as in example 2.
6. The number of infection lines was counted.
After rhizobium inoculation for 5d, digging out plants from vermiculite, cleaning roots of the plants, and respectively counting the numbers of infection lines of wild type plants and LjPRP1 over-expression plants under a NikonSMZ18 fluorescent microscope, wherein the numbers of the wild type plants are 15, and the numbers of the LjPRP1 over-expression plants are 16. The results are shown in FIG. 5 and Table 3.
TABLE 3 number of lines of infection for wild type and LjPRP1 overexpressing plants
From fig. 5 and table 3, it can be seen that the lines of infection by the overexpression of LjPRP1 are significantly reduced compared to the wild-type, and that upregulation of LjPRP1 gene expression will inhibit the formation of lines of infection.
Example 5
Effect of LjPRP1 overexpression on the Activity of Nitrogen fixing enzymes
LjPRP1 overexpressing seeds were from the stable transformation in example 4.
2. Seed germination was as in example 1.
3. Transplanting seedlings and inoculating rhizobia were as in example 1.
4. The activity of the BAIMAIGENERATION AZOGULAse was determined as in example 3.
5. Ethylene standard curve was prepared as in example 3.
6. The activity of the nitrogenase was calculated as in example 3.
The results are shown in FIG. 6 and Table 4, wherein the number of wild type plants was 36, and the number of LjPRP1 overexpressing plants was 40.
TABLE 4 Nitrogen fixation enzyme Activity of wild-type and LjPRP1 overexpressing plants
From fig. 6 and table 4, the activity of LjPRP1 overexpressing the nitrogen fixation enzyme was significantly reduced compared to the control, indicating that LjPRP1 overexpression reduced the root nodule nitrogen fixation efficiency.
Example 6
Effect of LjPRP1 expression on plant root nodule
In examples 3 and 5, nodule samples inoculated with Rhizobium MAFF for 3030993 weeks were collected, fixed in FAA fixative (Servicebio, G1103-500 mL), paraffin embedded and sectioned by Wohai Sieve Biotechnology Co., ltd., stained with toluidine blue, and observed with a stereomicroscope, and the results are shown in FIG. 7. From fig. 7, it can be seen that the LjPRP1 mutant has more infected cells and fewer infected cells in the LjPRP1 overexpressing plant, and that the expression of LjPRP1 inhibits root nodule development, as compared to the wild type.
In conclusion, the Baimaigen LjPRP1 gene has important functions in regulating and controlling nitrogen fixation efficiency. Mutation of the LjPRP1 gene obviously increases the number of infection lines, the activity of the azotase of the single plant seedling obviously increases, and overexpression of the LjPRP1 gene obviously reduces the number of the infection lines, and the activity of the azotase of the single plant seedling obviously reduces, so that the LjPRP1 gene has important significance in the aspect of improving the nitrogen fixation efficiency and yield of the Baimai 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 (10)
1. The LjPRP1 protein for regulating and controlling the nitrogen fixation efficiency of plant root nodule and/or regulating and controlling the plant yield is characterized in that the amino acid sequence of the LjPRP1 protein is shown as SEQ ID NO. 1.
2. An LjPRP1 gene encoding the LjPRP1 protein according to claim 1, wherein the nucleotide sequence of the LjPRP1 gene is shown in SEQ ID No. 2.
3. Use of the LjPRP1 protein of claim 1 or the LjPRP1 gene of claim 2 for regulating nitrogen fixation efficiency of plant nodules and/or regulating plant yield.
4. The use according to claim 3, wherein said modulating plant nodule nitrogen fixation efficiency comprises: regulating plant nodule development and/or regulating nitrogen fixation enzyme activity in plant nodule.
5. The use according to claim 3 or 4, wherein said modulating plant nodule nitrogen fixation efficiency comprises: the nitrogen fixation efficiency of plant root nodules is improved by negatively regulating the expression of the LjPRP1 gene, or the nitrogen fixation efficiency of plant root nodules is reduced by positively regulating the expression of the LjPRP1 gene.
6. The use according to claim 3, wherein said modulating plant yield comprises: plant yield is increased by negatively regulating expression of the LjPRP1 gene, or plant yield is decreased by positively regulating expression of the LjPRP1 gene.
7. Use of the LjPRP1 protein of claim 1 or the LjPRP1 gene of claim 2 for the cultivation of transgenic plants with increased nitrogen fixation efficiency and/or increased yield.
8. The use of claim 7, wherein the nitrogen fixation efficiency improvement comprises: promoting plant nodule development and/or increasing the activity of nitrogen fixation enzymes in plant nodules.
9. The use according to claim 8, wherein the plants comprise leguminous plants.
10. The use according to claim 9, wherein the leguminous plants comprise one or more of soybean, alfalfa and centella asiatica.
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