CN109337847A - One plant of Cassia rhizobium TXR2 and its application - Google Patents
One plant of Cassia rhizobium TXR2 and its application Download PDFInfo
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- CN109337847A CN109337847A CN201811480784.7A CN201811480784A CN109337847A CN 109337847 A CN109337847 A CN 109337847A CN 201811480784 A CN201811480784 A CN 201811480784A CN 109337847 A CN109337847 A CN 109337847A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/41—Rhizobium
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
Abstract
The invention discloses a kind of rhizobium TXR2 and its applications, belong to microorganisms technical field.The bacterial strain is separated and is purified from the fresh root nodule of legume forages of Chamaecrista spp, PCR detectionnodAGene, and obtained by 16S rDNA molecular biology identification, be determined as Bradyrhizobium (Bradyrhizobium) new strains system, and be named as day circle No. 2.China typical culture collection center is preserved on April 10th, 2018, deposit number is CCTCC NO:M2018193.Rhizobium TXR2 of the invention proves its characteristic strong with efficient dross and nitrogen fixing capacity by the test of laboratory potting tieback, root nodule number, nodule nitrogen fixation efficiency, biomass and the N content of crop tissue of legume forages of Chamaecrista spp can be significantly improved by being inoculated with the rhizobium, and then is achieved the effect that culture fertility and improved the ecological environment.
Description
Technical field
The invention belongs to microorganism fields, and in particular to one plant of Cassia rhizobium TXR2 and its application.
Background technique
There are about 56,900,000 hm in China2Acid soil accounts for 42.2% or more total area under cultivation.In recent years, with big quantization
The application of nitrogenous fertilizer is learned, soil acidification is on the rise.China's acid soil is concentrated mainly on the Changjiang river areas to the south, this area's light, heat,
Water etc. is resourceful, but soil is thin and weak, glue weight, pH value is low and nutrient availability is low, seriously constrains the quick of this area's agricultural
Development.In production, crop yield is improved often through a large amount of chemical fertilizer are applied.But the investment of a large amount of chemical fertilizer not only results in
Soil hardening and further acidification, but also will cause the pollution of water eutrophication and underground water, seriously destroy ecological environment.
By plantation leguminous plant combine legume inoculation technology, come improve soil, increase soil fertility be at present more
A kind of approach approved.Leguminous plant can form mutually beneficial symbiosis in conjunction with rhizobium.Rhizobium are from host plant
It obtains growing required carbohydrate and other nutrients, and host plant then obtains nitrogen during rhizobium biological nitrogen fixation
Nutrition to achieve the effect that improve crop yield and reduce fertilizer application amount, and then is kept while reducing water and soil pollution
The sustainable development of agricultural.
Cassia plant (Chamaecrista sppGreene.) it is the important leguminous plant in tropical and subtropical region,
Including three classes such as arbor, shrub and draft.Chamaecrista Rotundifolia (Chamaecrista rotundifolia (Pers.) Greene)
Australia is originated in, is the perennial leguminous herbaceous plant of half erect type of Cassia, there is nitrogen-fixing efficiency height, impoverishment tolerant, biology
It measures that larger, pattern is gorgeous, especially adapts to the characteristics such as acid soil.1996, Fujian Academy of Agricultural Sciences was from Australia Forage Grasses
Germ plasm resource center introduce Chamaecrista Rotundifolia, and as improvement acid soil leguminous green manure type herbage, Fujian, Guangdong and
The acid soils area such as Guangxi popularizing planting.But Chamaecrista Rotundifolia under field conditions (factors) dross slowly and less, nitrogen-fixing efficiency it is lower,
It seriously affects its plantation and promotes and applies.It therefore, is the dross rate and nitrogen-fixing efficiency that improve Chamaecrista Rotundifolia, urgent need filters out dross
Rate height and the strong rhizobium strains of nitrogen fixing capacity to improve the nitrogen-fixing efficiency of legume forages of Chamaecrista spp, and then reach culture fertility and change
The effect of kind ecological environment.
Summary of the invention
One of the objects of the present invention is to provide one plant of legume forages of Chamaecrista spp rhizobium TXR2, the classification naming roots of the bacterial strain
Tumor bacterium (BradyrhizobiumSp.) TXR2 was the new strains system of Bradyrhizobium, in preservation on April 10 in 2018
In China typical culture collection center, deposit number is CCTCC NO:M2018193, preservation address are as follows: Wuhan University.It should
Bacterial strain has the characteristics that dross rate height and nitrogen fixing capacity are strong.
Another object of the present invention is to apply above-mentioned rhizobium TXR2 in leguminosae cassia plant symbiosis fixed nitrogen,
By improving the biological nitrogen fixation ability of Chamaecrista Rotundifolia, promotes its growth, achieve the purpose that synergy of losing weight in Cassia production.
Above-mentioned technical purpose of the invention is achieved through the following technical solutions:
The separation and purifying of 1 rhizobium strains
1) it takes that Chamaecrista Rotundifolia is fresh, mature and a big full root nodule, is rinsed with water clean, and blot surface moisture with filter paper；
2) be put into 95%(w/v) alcohol in handle 3 ~ 5 minutes, place into 0.1% (w/v) HgCl2 In, it sterilizes 3 ~ 5 points
Clock is used aseptic water washing 5 ~ 6 times after taking-up；
3) it cuts in half on the glass slide of flame sterilization, clamps half of tumor with aseptic nipper, notch cultivates base table towards YMA
Face scribing line, is cultivated at 28 DEG C after inversion.The formula of YMA culture medium is as shown in table 1:
1 YMA(Yeast Mannitol Agar of table) culture medium prescription
4) after growing thallus, a small amount of rhizobium bacterium colony is scraped from plate with pipette tips, again in flat after adding 1mL sterile water to dilute
Lining out culture, 3 d observe bacterium colony situation later, observe 15d always (slow raw rhizobium need 7 ~ 15d bacterium colony occur).
Such as occur without monoclonal colonies, then needs to purify in flat lining out repeatedly, until there are monoclonal colonies.
5) after growing monoclonal thallus, judge whether it is rhizobium according to following two aspect:
1. colonial morphology: the bacterium colony of rhizobium is that circle, milky, translucent, neat in edge, cement are more or less.Culture 3 ~
5 d colony diameters are fast-growing Rhizobium up to 2 ~ 4 mm, and 7 ~ 10 d colony diameters of culture are that 1 mm is then slow raw type root
Tumor bacterium.
②nodAThe PCR of gene is detected: the above-mentioned form rhizobium monoclonal of picking carries outnodAGene PCR inspection
It surveys, primer is respectivelynodA- F andnodA- R, forward primer SEQ ID NO.1:nodA-F 5’-
TGCRGTGGARDCTRYGCTGGGAAA-3'；Reverse primer SEQ ID NO.2:nodA-R 5’-
GNCCGTCRTCRAASGTCARGTA-3'.Enzyme selects 2 × starMix, respectively with the genomic DNA of rhizobium BXYD3 and
Water is that template does positive control and negative control.nodAGene length be 666 bp, with electrophoretic image technology observe its
Whether there is band at 666 bp, the bacterium colony with band is rhizobium.Its PCR reaction system is as follows:
Table 2 nodA Gene 2 × starMix enzyme reaction system
Reaction condition: 94 DEG C of 5 min；(94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1 min) × 35 cycle；70℃ 5 min .
The property of 2 rhizobium TXR2
1) morphological feature
The bacterial strain is on colonial morphology, and for slow raw type, circle is smaller, milky, translucent, cement is more.On YMA plate
Diameter after growing 5 ~ 7 d is 0.8 ~ 2.0 mm (attached drawing 1).
2) cultural character
The optimum growing condition of the bacterium are as follows: pH=7.0,28 DEG C of temperature, 180 r/min of revolving speed, using mannitol and yeast
Powder is respectively as its carbon source and nitrogen source.
3) genetic characteristics
ThroughnodAGene PCR, electrophoretic image detection, have at 666 bp compared with bright wisp band (attached drawing 2).
4) functional characteristic
Rhizobium TXR2 has the characteristics that dross rate height and nitrogen fixing capacity are strong.Thallus is released in natural environment, to people, is moved
Object and non-phytotoxic, do not pollute the environment, and enrich the population diversity of rhizobium between soil instead, to the knot of legume forages of Chamaecrista spp
Tumor fixed nitrogen has apparent facilitation.
The 16S rDNA molecular biology identification of 3 rhizobium TXR2
For the Phylogenetic for identifying rhizobium strains, 16S rDNA's is carried out to the separated rhizobium strains arrived
Forward primer SEQ ID NO.3 is sequenced in PCR specific amplification:V4V5515-F 5'-GTGCCAGCMGCCGCGGTAA-3'；
Reverse primer SEQ ID NO.4 is sequenced:V4V5907-R 5'-CCGTCAATTCCTTTGAGTTT-3'；Sequencing gained rhizobium
The 16S rDNA sequence of JYN6 is as shown in SEQ ID NO.5；Its PCR reaction system is as follows:
3 16S rDNA2 × starMix enzyme reaction system of table
Reaction condition: 95 DEG C of 5 min；(95 DEG C of 20 s, 55 DEG C of 20 s, 72 DEG C of 50s) × 44 cycle；70℃ 5 min.
13 reference strains sequences are obtained from NCBI (GenBank) database, with software BioEdit and
MEGA6 analyzes the 16S rDNA partial sequence of isolated strains and reference strains, constructs isolated strains and reference strains
Phylogenetic tree.So that it is determined that rhizobium TXR2 be Bradyrhizobium (Bradyrhizobium) new strains system (attached drawing
3), and it is named as day circle 2.
The invention has the following beneficial effects:
1) rhizobium TXR2 of the invention has a dross rate height and the strong characteristic of nitrogen fixing capacity, tieback test discovery, and is not inoculated with
Rhizobium are compared, and are inoculated with 30 d of rhizobium and Chamaecrista Rotundifolia fresh weight and dry weight 223.16wt% and 293.33wt% is respectively increased, can
For actual production；2) Chamaecrista Rotundifolia is inoculated with rhizobium TXR2 of the invention, improve extremely significantly total nitrogen content (P<
0.01), to achieve the purpose that promote Chamaecrista Rotundifolia plant strain growth and culture fertility；3) rhizobium TXR2 of the invention is applicable in
It, can one of important measures as Chamaecrista Rotundifolia conventional cultivation in acid soil in the south area.
Detailed description of the invention
Fig. 1 is colonial morphology of the rhizobium TXR2 on YMA culture medium.
Fig. 2 is rhizobium TXR2 warpnodAGene PCR, electrophoretic image detection institute are at image；Wherein ,+represent and use
The DNA of rhizobium BXYD3 is that template does positive control, and-expression does negative control with water
Fig. 3 is the 16S rDNA Analysis of Partial phylogenetic tree of rhizobium TXR2；Wherein, the digital table in branch
Show confidence level；Accession number of the bracket content representation in Gene bank；Scale bar indicates have 2 to replace in 100 nucleotide
It changes.
Fig. 4 is rhizobium TXR2 vermiculite tieback effect picture；CK: blank control.
Chamaecrista Rotundifolia dross number and nodule nitrogenase activities after 30 d of Fig. 5 Rhizobium Inoculation TXR2；Wherein, A: roundleaf
Cassia dross number；B: Chamaecrista Rotundifolia nodule nitrogenase activities, CK: blank control.
The influence that 30 d of Fig. 6 Rhizobium Inoculation TXR2 grows Chamaecrista Rotundifolia；Wherein, A: Chamaecrista Rotundifolia fresh weight；B: circle
Leaf Cassia dry weight；C: Chamaecrista Rotundifolia plant height；D: Chamaecrista Rotundifolia SPAD value；E: Chamaecrista Rotundifolia plant nitrogen content；CK: blank pair
According to；*: 0.01 <P< 0.05, * *: 0.001 <P< 0.01, * * *:P<0.001。
Specific embodiment
The separation and purifying of 1 nodule nitrogen fixation bacteria strain TXR2 of embodiment
The separation of 1 rhizobium
It takes that Chamaecrista Rotundifolia is fresh, mature and a big full root nodule, is rinsed with water clean, blot surface moisture with filter paper.First put
Enter 95%(w/v) ethyl alcohol in handle 3 ~ 5 minutes, take out use aseptic water washing 5 ~ 6 times, place into 0.1%(w/v)
HgCl2 Sterilizing 3 ~ 5 minutes is taken out and is used aseptic water washing 5 ~ 6 times.Then it cuts in half on the glass slide of flame sterilization,
Half of tumor is clamped with aseptic nipper, notch is crossed towards YMA (table 1) media surface, cultivated at 28 DEG C after inversion.
2 purifying
After growing thallus, a small amount of rhizobium bacterium colony is scraped from plate with pipette tips, with 1 mL sterile water dilution after again in
Flat lining out culture, 3 d observe bacterium colony situation later, observe that (slow raw rhizobium need 7 ~ 15 d to occur to 15 d always
Bacterium colony).Such as occur without monoclonal colonies, then needs to purify in flat lining out repeatedly, until there are monoclonal colonies.
Rhizobium can be tentatively judged whether it is according to following two aspect:
1. colonial morphology: the bacterium colony of rhizobium is circle, milky, translucent, neat in edge, and cement is more or less.Culture 3 ~
5 d colony diameters are fast-growing Rhizobium up to 2 ~ 4 mm, and 7 ~ 10 d colony diameters of culture are then to give birth to type root slowly up to 1 mm
Tumor bacterium.
②nodAGene PCR detection: the above-mentioned form rhizobium monoclonal of picking carries outnodAGene PCR detection,
Primer is respectivelynodA- F andnodA- R, enzyme select 2 × starMix, use the genomic DNA of rhizobium BXYD3 respectively
It is that template does positive control and negative control with water.The band length of this and gene is 666 bp, with the electrophoretic image conceptions of technology
Examining bacterial strain TXR2 has at 666 bp compared with bright wisp band, preliminary judgement its be rhizobium.Then isometric 50% w/v is added)
Glycerol puts -80 DEG C of guarantor bacterium.
The present embodiment bacterial strain TXR2 is Slow_growing rhizobia, is round smaller, milky on colonial morphology, semi-transparent
It is bright, cement is more；Diameter after growing 5 ~ 7 d on YMA plate is 0.8 ~ 2.0 mm(attached drawing 1)；Optimum growing condition
Are as follows: pH=7.0,28 DEG C of temperature, 180 r/min of revolving speed, using mannitol and yeast powder respectively as its carbon source and nitrogen source；
ThroughnodAThe genomic DNA of gene PCR, electrophoretic image detection, band and positive control rhizobium BXYD3 are template
Band consistent (mrna length is 666 bp, such as attached drawing 2.
It with this isolation and purification method, is purified repeatedly by 4 generations, the scribing line training in YMA solid medium (table 1)
It supports, obtains more plants of pure bacterium, pure bacterium isolates and purifies dross rate through its nodulation and nitrogen fixation ability of vermiculite tieback verification experimental verification, the present embodiment
The high and strong bacterial strain TXR2 of nitrogen fixing capacity.
The 16S rDNA molecular biology identification of 2 rhizobium TXR2 of embodiment
The PCR specific amplification of 16S rDNA is carried out to rhizobium monoclonal bacterium solution, forward primer is SEQ ID NO.3:V4V5515-F 5'-GTGCCAGCMGCCGCGGTAA-3'；Reverse primer is SEQ ID NO.4:V4V5907-R 5’-
CCGTCAATTCCTTTGAGTTT-3'.Enzyme selects 2 × star Mix.PCR amplified production does electrophoretic image technology and is examined
It surveys, observes whether it has band, remaining PCR amplified production is used for sequencing.Sequencing result such as SEQ ID NO:5
It is shown.Its PCR reaction system such as table 4.
4 16S rDNA2 × starMix enzyme reaction system of table
Reaction condition: 95 DEG C of 5 min；(95 DEG C of 20 s, 55 DEG C of 20 s, 72 DEG C of 50s) × 44 cycle；70℃ 5 min.
13 reference strains sequences are obtained from NCBI (GenBank) database, with software BioEdit and
MEGA6 analyzes the 16S rDNA partial sequence of isolated strains and reference strains, constructs isolated strains and reference strains
Phylogenetic tree (attached drawing 3).So that it is determined that rhizobium TXR2 be Bradyrhizobium (Bradyrhizobium) new bacterium
Strain, and it is named as day circle 2.
The test of 3 tieback of embodiment
Test objective: filtering out has higher joint efficiency and the stronger rhizobium of nitrogen fixing capacity with legume forages of Chamaecrista spp.
Test material: test plant: No. 1 Chamaecrista Rotundifolia is educated in Fujian；Strains tested: the rhizobium isolated and purified.
Main experimental instrument and equipment: artificial climate growth room, superclean bench, high-pressure sterilizing pot, constant incubator, 15
The plant pot 25 of × 15 cm, 2, beaker, tweezers, culture dish, filter paper, glass bar, scissors and the gauze of 1 L etc..
Test drug and reagent
(1) test drug: mannitol, MgSO4∙7H2O, NaCl, yeast powder, K2HPO4、KH2PO4、CaCO3、Ca(NO3)2∙
4H2O、MgSO4·7H2O、CaCl2·2H2O、Na2HPO4·12H2O、C10H12N2O8FeNa·3H2O、Na2MoO4、MnSO4、
H3BO3、CuSO4·5H2O and ZnSO4·7H2O。
Test reagent:
1) 10 g of mannitol, MgSO YMA (Yeast Mannnitol Agar) fluid nutrient medium: are weighed4∙7H2O 0.2g,
0.1 g of NaCl, yeast powder 3 g, K2HPO40.25 g, KH2PO40.25 g, CaCO33 g (are added) when preservation, agar 15
G is dissolved in 1 L pure water.
2) plant Poor nitrogen nutrition liquid: Ca (NO3)2∙4H2O 0.03 g, MgSO4·7H2O 0.28 g, CaCl2·2H2O
0.10 g, KH2PO40.10 g, Na2HPO4·12H2O 0.15 g, C10H12N2O8FeNa·3H20.0075 g of O, microelement
1 mL, distilled water 1000 mL, pH 5.5.Trace element formula (g L-1): Na2MoO40.03 g, MnSO41.81 g,
H3BO32.86 g, CuSO4·5H2O 0.8 g, ZnSO4·7H2O 0.22 g。
Test procedure
(1) strain culturing
The strains tested switching that -80 DEG C are saved is in YMA Liquid Culture, culture to logarithmic phase.That is OD value reaches 1 ~ 1.2.
(2) sterilization treatment
Plant pot required by testing, beaker, tweezers, culture dish, filter paper, glass bar, gauze, scissors are wrapped, and vermiculite is used
Freshness protection package installs, and 121 DEG C, sterilize 30 min.YMA fluid nutrient medium, distilled water are installed with bottle, and 121 DEG C, sterilizing 20
min。
Test plant seed is immersed in 80 DEG C of hot water and impregnate 3 min, make kind of soft and soggyization, jelly is precipitated, is used in combination
Clear water repeated flushing is clean, later by Chamaecrista Rotundifolia seed through 75%(w/v) 15 min of ethanol postincubation, with aseptic water washing 5 ~ 6
It is secondary, with 10%(w/v) H2O2 4 min of surface sterilization is carried out, then with aseptic water washing 7 ~ 8 times, is finally putting into filter paper
10 h of vernalization at 28 DEG C of culture dish, grows 0.2 cm to radicle.
(3) vermiculite culture and inoculation
(1) sterilized vermiculite, is then contained in sterilized potting by the bottom that meche is passed through to sterilized plant pot
In basin, plant Poor nitrogen nutrition liquid is made to guide to matrix and plant root by meche, to guarantee that Chamaecrista Rotundifolia herbage growth is developed
Demand to nutrition and moisture.Then plant pot is covered in the beaker equipped with nutrient solution.This upper layer and lower layer is wrapped up with masking foil
Device, to avoid nutrient solution under illumination condition long moss.
(2) by processed Chamaecrista Rotundifolia seed bunch planting in vermiculite, by Chamaecrista Rotundifolia seed Rhizobium Inoculation bacterium solution,
Every 2 mL of basin, bacterial content are 1,000,000,000 every milliliter, finally cover the vermiculite of 1 cm of thickness.Be placed in growth room (photoperiod are as follows:
Day night=16h/8h, temperature are as follows: day night=26 DEG C/24 DEG C) culture.
Using not Rhizobium Inoculation bacterium solution as blank control, do 3 repetitions, harvested after being inoculated with 30 d, with plant fresh weight,
Dry weight, dross number, plant nitrogen content, rhizobium nitrogenase activity, SPAD value and plant height are as measurement rhizobium associativity and admittedly
The judge index of nitrogen ability.
Interpretation of result
As shown in Fig. 5, without dross phenomenon, average every 5 Chamaecrista Rotundifolias of tieback processing can dross for blank control (CK)
44.33.Root nodule discovery is cut, is red inside root nodule, primitive decision strains tested TXR2 is the root with nitrogen-fixing efficiency
Tumor bacterium (attached drawing 4), and its nodule nitrogenase activities is measured, value is 0.47 umol (g h)-1。
By Fig. 6 it is found that compared with the blank control of not Rhizobium Inoculation, No. 1 Rhizobium Inoculation is educated in Chamaecrista Rotundifolia Fujian
TXR2 can extremely significant its plant fresh weight of raising and plant weights (P< 0.01), extremely extremely significant raising plant height, SPAD value and plant contain
Nitrogen quantity (P<0.001).Illustrate that associativity and the nitrogen-fixing efficiency of rhizobium TXR2 are higher.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
<120>one plants of Cassia rhizobium TXR2 and its applications
<130> 5
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213> nodA-F
<400> 1
tgcrgtggar dctrygctgg gaaa 24
<210> 2
<211> 22
<212> DNA
<213> nodA-R
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, or t
<400> 2
gnccgtcrtc raasgtcarg ta 22
<210> 3
<211> 19
<212> DNA
<213> V4V5515-F
<400> 3
gtgccagcmg ccgcggtaa 19
<210> 4
<211> 20
<212> DNA
<213> V4V5907-R
<400> 4
ccgtcaattc ctttgagttt 20
<210> 5
<211> 388
<212> DNA
<213>the 16S rDNA sequence of rhizobium TXR2
<400> 5
ctcctaagac gttgcctcgg atcactgggc gtaagggtgc gtaggcgggt cctttaagtc 60
agaggtgaaa tccctggagc tcaactccag aactgccttt gatactgaag atcttgagtt 120
cgggagaggt gagtggaact gcgagtgtag aggtgaaatt cgtagatatt cgcaagaaca 180
ccagtggcga aggcggctca ctggcccgat actgacgctg aggcacgaaa gcgtggggag 240
caaacaggat tagataccct ggtagtccac gccgtaaacg atgaatgcca gccgttagtg 300
ggtttactca ctagtggcgc agctaacgct ttaagcattc cgcctgggga gtacggtcgc 360
aagattaaaa ctcaaaggaa ttgacgga 388
Claims (5)
1. one plant of Cassia rhizobium TXR2, it is characterised in that: the rhizobium (BradyrhizobiumSp.) TXR2 is
It separates and purifies from the fresh root nodule of legume forages of Chamaecrista spp, PCR detectionnodAGene, and through 16S rDNA molecular biology
Identification gained, is preserved in China typical culture collection center, deposit number CCTCC on April 10th, 2018
NO:M2018193.
2. one plant of Cassia rhizobium TXR2 according to claim 1, it is characterised in that: the PCR detectionnodA
The primer sequence of gene, as shown in SEQ ID NO.1 and SEQ ID NO.2.
3. one plant of Cassia rhizobium TXR2 according to claim 1, it is characterised in that: the 16S rDNA molecule
Biology identification the primer sequence, as shown in SEQ ID NO.3 and SEQ ID NO.4.
4. one plant of Cassia rhizobium TXR2 according to claim 1, it is characterised in that: the 16S rDNA sequence
Column, as shown in SEQ ID NO.5.
5. one plant of Cassia rhizobium TXR2 as described in claim 1 improves biological nitrogen fixation energy in species Cassia platymiscium
Application in power.
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| CN105274030A (en) * | 2015-11-12 | 2016-01-27 | 领先生物农业股份有限公司 | Rhizobium and application thereof |
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|---|---|---|---|---|
| CN101748088A (en) * | 2009-12-29 | 2010-06-23 | 刘国道 | Bacterial strain of root nodule nitrogen-fixing strain series RY3 and application thereof |
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