CN116606361B - LjPRP1 protein for regulating nitrogen fixation efficiency of plant root nodule and/or regulating plant yield and application thereof - Google Patents
LjPRP1 protein for regulating nitrogen fixation efficiency of plant root nodule and/or regulating plant yield and application thereof Download PDFInfo
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- 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 LjPRP protein for regulating and controlling nitrogen fixation efficiency of plant root nodules and/or regulating and controlling plant yield and application thereof. Compared with the wild type, the invention proves that the activity of LjPRP gene mutant plant nodule nitrogen fixation enzyme is obviously improved, which proves that the LjPRP1 protein coded by LjPRP gene regulates the nitrogen fixation efficiency of the nodule; on the basis, the invention uses stable transformation technology to overexpress LjPRP gene to obtain the overexpressed material, compared with wild type, the activity of the overexpressed material root nodule nitrogen fixation enzyme is obviously reduced, which shows that LjPRP gene and LjPRP protein expressed by the gene regulate nitrogen fixation efficiency of the root nodule, inhibit the expression of the gene, and improve nitrogen fixation efficiency of leguminous plants, thereby increasing biomass and yield of plants, and having application prospect in 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 LjPRP 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 radix et rhizoma Barbati (Lotusjaponicas) is one of leguminous plants, improves nitrogen fixation efficiency of the radix et rhizoma Barbati, and is helpful for genetic improvement of leguminous crops suitable for cultivation in various places throughout the country, such as grains, oil crops, 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 LjPRP protein in regulating nitrogen fixation and/or regulating yield of plant root nodule in Baimaigen.
Disclosure of Invention
In order to solve the problems, the invention provides LjPRP protein for regulating and controlling nitrogen fixation efficiency of plant root nodule and/or regulating and controlling plant yield and application thereof. The LjPRP 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 LjPRP protein for regulating nitrogen fixation efficiency of plant root nodule and/or regulating plant yield, wherein the amino acid sequence of the LjPRP protein is shown as SEQ ID NO. 1.
The invention also provides a LjPRP gene for encoding the LjPRP protein according to the technical scheme, and the nucleotide sequence of the LjPRP gene is shown as SEQ ID NO. 2.
The invention also provides application of LjPRP protein described in the technical scheme or LjPRP gene described in the technical scheme in regulating nitrogen fixation efficiency of plant root nodule and/or regulating 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 the plant root nodule is improved by negative regulation and control of LjPRP gene expression, or the nitrogen fixation efficiency of the plant root nodule is reduced by positive regulation and control of LjPRP gene expression.
Preferably, the regulating plant yield comprises: plant yield is increased by negative regulation of LjPRP gene expression or reduced by positive regulation of LjPRP gene expression.
The invention also provides application of LjPRP protein described in the technical scheme or LjPRP gene described in the technical scheme in culturing 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 LjPRP protein for regulating nitrogen fixation efficiency of plant root nodule and/or regulating plant yield, wherein the amino acid sequence of the LjPRP protein is shown as SEQ ID NO. 1. According to the invention, the fact that the expression of LjPRP gene is hardly detected in the plant of the zebra crossing LjPRP gene mutant is proved, compared with a wild type, the activity of the nitrogen fixation enzyme of the root nodule of the plant of the LjPRP gene mutant is obviously improved, which proves that the LjPRP1 protein coded by LjPRP1 gene regulates the nitrogen fixation efficiency of the root nodule, and the LjPRP protein has an important function in regulating the nitrogen fixation efficiency of the root nodule of the plant; on the basis, the invention uses stable transformation technology to overexpress LjPRP gene to obtain the overexpressed material, compared with wild type, the activity of the overexpressed material root nodule nitrogen fixation enzyme is obviously reduced, which shows that LjPRP gene and LjPRP protein expressed by the gene regulate nitrogen fixation efficiency of the root nodule, inhibit the expression of the gene, and improve nitrogen fixation efficiency of leguminous plants, thereby increasing biomass and yield of plants, and having application prospect in 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 results of LjPRP mutant plants and wild-type LjPRP gene;
FIG. 2 is a graph of the number of lines of infection for LjPRP mutant plants and wild type;
FIG. 3 is a graph of ethylene standard curve;
FIG. 4 is a graph showing the activity of the individual seedling nitrogen fixation enzymes of LjPRP mutant plants;
FIG. 5 is a plot of the number of lines of infection for 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 Ljprp-1 is a mutant plant; ljPRP1-OX-7 is an overexpressing 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 LjPRP protein for regulating nitrogen fixation efficiency of plant root nodule and/or regulating plant yield, wherein the amino acid sequence of LjPRP protein is shown as SEQ ID NO.1, and the method is as follows ：MASQVASVEEVAGDKYRSFIHEEADTTHWRHGGPPTYDVVNHLFEQGRTKEWPKGSLEETVQNAIKSWEMEVSHKTRLQDFRTINPEKFKLFVNGREGLSAEETLSIGSYNALLKSSLPEEFKYYKSEEETFESSHEAFRSAFPRGFAWEVIKVYTGPPEIAYKFRHWGFFEGPFKGHAPTGKMVEFYGLGTLKVDNARKGEEVEIYYDPEEWRGDLLPASGTATEDPTKTTPTSQACPFSK.
The invention proves that LjPRP gene and LjPRP protein expressed by the gene regulate nitrogen fixation efficiency of the root nodule by inhibiting LjPRP protein encoding gene expression and promoting LjPRP protein encoding gene expression, and can improve nitrogen fixation efficiency of leguminous plants by inhibiting LjPRP gene expression, thereby increasing biomass and yield of the plants.
The invention also provides a LjPRP gene for encoding the LjPRP1 protein in the technical scheme, the nucleotide sequence of the LjPRP gene is shown as SEQ ID NO.2, and the method is as follows ：5'-ATGGCCAGCCAAGTAGCCTCCGTTGAAGAAGTTGCAGGAGACAAATAC AGATCTTTCATTCATGAAGAGGCTGACACAACCCACTGGAGACATGGTGGACCACCCACCTATGATGTTGTGAACCACCTTTTTGAACAAGGTCGAACCAAGGAATGGCCTAAAGGTTCATTAGAGGAGACAGTGCAAAACGCCATAAAATCATGGGAGATGGAGGTTTCTCACAAAACCCGCTTGCAGGATTTCAGAACCATAAATCCTGAAAAGTTCAAGCTCTTTGTTAATGGGAGGGAGGGGTTATCTGCAGAGGAAACTCTGAGCATAGGAAGTTATAATGCTTTGCTAAAAAGCTCTCTACCAGAAGAATTCAAGTATTACAAATCTGAAGAAGAGACTTTTGAATCATCTCATGAAGCTTTCAGATCAGCTTTTCCTCGTGGATTTGCATGGGAAGTGATCAAAGTTTATACCGGACCCCCTGAAATTGCTTACAAGTTTAGGCACTGGGGATTCTTTGAAGGTCCTTTCAAGGGACATGCCCCTACTGGGAAGATGGTTGAGTTCTATGGCTTGGGAACTCTCAAGGTTGACAACGCGCGGAAAGGGGAGGAGGTGGAGATCTACTACGACCCAGAAGAGTGGCGGGGTGATCTTCTACCTGCGAGTGGGACCGCCACAGAGGACCCCACTAAAACTACACCAACTTCTCAAGCATGTCCTTTCTCCAAATAA-3'.
The invention also provides application of LjPRP protein described in the technical scheme or LjPRP gene described in the technical scheme in regulating nitrogen fixation efficiency of plant root nodule and/or regulating 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 the plant root nodule is improved by negative regulation and control of LjPRP gene expression, or the nitrogen fixation efficiency of the plant root nodule is reduced by positive regulation and control of LjPRP gene expression; more preferably comprises promoting plant nodule development and/or enhancing nitrogen fixation enzyme activity in plant nodules by negative regulation of LjPRP gene expression or inhibiting plant nodule development and/or inhibiting nitrogen fixation enzyme activity in plant nodules by positive regulation of LjPRP gene expression.
In the present invention, the controlling plant yield preferably comprises: plant yield is increased by negative regulation of LjPRP gene expression or reduced by positive regulation of LjPRP gene expression.
In the present invention, the means of negative regulation preferably comprises a mutation LjPRP gene.
In the present invention, the means of up-regulation preferably comprises over-expression LjPRP gene.
The invention also provides application of LjPRP protein described in the technical scheme or LjPRP gene described in the technical scheme in culturing 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. The invention can promote plant nodule development and improve the activity of the nitrogen fixation enzyme in the plant nodule by negatively regulating and controlling LjPRP gene expression, thereby improving the nitrogen fixation efficiency of the nodule, leading the nodule to fix more nitrogen to provide the plant and achieving the effect of improving the yield.
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, a LjPRP protein for regulating nitrogen fixation efficiency of plant nodule and/or regulating plant yield and its application are described in detail below 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 LjPRP gene
1. Seed germination
The Baimaigen LjPRP gene mutant seed Ljprp-1 was purchased from LotusBase (https:// lotus.au. Dk/lore 1/search), plantID:30037066 the insertion site of LORE1 of this mutant seed was on the first exon of LjPRP, 168bp after the start codon ATG, and the LjPRP gene was found in the Bai Mai Gen genome database miyakogusa under the gene number Lj0g3v0096089.1. After the seeds are purchased, heterozygous and homozygous mutants are identified through PCR, the homozygous mutants are used for subsequent experiments, the wild type seeds of the Baimaigen are disclosed in 【FengY,WuP,LiuC,PengL,WangT,WangC,TanQ,LiB,OuY,ZhuH,YuanS,HuangR,StaceyG,ZhangZ,CaoY.SuppressionofLjBAK1-mediatedimmunitybySymRKpromotes rhizobialinfectioninLotusjaponicus.MolecularPlant,2021,14:1935-1950】., and the wild type seeds of the Baimaigen LjPRP1 gene mutant seeds Ljprp-1 and the wild type seeds of the Baimaigen are germinated 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 in full contact with the concentrated sulfuric acid, treating for 10min, and enabling the color of the seeds to be slightly light and the color of the concentrated H 2SO4 to be yellow. Carefully draw concentrated sulfuric acid into glassware with a small amount of water, add a large amount of MiliQ-H 2 O into the flask to quickly rinse the seeds, discard the waste. The rinsing was repeated 4 times with a small amount of MiliQ-H 2 O. 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. The waste solution was discarded, and seeds were rinsed with MiliQ-H 2 O and the rinsing was repeated 4 times. Seeds were submerged with 20mL of MiliQ-H 2 O and vernalized 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 constant volume to 1L, solid added 1.5wt.% agar powder, 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. Rhizobium Mesorhizobium loti MAFF303099 is shake-cultured at 28 ℃ by using a TY liquid culture medium, the bacterial cells are collected after centrifugation for 5min at an OD 600 value of 0.8 and 4000r/min, diluted to an OD 600 =0.01 by using a B & D nitrogen-free culture medium for resuspension, and inoculated to the roots of the Baimaigen seedlings, wherein the inoculation amount of each seedling is 3ml.
The TY culture medium is prepared by the following method: 3g of yeast extract, 5g of tryptone, 0.69gCaCl 2, pH adjusted to 7.0, miliQ-H 2 O constant volume to 1L and sterilized at 121℃for 30min.
The B & D nitrogen-free liquid culture medium consists of ：1mmol/L CaCl2、0.5mmol/L KH2PO4、10μmol/L Fe(C6H5O7)、0.25mmol/L MgSO4、0.25mmol/L K2SO4、1.0μmol/L MnSO4、2.0μmol/L H3BO3、0.5μmol/L ZnSO4、0.2μmol/L CuSO4、0.1μmol/L CoSO4 and 0.1 mu mol/L Na 2MoO4 with the following concentration components; sterilizing at 121deg.C for 30 min.
Mesorhizobium loti MAFF303099 is 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.Suppression of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonicus.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 from monatin. PCR reaction procedure using Applied Biosystems ViiATM A 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 dipyridin (Genbank ID: AW 720576) and ATPase (Genbank ID: AW 719841) as internal controls;
the primers for amplifying LjPRP gene, ubiquitin and 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 the expression of LjPRP1 was hardly detected in LjPRP-1 homozygous mutant plants relative to wild type plants (FIG. 1). This result indicated that LORE1 insertion in the seed of the zebra crossing LjPRP1 gene mutant caused a deletion mutation in the LjPRP1 gene, resulting in an inability to express the LjPRP1 gene.
Example 2
Effect of mutation of the Baomai root LjPRP 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. Rhizobia MesorhizobiumlotiMAFF303099/pHC60 is shake-cultured at 28 ℃ with TY liquid medium, the bacterial cells are collected after centrifugation for 5min at 4000r/min until the OD 600 value is 0.8, diluted to OD 600 =0.01 by B & D nitrogen-free medium resuspension, inoculated to the roots of the Baimai root seedlings, and the inoculation amount is 3ml.
TY medium: 3g of yeast extract, 5g of tryptone, 0.69gCaCl 2, pH adjusted to 7.0, miliQ-H 2 O constant volume to 1L and sterilized at 121℃for 30min.
The B & D nitrogen-free liquid medium consisted of ：1mmol/LCaCl2,0.5mmol/LKH2PO4,10μmol/LFe(C6H5O7),0.25mmol/LMgSO4,0.25mmol/LK2SO4,1.0μmol/LMnSO4,2.0μmol/LH3BO3,0.5μmol/LZnSO4,0.2μmol/LCuSO4,0.1μmol/LCoSO4 and 0.1. Mu. Mol/LNa 2MoO4 in the following concentrations and was sterilized at 121℃for 30min.
MesorhizobiumlotiMAFF303099/pHC60 is derived from literature 【FengY,WuP,LiuC,PengL,WangT,WangC,TanQ,LiB,OuY,ZhuH,YuanS,HuangR,StaceyG,ZhangZ,CaoY.SuppressionofLjBAK1-mediatedimmunitybySymRKpromotes rhizobialinfectioninLotusjaponicus.MolecularPlant,2021,14:1935-1950】,, i.e. "M.lotimaFF303099expressingGFP".
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 number of infection lines of Wild Type (WT) and LjPRP1 mutant plants under a NikonSMZ fluorescent microscope, wherein the number of the wild type and LjPRP mutant plants is 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
As can be seen from fig. 2 and table 1, the infection line of LjPRP a mutant plants was significantly increased compared to the wild type, and it was seen that negative regulation of LjPRP1 gene expression would promote the formation of the infection line.
Example 3
Effect of mutation of the Barbamate LjPRP 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 bottle was placed in a plastic box containing water, and after reacting for 2 hours at 28 ℃, the glass bottle was taken out and detected by a gas chromatograph, wherein the number of wild type plants and LjPRP 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 by the present invention is y=2362.5x+5600 (R 2 =0.9993), see fig. 3. Wherein R 2 represents a linear regression coefficient of determination, reflecting the correlation between the two variables. It is generally believed that the closer the R 2 value is to 1, the stronger the correlation and the more accurate the analysis of the results. The R 2 value of the invention reaches 0.9993, which completely accords with the description of linear regression, so that the following determination of the activity of the root nodule nitrogen fixation enzyme 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 per reaction time (unit of enzyme activity is typically nmolC 2H4/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 2H4(μmol)＝C2H4 volumes (. 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 Nitrogen fixing enzyme Activity of wild type and LjPRP mutant plants
As can be seen from fig. 4 and table 2, the activity of the nitrogen fixation enzyme of the LjPRP mutant single plant seedling is significantly increased compared with the wild type, which indicates that the mutation of LjPRP (i.e. the negative regulation of LjPRP1 gene expression) 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 rhizomes were harvested at 3030993 weeks of 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 primers according to LjPRP1 full-length cDNA, and using the hundred vein root nodule cDNA as a template, and amplifying LjPRP1 full-length cDNA fragments by using the primers LjPRP-cDNA-F and LjPRP-cDNA-R respectively; then using LjPRP full-length cDNA fragment as template, using primer LjPRP-pub-F and LjPRP1-pub-R to make second round amplification so as 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 used Max Super-FIDELITY DNA Polymerase (Vazyme), wherein the two PCR amplification systems are 1 mu L of (20μL)：2×Phanta Max Buffer 10μL、dNTP(10mM earch)0.5μL、F-primer(10μM)0.5μL、R-primer(10μM)0.5μL、Phanta Max Super-Fidelity DNA Polymerase 0.5μL、 template DNA and 7 mu L of MiliQ-H 2 O;
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.
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 nodules.New Phytol,2018.220(2):p.425-434.), and digested with restriction enzymes StuI (Thermo, ER 0421) and XbaI (Thermo, FD 0684) and ligated to LjPRP fragment respectively by Ginson reaction; the obtained recombinant vector was transformed into Agrobacterium tumefaciens Agrobacterium Rhizogenes EHA.
The cleavage system was 50. Mu.L: 2. Mu.g of vector, 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 LjPRP. Mu.L of each of the supports using Gibson reaction method (Yeasen, 10911ES 20): 2X Hieff Enzyme Premix 2.5. Mu.L, gene fragment 0.2. Mu.L, vector 0.5. Mu.L and MiliQ-H 2 O1.8. Mu.L;
Gibsion reaction conditions are: 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 in full contact with the concentrated sulfuric acid, treating for 10min, and enabling the color of the seeds to be slightly light and the color of the concentrated H 2SO4 to be yellow. Carefully draw concentrated sulfuric acid into glassware with a small amount of water, add 60mL of MiliQ-H 2 O to the flask to quickly rinse the seeds, discard the waste. Rinsing was repeated 4 times with 40mL of MiliQ-H 2 O until there was no change in MiliQ-H 2 O color. 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. The waste solution was discarded, and seeds were rinsed with MiliQ-H 2 O until no change in color was observed in the sterile water. Seeds were submerged with 20mL of MiliQ-H 2 O 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 4d.
(2) Strains: when the cotyledon of the seedling grows up and the true leaf does not grow out, the agrobacterium Agrobacterium tumefaciensEHA is inoculated into LB and cultured at 28 ℃ so that 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: and adding acetosyringone (Shanghai, A601111) with a final concentration of 20 mug/mL into the bacterial liquid, cutting seedling cotyledons, cutting green stems into small sections of 0.3cm, and placing the cut stem sections 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 LjPRP-OX-7.
Co-culture medium: 0.387g B5 salt, miliQ-H 2 O to 1L, pH to 5.5, and sterilizing at 115℃for 20min. 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 constant volume to 1L, pH adjusted to 5.5, and 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 constant volume to 1L, pH adjusted to 5.5, and 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 constant volume to 1L, pH adjusted to 5.5, and quenched at 115℃for 20min. 1mL of 0.5mg/mL6-BA,2.5mL of 2mol/L (NH 4)2SO4, 0.1mL of 0.5mg/mLNAA,1mL of 1000 XB 5 vitamin, 1mL of 300mg/mL cefotaxime and 0.15mL of 50mg/mL hygromycin were added before use.
Bud growth medium: 3.87g B5 salt, 20g sucrose, 4g plant gel, miliQ-H 2 O constant volume to 1L, pH adjusted to 5.5, and 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 constant volume to 1L, pH adjusted to 5.5, and 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 of B5 salt, 10g of sucrose, 4g of plant gel, miliQ-H 2 O to a volume of 1L, pH to 5.5, and sterilization at 115℃for 20min. 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 of B5 salt, 10g of sucrose, 4g of plant gel, miliQ-H 2 O to a volume of 1L, pH to 5.5, and sterilization 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 adjustment to 7.0, miliQ-H 2 O constant volume to 1L, solid addition of 1.8% agar powder, sterilization at 121℃for 30min.
MS medium: 4.43g MS salt (Sigma, M5519-1L), 20g sucrose, pH adjusted to 6.0, miliQ-H 2 O constant volume to 1L, solid added 1.5% agar powder, sterilized at 115℃for 20min.
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 LjPRP over-expressed plant seeds obtained as described above and wild type seeds in 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 counting the numbers of infection lines of wild type plants and LjPRP1 over-expression plants under a NikonSMZ fluorescent microscope, wherein the numbers of the wild type plants are 15 plants, and the numbers of the LjPRP1 over-expression plants are 16 plants. The results are shown in FIG. 5 and Table 3.
TABLE 3 number of lines of infection for wild type and LjPRP.sup.1 overexpressing plants
From fig. 5 and table 3, it can be seen that the lines of infection by LjPRP a1 overexpression are significantly reduced compared to wild-type, and that positive regulation of LjPRP1 gene expression will inhibit the formation of the lines of infection.
Example 5
Effect of LjPRP1 overexpression on the activity of azotase
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 plants and the number of LjPRP1 overexpressing plants was 40 plants.
TABLE 4 Nitrogen fixation enzyme Activity of wild-type and LjPRP.sup.1 overexpressing plants
From fig. 6 and table 4, the activity of LjPRP1 overexpressing the azotase was significantly reduced compared to the control, indicating that LjPRP1 overexpression reduced the nitrogen fixation efficiency of the nodules.
Example 6
Effect of LjPRP on plant nodule expression
In examples 3 and 5, nodule samples inoculated with Rhizobium MAFF3030993 weeks were collected, fixed with FAA fixative (Servicebio, G1103-500 mL), paraffin embedded and sectioned by Wohan Sieve Biotechnology Co., ltd., stained with toluidine blue, and observed with a stereomicroscope, and the results are shown in FIG. 7. As can be seen from fig. 7, the LjPRP mutant showed more infected cells and less infected cells in LjPRP1 overexpressing plants compared to the wild type, and the LjPRP1 expression inhibited root nodule development.
In conclusion, the Baimaigen LjPRP gene has important functions in regulating and controlling nitrogen fixation efficiency. Mutation of LjPRP gene obviously increases the number of infection lines, overexpression of LjPRP gene obviously reduces the number of infection lines, and obviously reduces the activity of the nitrogen fixation enzyme of single seedling, which shows that LjPRP gene has important significance in improving nitrogen fixation efficiency and yield of Baimaigen.
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 (5)
- The application of LjPRP1 protein or LjPRP gene in regulating nitrogen fixation efficiency of Baimaigen nodule is disclosed, wherein the amino acid sequence of LjPRP protein is shown as SEQ ID NO.1, and the nucleotide sequence of LjPRP gene is shown as SEQ ID NO. 2.
- 2. The use according to claim 1, wherein the regulation of the nitrogen fixation efficiency of the baimai root nodule is: regulating the growth of the root nodule of the Baimai root and/or regulating the activity of the nitrogen fixation enzyme in the root nodule of the Baimai root.
- 3. The use according to claim 1 or 2, wherein the regulatory hundred root nodule nitrogen fixation efficiency is: the nitrogen fixation efficiency of the hundred-vein root nodule is improved by negative regulation and control of LjPRP gene expression, or the nitrogen fixation efficiency of the hundred-vein root nodule is reduced by positive regulation and control of LjPRP gene expression.
- The application of LjPRP1 protein or LjPRP gene in culturing transgenic Baimaigen with raised nitrogen fixation efficiency, the LjPRP protein has amino acid sequence shown in SEQ ID No.1, and the LjPRP gene has nucleotide sequence shown in SEQ ID No. 2.
- 5. The use according to claim 4, wherein the nitrogen fixation efficiency is improved by: promote the growth of the root nodule and/or increase the activity of the nitrogen fixation enzyme in the root nodule.
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