CN114292863B - Nitrate transport protein and application of encoding gene thereof in improving quantity of hairy root nodules of crops - Google Patents
Nitrate transport protein and application of encoding gene thereof in improving quantity of hairy root nodules of crops Download PDFInfo
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Abstract
The invention discloses an application of nitrate transport protein and a coding gene thereof in improving the quantity of hairy root nodules of crops, and the application of the nitrate transport protein and the coding gene thereof in over-expressing SEQ ID NO in a plant body: 1 or a nucleotide sequence as set forth in SEQ ID NO:1 to obtain a transgenic plant with increased number of nodules on hairy roots compared with wild plants. The invention has important scientific research and practical significance for improving the nodulation nitrogen fixation capability of the soybean and the soybean yield.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to nitrate transport protein and application of a coding gene thereof in improving the quantity of hairy root nodules of crops, thereby improving the nodulation and nitrogen fixation capacity of the crops.
Background
Plants acquire the required nitrogen source from nature mainly by 3 ways: NO to be absorbed 3 - Reducing to organic nitrogen, directly absorbing and utilizing ammonium or organic nitrogen in soil, or fixing N in air by azotobacter 2 And converted into a plant-usable form.
For most non-leguminous plants, NO 3 - Is the most dominant nitrogen utilization form. Plants have evolved both a Low Affinity Transport System (LATS) and a High Affinity Transport System (HATS) to accommodate changes in nitrogen in the soil. NRT1.5 is a low affinity bi-transport protein that localizes in the plasma membrane and is expressed in pericycle cells near the xylem. Reducing or inhibiting NRT1.5 expression reduces nitrate transport from root to stem, indicating that NRT1.5 is involved in nitrate transport in root xylem. The leguminous plants and the rhizobia establish a good symbiotic relationship in the long-term evolution process, and the leguminous plants carry out symbiotic nitrogen fixation by forming a unique rhizobium structure. In agricultural production, the high-efficiency symbiotic nitrogen fixation system can provide 50% -60% of total nitrogen required for growth and development for soybeans.
The rhizobium-leguminous plant symbiotic nitrogen fixation system is a very efficient nitrogen utilization system in nature, the analysis of the operation mode of the system is always a hot spot and a difficult point in scientific research, and the identification of the functional genes in soybean nodulation and symbiotic nitrogen fixation has important significance for enhancing the symbiotic nitrogen fixation capability of leguminous plants, especially soybean of important grain and oil economic crops. And simultaneously, the method provides a larger possibility for reducing the application amount of nitrogen fertilizer and increasing the yield of soybeans in production.
Current studies have found NRT1.5 as low affinity bi-transport protein mediated NO 3 - The loading into xylem and its long distance transport to the upper part of the ground, the results of the study in arabidopsis show that NRT1.5 is also a k+/h+ antiporter, directly involved in the efflux of cell k+. However, there are no studies and reports on increasing the number of root nodules of leguminous plants.
Disclosure of Invention
The invention aims to provide a method for increasing the number of hairy root nodules of crops.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
A method for increasing the number of hairy root nodules of crops, which over-expresses SEQ ID NO in a sequence table in plants: 1 or a nucleotide sequence as set forth in SEQ ID NO:1 to obtain a transgenic plant with increased number of nodules on hairy roots compared with wild plants.
As a preferred embodiment of the present invention, a nucleic acid sequence comprising SEQ ID NO:1, then constructing a transformant by using the overexpression vector, infecting the root system of a receptor plant by using the obtained transformant, screening positive plants, and obtaining transgenic plants with the increased number of root nodules on hairy roots compared with normal plants.
As a preferable technical scheme of the invention, the over-expression vector takes pEGAD as a framework vector.
As a preferable technical scheme of the invention, the over-expression vector is connected with a 35S promoter.
As a preferred embodiment of the present invention, the sequence set forth in SEQ ID NO:1, and the forward amplification primer is shown as SEQ ID NO in the sequence table: 2, and the reverse amplification primer is shown as SEQ ID NO: 3.
As a preferred technical scheme of the invention, agrobacterium rhizogenes K599 is transformed by using the over-expression vector to obtain a transformant, then a hairy root transformation method mediated by agrobacterium rhizogenes K599 is used to obtain a transgenic plant, and positive plants are screened to obtain the transgenic plant with increased number of root nodules on the hairy root compared with normal plants.
As a preferable technical scheme of the invention, the target plant is a butterfly flower plant.
As a preferable embodiment of the present invention, the target plant is soybean.
SEQ ID NO:1 and the application thereof in improving the number of hairy root nodules of crops.
Comprising SEQ ID NO:1 or recombinant bacteria and application thereof in improving the number of hairy root nodules of crops.
The beneficial effects of adopting above-mentioned technical scheme to produce lie in: experiments prove that the transgenic plant is obtained by transforming the receptor soybean plant by using the overexpression vector constructed by the invention, so that the quantity of hairy root nodules of the soybean can be obviously increased, and the transgenic plant has important scientific research and practical significance for improving the nodulation and nitrogen fixation capacities of the soybean and improving the soybean yield.
Drawings
FIG. 1 is a graph showing test results of the correlation of the relative expression amount of GmNRT1.5c and GmNRT1.5c of roots of soybean hairy root transformed plants of empty vector, nodulation phenotype, nodulation number and the like in 35S. In the figure, A is the relative expression amount of GmNRT1.5c in the soybean GmNRT1.5c over-expression and empty vector transformation hairy root material; b is the underground nodulation phenotype of soybean gmnrt1.5c over-expression and empty vector transformation of hairy root material; panel C is a statistic of soybean GmNRT1.5c overexpression and empty vector transformed hairy root material root nodulation number.
Detailed Description
The following examples illustrate the invention in detail. The raw materials and the equipment used by the invention are conventional commercial products, and can be directly obtained through market purchase. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies. In the following examples, the percentages are by mass unless otherwise indicated. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Example 1 Gene
This example relates to the soybean gmnrt1.5c gene whose cDNA reading frame has the nucleotide sequence set forth in SEQ ID NO: 1. Corresponding to this gene is the nucleotide sequence of SEQ ID NO:1, the sequence of the complete complementary pair, in SEQ ID NO:1, and a nucleotide sequence which is substituted, deleted or added with one or more nucleotides based on the sequence shown in the formula 1 and has the same function. Taking the degeneracy of codons into consideration, the base sequence of the cDNA reading frame is appropriately modified on the premise of not changing the amino acid sequence, which is also an embodiment of the technical scheme of the invention.
Example 2, proteins
This example relates to a protein corresponding to the nucleotide sequence of example 1. On the premise of not affecting the structure and activity of the soybean GmNRT1.5c protein, various substitutions, deletions or additions of one or more amino acids or terminal modifications are carried out on the amino acid sequence of the soybean GmNRT1.5c protein, which is also a embodying mode of the technical scheme of the invention.
Example 3 acquisition of Gene sequences
The cDNA reading frame of the soybean gmnrt1.5c gene can be obtained by the following method: extracting RNA (ribonucleic acid) of a soybean variety Williams82 in a laboratory as a material, reversely transcribing the RNA into cDNA, designing a primer according to the existing Williams82 genome sequence to clone GmNRT1.5c gene CDS in soybean, wherein the primer comprises a sequence shown as SEQ ID NO:2 and the sequence of SEQ ID NO:3, and a downstream primer shown in 3. The DNA high-fidelity enzyme is utilized for PCR amplification, and a PCR reaction system is as follows: cDNA template: 1 μl; front primer (F): 0.5. Mu.L; rear primer (R): 0.5. Mu.L; buffer: 15. Mu.L; ddH 2 O：19μL；dNTP: mu.L of the total reaction system was 30. Mu.L. Reaction conditions: 94 ℃ C:: 2min,98 ℃ C: 20s,56 ℃ C: 30s,68 ℃ C: 2min,68 ℃ C: 10min,16 ℃ C: infinity; wherein, 98 ℃ is: 20s,56 ℃ C: 30s,68 ℃ C: three steps of 2min set 38 cycles. The 30. Mu.LPCR amplified product was analyzed by 1% agarose gel electrophoresis. The band of about 1791bp is recovered and purified by using a Shanghai industrial gel recovery kit, and the CDS sequence of the obtained GmNRT1.5c gene is shown as SEQ ID NO: 1.
Example 4 preparation of transgenic hairy roots of Soybean
The example provides a preparation method of soybean transgenic hairy roots with the overexpression of GmNRT1.5c genes. The specific operation steps are as follows.
(1) The agrobacterium rhizogenes is transformed by adopting a liquid nitrogen freeze thawing method, and the specific operation is as follows: a. taking out 200 mu L of frozen competent cells, melting, adding 5-10 mu L of plasmid DNA, mixing the wall of the flick tube uniformly, and placing on ice for 20-30min; b. placing in liquid nitrogen for 5min, taking out, transferring the tube to 37 deg.C, melting for 5min, adding 1mLLB (non-resistant) liquid culture medium, and oscillating at 28 deg.C at low speed (150 r/min) for 1.5-2 hr; 4000r/min,30sec, removing supernatant, adding 100 μLLB liquid culture medium, suspending thallus, and plating (containing 50mg/mL kanamycin and 50mg/mL streptomycin); d. culturing at 28deg.C until monoclonal is grown for hairy root transformation.
(2) Agrobacterium rhizogenes K599 mediated hairy root transformation: a. germinating soybean variety Williams82 in vermiculite for 72 hr, cutting the hypocotyl of cotyledon with the lower end of 0.1-0.2cm when the cotyledon is just opened, soaking in activated Agrobacterium K599 for 1 hr (OD 600 =0.8); b. the explants were transferred to 1/10MS medium (Medium formulation: 2% sucrose, 0.39% MES,0.044% Murashige)&Skoog ModifiedBasalMediumwithGamborgVitamins (cat# M404), pH was adjusted to 5.4) and grown co-cultivated on filter paper for 3-4 days; c. after the hairy roots grow out of the calli, the explants are transplanted into vermiculite.
Example 5 Positive identification of transgenic Soy hairy root Material
The example provides a positive identification method of soybean transgenic hairy root material with over-expressed GmNRT1.5c gene. The specific operation steps are as follows.
(1) Root tissue total DNA extraction of gmnrt1.5c transgenic hairy roots: a. placing 0.1-0.2g of plant tissue into a 2mLEP tube, adding steel balls, quick-freezing with liquid nitrogen, and grinding into powder with a proofing machine. Adding 500 μLCTAB, standing at 65deg.C for 30min to allow full cleavage; b. 200 mu L of chloroform is added, and after being mixed by intense shaking, the mixture is centrifuged at 11000rpm for 15min; c. adding the supernatant into a new 1.5mLEP tube, adding anhydrous ethanol with twice volume, shaking, mixing, standing at-20deg.C for more than half an hour, and centrifuging at 11000rpm for 15min; d. removing supernatant, centrifuging, drying, dissolving in 40 μl water, and storing in refrigerator at 4deg.C. The existence of Bar gene in hairy root DNA is verified by Polymerase Chain Reaction (PCR) with DNA as template, and hairy root transformed by corresponding empty vector is used as negative control.
The amplification primers of the Bar gene are as follows:
Bar-F：CTACATCGAGACAAGCACGGTCAA(SEQ ID NO：4)
Bar-R：AGAAACCCACGTCATGCCAGTTC(SEQ ID NO：5)
example 6 Gene relative expression level of Soy transgenic hairy root Material
The present example provides a method for measuring the relative expression of the gmnrt1.5c gene of a soybean transgenic hairy root material in which the gmnrt1.5c gene is overexpressed. The specific operation steps are as follows.
(1) Root tissue total RNA extraction of gmnrt1.5c transgenic hairy root: the EP tube and gun head involved in this process are both imported consumables using the purchased rnasefile. a. And (5) quick freezing and grinding plant tissues by liquid nitrogen. Placing 0.1-0.2g of plant tissue into a 2mLEP tube, adding steel balls, quick-freezing in liquid nitrogen, and grinding into powder; b. adding 1ml of LTrilzol reagent, shaking and mixing uniformly, and standing at room temperature for 5-10min to facilitate separation of nucleic acid protein complex; c. adding 200 mu L of chloroform, shaking vigorously for 15s, and standing at room temperature for 2-3min; d.4deg.C 12000rpm centrifugal 10-15min; e. the supernatant was pipetted into a 1.5mLEP tube of RNasetree and an equal volume of isopropanol (typically 400-450. Mu.L) was added; f. after being mixed gently, the mixture is placed at the temperature of minus 20 ℃ for more than half an hour; centrifuging at 12000rpm for 10min at 4 ℃, discarding supernatant, and depositing RNA at the bottom of the tube; h. adding 1mL of precooled 70% ethanol to wash the precipitate, and centrifuging at 12000rpm at 4 ℃ for 3-5min; i. the supernatant was discarded, dried at room temperature and the pellet was dissolved in 20. Mu. LDEPCH 2 In OThe method comprises the steps of carrying out a first treatment on the surface of the j. Electrophoresis detects whether the extracted RNA is intact. Taking 1 mu LRNA,2 mu Lloadingbuffer, electrophoresis at 200V for 5min, and preserving the rest RNA sample at-80 ℃ for later use.
(2) Reverse transcription PCR: a. mu.LRNA, 2. Mu.L 5xgDNAdigester Buffer, 1. Mu.L DNAdigester, 2. Mu.LRNaseFreeddH were added sequentially to 200. Mu.LPCR tubes treated with DEPC 2 O, incubating at 42 ℃ for 2min, and rapidly cooling on ice; b. to the tube of the above reaction was directly added 10. Mu.L of 2 xHorlI superMixplus (available from Novogene Co.); c. mixing the above reaction solutions, reacting at 25deg.C for 5min, at 42deg.C for 30min, and at 85deg.C for 5min; d. after the reaction is completed, the cDNA synthesized by the reaction can be used as a qPCR reaction template.
(3) Real-time fluorescent quantitative PCR: the system was mixed according to the following reaction system, and then was put into 96-well optical plates separately and covered with a dedicated optical film. The amplification instrument used was a fluorescent quantitative PCR instrument from Bio-Rad company.
The amplification procedure was: 95 ℃ for 5min;95℃for 10s,55℃for 20s,72℃for 20s,40cycles; the reaction system of 15s at 95℃and 1min at 60℃and 15s at 95℃and 10. Mu.L was prepared as follows: 2 XSBRGreenMix 5. Mu.L, forward primer (10. Mu.M) 0.2. Mu.L, reverse primer (10. Mu.M) 0.2. Mu.L, cDNA0.2. Mu.L, ddH 2 O4.4μL。
The quantitative primer sequences of the gmnrt1.5c gene are as follows:
GmNRT1.5c-F：GCCTTGACATCGATAGACTTGATCGC(SEQ ID NO：6)
GmNRT1.5c-R：GTCTCATCAATCTCTCCAGTTTTGG(SEQ ID NO：7)
the results are shown in fig. 1, which demonstrate that the relative expression level of gmnrt1.5c was significantly up-regulated by preparing transgenic soybean hairy root lines overexpressing gmnrt1.5 c. Under the identical experimental environment, compared with the transgenic root system (EV) transformed by the empty vector, the number of the root nodules of the transgenic soybean hairy root system which up-regulates and expresses GmNRT1.5c is obviously increased. The up-regulation expression of GmNRT1.5c gene can effectively enhance the nitrogen fixation capacity of soybean nodulation.
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.
Sequence listing
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tactcagtag ttttcacaca aatggcttca ctttttgtgg agcaaggtgc tgccatgaaa 1140
accaaggttt ccaacttcag aataccacca gctagcatgt ccagctttga tatcctcagt 1200
gtggctgttt tcattttctt ttaccgtcga gttcttgatc catttgtggg aaaacttaaa 1260
aagacagatt ctaagggact tacagagctt cagagaatgg gagttggact tgttatagct 1320
gtactggcaa tggtttcggc tggattagtt gaatgctata ggctcaagta tgcaaaacaa 1380
ggatgcctac actgcaatga ctcgagcact ttaagcatct tctggcaaat ccctcagtat 1440
gcatttatag gagcttctga ggtttttatg tacgtaggtc agttggagtt cttcaacgct 1500
cagacaccag acggcttaaa gagctttgga agtgctcttt gcatgacatc catttccctt 1560
gggaactatg tgagtagttt acttgttagt gtggttatga agatatccac cgaagatcac 1620
atgccggggt ggatccctgg acacctcaac aaaggccact tagataggtt ttacttcctc 1680
ttagctgcct tgacatcgat agacttgatc gcttatattg catgtgcaaa atggtacaag 1740
tctatacagc tagaggccaa aactggagag attgatgaga cacaagtata a 1791
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atgtcttgct tagagtctca 20
<210> 3
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ttatacttgt gtctcatcaa tctctccagt t 31
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ctacatcgag acaagcacgg tcaa 24
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
agaaacccac gtcatgccag ttc 23
<210> 6
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gccttgacat cgatagactt gatcgc 26
<210> 7
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gtctcatcaa tctctccagt tttgg 25
Claims (6)
1. A method for increasing the number of hairy root nodules of a plant, comprising the steps of: overexpression of SEQ ID NO in the sequence Listing in plants: 1, obtaining a transgenic plant with an increased number of nodules on hairy roots compared to wild plants; the plant is soybean.
2. A method of increasing the number of hairy root nodules of a plant as claimed in claim 1 wherein: first construct a nucleic acid sequence comprising SEQ ID NO:1, then constructing a transformant by using the overexpression vector, infecting the root system of a receptor plant by using the obtained transformant, screening positive plants, and obtaining transgenic plants with the increased number of root nodules on hairy roots compared with normal plants.
3. A method of increasing the number of hairy root nodules of a plant as claimed in claim 2 wherein: the over-expression vector takes pEGAD as a framework vector.
4. A method of increasing the number of hairy root nodules of a plant as claimed in claim 3 wherein: the over-expression vector is connected with a 35S promoter.
5. A method of increasing the number of hairy root nodules of a plant as claimed in claim 2 wherein: for SEQ ID NO:1, and the forward amplification primer is shown as SEQ ID NO in the sequence table: 2, and the reverse amplification primer is shown as SEQ ID NO: 3.
6. A method of increasing the number of hairy root nodules of a plant as claimed in claim 2 wherein: and transforming Agrobacterium rhizogenes K599 by using the over-expression vector to obtain a transformant, then obtaining a transgenic plant by using a hairy root transformation method mediated by the Agrobacterium rhizogenes K599, and screening positive plants to obtain the transgenic plant with increased root nodule number on the hairy roots compared with normal plants.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841863A (en) * | 2018-07-24 | 2018-11-20 | 华中农业大学 | A method of cultivating high nodulation and nitrogen fixation plant |
| CN109852634A (en) * | 2019-01-03 | 2019-06-07 | 华中农业大学 | A method of cultivating high nodulation and nitrogen fixation genetically modified plants |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841863A (en) * | 2018-07-24 | 2018-11-20 | 华中农业大学 | A method of cultivating high nodulation and nitrogen fixation plant |
| CN109852634A (en) * | 2019-01-03 | 2019-06-07 | 华中农业大学 | A method of cultivating high nodulation and nitrogen fixation genetically modified plants |
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