CN111471702A - Slow-growing rhizobium stable red fluorescent labeling vector and application thereof - Google Patents
Slow-growing rhizobium stable red fluorescent labeling vector and application thereof Download PDFInfo
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Abstract
The invention provides a stable red fluorescent marking carrier of slow rhizobium and application thereof. The fluorescence labeling of rhizobia depends on a vector which is stably present in bacterial cells and efficiently expresses a fluorescent protein. However, the existing rhizobium marking method has the defects of weak fluorescence signal, loss of expression plasmid and the like. Based on the existing defects of the fluorescence labeling of rhizobia. The present application constructs a slow-growing rhizoma bacteriumBradyrhizobium elkaniiA rhizobium red fluorescence expression vector which stably exists in the rhizobium, and has strong fluorescence signals. Can provide an effective method and a tool for visualizing the interaction between the rhizobia and the leguminous plants and deeply researching the interaction between the leguminous plants and the rhizobia.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a slow-growing rhizobium stable red fluorescent labeling vector and application thereof.
Background
The interaction of plants and beneficial microorganisms is researched, and the beneficial microorganisms are utilized, so that the method has an important effect on agricultural sustainable development. Symbiosis of legumes and rhizobia for biological nitrogen fixation is a classic example of plant and beneficial microbial work, and the use of rhizobia interaction with legumes is an important way to increase soybean yield with less nitrogen fertilizer application. However, how to conveniently and visually study the interaction between rhizobia and leguminous plants requires stable fluorescent labeling of rhizobia. However, the stable fluorescent labeling of the bacteria depends on the application of a vector which can stably exist in the bacteria and can efficiently express the fluorescent protein, and although some reported vectors are suitable for labeling bradyrhizobium, the soybean has the defects of easy plasmid loss, fluorescence intensity and the like. Therefore, the construction and the reconstruction of the vector which stably exists in the slow rhizobium and efficiently expresses the fluorescent proteins with various colors are the basis for visually researching the interaction of the soybean plant and the rhizobium.
Disclosure of Invention
The invention aims to provide a stable red fluorescent marking carrier of bradyrhizobium and application thereof.
In order to realize the purpose, the following technical scheme is adopted:
the slow rhizobium stable red fluorescent marker carrier is Kanna resistance, and Tdtomato red fluorescent protein with the fluorescent intensity about 6 times that of the common GFP fluorescent protein is selected as the fluorescent protein of the carrier. The promoter of the fluorescent protein is NPTII promoter (the promoter sequence is shown in figure 2), and a ribosome binding site sequence GGAGGAAGAAAAA (RBS site) is added between the promoter and the fluorescent protein to improve the translation efficiency of the protein and enhance the final fluorescent protein intensity. The specific modification method of the carrier is to utilizeSphI andEcoRi pRBC vector is cut (pRBC is added on the basis of the original vector pMG103 by modifying the multiple cloning site of pMG103SphI (GCATGC) andEcoRi (GAATTC) enzyme cutting site is added with SpeI (ACTAGT) enzyme cutting site, and the gene expression frame of the fluorescent protein Tdtomato connected with the Kaner promoter NPTIIpro is inserted intoSphI (GCATGC) andEcoRi (GAATTC) between the two cleavage sites, and a ribosome binding site sequence GGAGGAAGAAAAA (RBS site: in bold italics) between the promoter and the fluorescent proteinA formal representation; NPTII promoter is shown in a bold and positive form), the size of the vector is 7427bp, and the sequence is shown as SEQ ID NO. 1. The map of the entire vector successfully constructed is shown in FIG. 1.
The invention has the advantages that:
first, the vector used has: the two vector replication initiation sites pHSG298 ori and pMG101 ori not only ensure the copy number of the plasmid in the strain, reduce the plasmid loss caused by bacterial division as much as possible, but also increase the copy number of the fluorescent protein gene Tdtomato in the bacteria, and are beneficial to the increase of the fluorescence intensity. Secondly, the fluorescent protein used by the carrier is Tdmomato, the fluorescence intensity of the fluorescent protein is about 6 times of that of the common GFP protein, and the fluorescent signal of the bacteria can be enhanced to the maximum extent. Thirdly, the promoter used is the promoter NPTII promoter of the bacteria Carna resistance gene, and the NPTII promoter is a strong promoter in the bacteria and can continuously activate the transcription of downstream genes. Fourthly, a ribosome binding sequence is integrated between the NPTII promoter and the downstream gene of the vector, messenger RNA can be promoted to be bound to the ribosome, translation of downstream fluorescent protein is obviously improved, and the quantity of the fluorescent protein of bacteria is increased. Therefore, the method has important significance for ensuring the normal growth of the soybeans under the condition of moderate low phosphorus in the field and improving the tolerance of the soybeans to the moderate low phosphorus.
Drawings
FIG. 1 is a schematic diagram of a red fluorescence-stable expression vector pRBC-NPTIIpro-Tdtomato of Rhizobium bradyrhizobium.
FIG. 2 labeling of bradyrhizobium with pRBC-NPTIipro-Tdtomato vectorBradyrhizobium elkaniiThe fluorescence analysis of (3).
FIG. 3. colonization of soybean roots with labeled bradyrhizobia.
FIG. 4 shows the results of fluorescence observation of fluorescently labeled bradyrhizobia within nodules.
Detailed Description
Example 1 construction of vectors
The original vector was first used, and the NPTII promoter from pET-28a was used with the primer NPTIipro-F: CAGTGCCAAGCTTGCATGCCACGCTGCCGCAAGCA and NPTIIpro-R: CCATGATTACGAATTCACTAGTATCCTGTCTCTTGATC, and fusing the PCR product to the PCR product by a one-step cloning methodSphI andSpeIan endonuclease cut pRBC vector (pRBC is added on the basis of an original vector pMG103 by modifying a multiple cloning site of the pMG103SphI andEcoRenzyme cutting site of SpeI is added to the enzyme cutting site of SpeI), and an intermediate vector pRBC-NPTIIpro is obtained. Meanwhile, a fluorescent protein gene Tdtomato is cloned from the carrier pCMV-Tdtomato by using a primer Tdtomato-F: GATCAAGAGACAGGATACTAGTGGAGGAAGAAAAAATGGTGAGCAAGGGCG and a primer Tdtomato-F: AGCTATGACCATGATTACGAATTCTTACTTGTACAGCTCGTC, and a segment of ribosome binding site GGAGGAAGAAAAA is added in front of the Tdtomato gene. Simultaneously, Tdtomato is connected to the TdtomatoSpeI andEcoRand (3) obtaining a final vector pRBC-NPTIIpro-Tdtomato on a pRBC-NPTIIpro vector cut by the endonuclease I, transferring the constructed vector into Escherichia coli DH5 α by an Escherichia coli heat shock transformation method (42 ℃ for 90 seconds and 3 minutes on ice), and carrying out sequencing identification on the vector, wherein the sequence of the vector is shown as SEQ ID NO. 1.
Example 2 labeling of bradyrhizobium with pRBC-NPTIipro-Tdtomato vectorBradyrhizobium elkanii
1) Escherichia coli DH5 α harboring pRBC-NPTIIpro-Tdtomoto vector was cultured in an expanded state on a medium containing kanamycin antibiotic (100 mg/L), and DNA of the plasmid vector pRBC-NPTIIpro-Tdtomoto was extracted using a plasmid extraction kit, and the DNA concentration was detected at about 300-500 ng.
2) Preparing slow-growing rhizomatous competence: YMA solid medium plate activated bradyrhizobiumBradyrhizobium elkaniiCulturing at 30 deg.C for 18-36 hr, picking single colony on plate, transferring into test tube containing 2m L YMA liquid culture medium, shake culturing at 30 deg.C (180 rmp) for 12 hr, diluting the bacterial liquid cultured in the previous step into 100m L liquid YMA culture medium (triangular flask) at 1:100 volume ratio under aseptic condition, shake culturing at 30 deg.C for about 6-12 hr (200 rpm) to OD value of 0.5-0.6 (OD 600), transferring to aseptic conditionThe cells were collected by centrifugation at 4 ℃ for 8 minutes at 7000 rpm, pouring the supernatant medium after centrifugation at 4 ℃ for 8 minutes, collecting the cells by centrifugation at 6000rpm for 10 minutes at 4 ℃ after the cells at the bottom of the tube were resuspended in ice-precooled 10 m L deionized water, washing the cells repeatedly for 2-3 times (to minimize the ion residues in the cells), resuspending in 2m L-precooled 10% glycerol (2 m L per 50 m L of the initial culture), and finally, the cells were dispensed into 1.5 m L centrifuge tubes (50 μ L per tube), frozen in liquid nitrogen and stored in a refrigerator at-80 ℃ for later use.
3) When in electric shock transformation, competent cells are taken and placed on ice to be dissolved, 3-5 mu L plasmid and plasmid DNA with the concentration of about 300 ng are added, the mixture is placed in an electric shock transformation cup after being pre-cooled at the temperature of-20 ℃ after 3 minutes, after electric shock, the bacterial liquid in the electric shock transformation cup is transferred to a sterile centrifuge tube with the concentration of 2.0M L, M408 liquid culture medium with the concentration of 800 mu L is additionally added, the mixture is cultured for 45 minutes at the temperature of 30 ℃ and then coated on a flat plate containing corresponding antibiotics, the culture is cultured for 36 hours at the temperature of 30 ℃, then the growth condition of colonies is observed, the flat plate is placed in a body type fluorescence microscope, and positive clones are selected according to the fluorescence brightness, as shown in figure 2, the rhizobium clone on the flat plate emits obvious red fluorescence under the excitation wavelength of the red fluorescence, and meanwhile, the bacterial clone of the slow rhizobium with the pRBC-NPTIIpomo-Tdtomotor vector under white light shows the macroscopic red color, which indicates that the pRBC-NPTIIpomo-TBradyrhizobium elkaniiA large amount of red fluorescent protein was produced.
Example 3 Slow Rhizobium Using fluorescent labelingBradyrhizobium elkaniiObserve the colonization of the soybean root system
Colonization of fluorescently-labeled bradyrhizobia on soybean root systems: culturing of fluorescently labeled bradyrhizobiaBradyrhizobium elkaniiCentrifuging at 6000rpm until OD600 = 1.0, collecting thallus, re-suspending thallus with sterile water until thallus OD600 = 0.3, soaking soybean root system of 5 days old in thallus re-suspending liquid, and standing for 2 hr. Low nitrogen contentCulturing with sand for 7-10 days, digging out soybean root system, removing sand, and observing rhizobium colonization on soybean root system under laser confocal microscope. As a result, as shown in FIG. 3, the obvious red fluorescence emitted by the rhizobia was observed on the root hair of soybean and on the root surface of the root system of soybean, indicating that the bradyrhizobia marked with pRBC-NPTIIpro-Tdtomato vector can be directly used for observing the colonization study of the rhizobia on the plant root system.
Example 4 study of interaction between Rhizobium and Soybean Using fluorescently labeled bradyrhizobium
Culturing of fluorescently labeled bradyrhizobiaBradyrhizobium elkaniiCentrifuging at 6000rpm until OD600 = 1.0, collecting thallus, re-suspending thallus with sterile water until thallus OD600 = 0.3, soaking soybean root system of 5 days old in thallus re-suspending liquid, and standing for 2 hr. The culture was continued for 30 days under low nitrogen. And collecting mature nodules, slicing the nodules, and observing the rhizobia with the fluorescence marks in the nodules by using a laser confocal microscope. As shown in FIG. 4, after soybean infection with the rhizobium labeled with red fluorescence and propagation for multiple generations, obvious red fluorescence can be observed in the formed soybean rhizobium through a fluorescence microscope, which indicates that the pRBC-NPTIipro-Tdtomato vector can stably exist in the bradyrhizobium and still has strong red fluorescence after passage for multiple generations. The pRBC-NPTIipro-Tdtomato vector can be used for researching the interaction between the bradyrhizobium and the leguminous plants.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> slow-growing rhizobium stable red fluorescent labeling vector and application thereof
<130>5
<160>5
<170>PatentIn version 3.3
<210>1
<211>7427
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gttgcgctat cgaccagctc aattaactgc tcgggctcgg acgcgaacca cgcgaagctg 3060
ccccaagcca aggagtcgag ggagccacgg ttgatgagag ctttgttgta ggtggaccag 3120
ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg 3180
atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccacgt tgtgtctcaa 3240
aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct 3300
gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc 3360
tcgaagccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc 3420
gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca 3480
gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc 3540
agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact 3600
cctgatgatg catggttact caccactgcg atccccggga aaacagcatt ccaggtatta 3660
gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg 3720
ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct 3780
caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt 3840
aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg 3900
gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa 3960
ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc 4020
atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa 4080
tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt 4140
ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg 4200
gacggcggct ttgttgaata aatcgcattc gccattcagg ctgcgcaact gttgggaagg 4260
gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag 4320
gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag 4380
tgccaagctt gcatgccacg ctgccgcaag cactcagggc gcaagggctg ctaaaggaag 4440
cggaacacgt agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac 4500
tgggctatct ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg 4560
cttacatggc gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca 4620
gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg 4680
ccgccaagga tctgatggcg caggggatca agatctgatc aagagacagg atactagtgg 4740
aggaagaaaa aatggtgagc aagggcgagg aggtcatcaa agagttcatg cgcttcaagg 4800
tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc 4860
gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct 4920
tcgcctggga catcctgtcc ccccagttca tgtacggctc caaggcgtac gtgaagcacc 4980
ccgccgacat ccccgattac aagaagctgt ccttccccga gggcttcaag tgggagcgcg 5040
tgatgaactt cgaggacggc ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg 5100
gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt cccccccgac ggccccgtaa 5160
tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg 5220
tgctgaaggg cgagatccac caggccctga agctgaagga cggcggccac tacctggtgg 5280
agttcaagac catctacatg gccaagaagc ccgtgcaact gcccggctac tactacgtgg 5340
acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc 5400
gctccgaggg ccgccaccac ctgttcctgg ggcatggcac cggcagcacc ggcagcggca 5460
gctccggcac cgcctcctcc gaggacaaca acatggccgt catcaaagag ttcatgcgct 5520
tcaaggtgcg catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg 5580
agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc 5640
tgcccttcgc ctgggacatc ctgtcccccc agttcatgta cggctccaag gcgtacgtga 5700
agcaccccgc cgacatcccc gattacaaga agctgtcctt ccccgagggc ttcaagtggg 5760
agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt gacccaggac tcctccctgc 5820
aggacggcac gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc 5880
ccgtaatgca gaagaagacc atgggctggg aggcctccac cgagcgcctg tacccccgcg 5940
acggcgtgct gaagggcgag atccaccagg ccctgaagct gaaggacggc ggccgctacc 6000
tggtggagtt caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact 6060
acgtggacac caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt 6120
acgagcgctc cgagggccgc caccacctgt tcctgtacgg catggacgag ctgtacaagt 6180
aagaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 6240
ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 6300
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 6360
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tggcgaactt 6420
ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa 6480
agcaaaagtt caaaatcagt aaccgtcagt gccgataagt tcaaagttaa acctggtgtt 6540
gataccaaca ttgaaacgct gatcgaaaac gcgctgaaaa acgctgctga atgtgcgagc 6600
ttcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 6660
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 6720
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 6780
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 6840
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 6900
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 6960
aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg 7020
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 7080
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 7140
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 7200
gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 7260
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 7320
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 7380
tttgatcttt tctacggggt ctgacgctca gtggaacgat ccgtcga 7427
<210>2
<211>35
<212>DNA
<213> Artificial sequence
<400>2
cagtgccaag cttgcatgcc acgctgccgc aagca 35
<210>3
<211>38
<212>DNA
<213> Artificial sequence
<400>3
ccatgattac gaattcacta gtatcctgtc tcttgatc 38
<210>4
<211>51
<212>DNA
<213> Artificial sequence
<400>4
gatcaagaga caggatacta gtggaggaag aaaaaatggt gagcaagggc g 51
<210>5
<211>42
<212>DNA
<213> Artificial sequence
<400>5
agctatgacc atgattacga attcttactt gtacagctcg tc 42
Claims (2)
1. A slow rhizobium stable red fluorescent labeling vector is characterized in that the sequence of the vector is shown as SEQ ID NO. 1.
2. An application of a slow rhizobium stable red fluorescent labeling vector in researching interaction of slow rhizobium and soybean.
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Citations (7)
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| WO1988000617A1 (en) * | 1986-07-22 | 1988-01-28 | Boyce Thompson Institute For Plant Research | Use of bacterial luciferase structural genes for cloning and monitoring gene expression in microorganisms and for tagging and identification of genetically engineered organisms |
| US5137816A (en) * | 1983-06-22 | 1992-08-11 | The Lubrizol Corporation | Rhizobial diagnostic probes and rhizobium trifolii nifH promoters |
| US5232840A (en) * | 1986-03-27 | 1993-08-03 | Monsanto Company | Enhanced protein production in bacteria by employing a novel ribosome binding site |
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| US5137816A (en) * | 1983-06-22 | 1992-08-11 | The Lubrizol Corporation | Rhizobial diagnostic probes and rhizobium trifolii nifH promoters |
| US5232840A (en) * | 1986-03-27 | 1993-08-03 | Monsanto Company | Enhanced protein production in bacteria by employing a novel ribosome binding site |
| WO1988000617A1 (en) * | 1986-07-22 | 1988-01-28 | Boyce Thompson Institute For Plant Research | Use of bacterial luciferase structural genes for cloning and monitoring gene expression in microorganisms and for tagging and identification of genetically engineered organisms |
| CN103409406A (en) * | 2013-08-22 | 2013-11-27 | 甘肃农业大学 | Method for constructing fluorescence labeled rhizobia |
| CN105453923A (en) * | 2015-12-08 | 2016-04-06 | 甘肃农业大学 | Method for promoting target rhizobium to migrate and colonize in alfalfa plant |
| US20170233756A1 (en) * | 2016-02-15 | 2017-08-17 | Benson Hill Biosystems, Inc. | Compositions and methods for modifying genomes |
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| CN111471702B (en) | 2022-07-26 |
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