CN104830692B - Sphingomonas gracilis scsio T-2 and application thereof - Google Patents
Sphingomonas gracilis scsio T-2 and application thereof Download PDFInfo
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Abstract
The invention discloses sphingomyelina gracilis scsio T-2 and application thereof. Sphaeria leptocyani (Leptolyngbya sp.) scsio T-2, accession number: CCTCC NO: m2015061. The invention screens a new blue algae strain namely the Sphingomonas leptospira (Leptolyngbya sp.) scsio T-2 from the leaf surface of Thalassia (Thalassia hemerichi), which has good nitrogen fixation activity and can secrete and generate 3-indoleacetic acid, so that the Sphingomonas leptospira (Leptolyngbya sp.) scsio T-2 has higher value and wide application prospect in the aspect of marine microbial fertility preparation.
Description
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to sphingomyelina gracilis scsio T-2 and application thereof.
Background art:
seaweeds are photoautotrophic, have a wide distribution in the world's temperate, tropical and subtropical coastal ecosystems, are an important component of the typical tropical marine ecosystems, and are one of the highest productive ecosystems in nature.
Sea grass is a flowering plant growing in the sea, and is extremely rare in species compared with terrestrial higher plants, and only 72 species are known globally, wherein 15 species are already in an endangered or extincted state; waycott et al (2009) analyzed data from 1879 on 215 monitoring points worldwide, found that about 58% of seaweeds are in a decline state, the speed of disappearance of seaweed area is increasing with time, and from less than 1% before 1940 to 7% since 1990, loss of habitat, loss of biodiversity and global climate change are all important reasons, so it is important to increase the research strength on ecological function of seaweed beds and expand the research field of seaweed beds (Short et al, 2011).
Most of the seaweed is distributed in oligotrophic sea areas, nitrogen is usually the main limiting factor of productivity, and nitrogen-fixing microorganisms provide a new nitrogen source for an ecosystem by performing biological nitrogen fixing action, so that nitrogen limitation is slowed to a certain extent, and the growth of the seaweed is promoted. The nitrogen-fixing microbial community mainly comprises bacteria such as blue algae (cyanobacteria), actinomycetes and proteobacteria, methanogenic archaea and the like. Hamisi et al (2009) studied nitrogen-fixing microorganisms in the beds of Haematococcus tanzanioides, the west Indian ocean coast found that blue-green algae are a major component of the nitrogen-fixing microbial community, such as Oscillatoria, Sphaerotheca, Pseudoanabaena, Microcoleus, Coccomyxococcus, Chroococcum, and Chroococcoid.
Blue algae of the genus leptospira (Leptolyngbya) are thin filamentous (<3 μm) blue algae, xeno-free, characterized by loose coil-like clusters (Rippka & Herdman,1992), with a wide distribution of blue algae in different ecoenvironments, such as oceans, fresh water, sediments, archaeological sites, etc. (Wilmotte & Herdman, 2001). The classification status of Sphingomonas leptospira is still controversial and is difficult to identify (Bruno et al,2009). The blue algae of the genus has reports (Ahmed et al, 2014) about high biological nitrogen fixation activity and capability of producing indoleacetic acid (IAA), the indoleacetic acid (IAA) can participate in regulation and control of a plurality of physiological and biochemical processes of plant cell elongation growth, cambium cell division, vascular tissue differentiation, leaf and flower abscission and the like, and also has regulation effects on apical dominance, tropism, transportation of assimilates and the like of plants (Asneboro et al, 2005).
Indian De uses nitrogen-fixing blue-green algae as fertilizer in rice paddy in 1939, then more than 10 countries such as the United states, the United kingdom, Japan and Egypt also develop related research, the research in the aspect of China started at the earliest is Rishanhao and the like (1959), and the fact that the nitrogen-fixing blue-green algae inoculated in the rice paddy can effectively provide nitrogen fertilizer and fertility soil for rice expenditure and increase the yield of rice by 10-30% is proved; the application of nitrogen-fixing blue algae can effectively promote the growth of wheat roots and buds and the field emergence rate (Wangmai et al, 1991), but the application of nitrogen-fixing blue algae as bacterial manure in a marine ecosystem is rarely reported.
The invention content is as follows:
the first purpose of the invention is to provide a new blue algae Sphingomonas leptospira (Leptolyngbya sp.) scio T-2 which is separated from leaves of Thalassia gulfweed (Thalassia hemperimichii) of New village of Lingshui, Hainan province, China, has high nitrogen-fixing biological activity and can produce plant growth hormone indoleacetic acid, and is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 22 days 2015, and the address: wuhan university, the preservation number: CCTCC NO: m2015061.
According to Acetylene Reduction Activity (ARA), a gas chromatograph is adopted to measure the nitrogen fixation Activity of the scsio T-2 (Leptolyngbya sp.), and research results show that the scsio T-2 (Leptolyngbya sp.) has high nitrogen fixation Activity which is 45 +/-12.9 nmol N2μg-1Chla。
The culture solution of Sphaeria gracilis (Leptolyngbya sp.) scsio T-2 is used for culturing seeds of the mangrove plant, namely the Tinospora crassipes (oliv.) Merr, and the culture solution is found to effectively promote the germination, the budding and the growth of the seeds and has good growth promoting effect on the seeds. Therefore, the mystus leptocyani (Leptolyngbya sp.) scsio T-2 provided by the invention can be further prepared into biological nitrogen-fixing bacterial manure and applied to the protection and restoration of typical marine ecosystems such as mangrove forest ecosystems and the like.
Therefore, the second purpose of the invention is to provide the application of Sphingomonas leptocyclina (Leptolyngbya sp.) scsio T-2 in preparing the growth-promoting azotobacter fertilizer.
The Salkowski colorimetry is utilized to measure the secretion of plant growth hormone substances of the scsio T-2 (Leptolyngbya sp.), a standard curve is made by pure 3-IAA (3-indoleacetic acid), and the yield is 9.5 +/-0.71 mu g/mg.
Therefore, the third purpose of the invention is to provide the application of Sphingomonas leptospira (Leptolyngbya sp.) sciio T-2 in the preparation of 3-indoleacetic acid.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates and screens a new blue algae strain-the lean sphingomyelinia sp scsio T-2 from the leaf surface of the Thalassia (T.hemperichi), which has good nitrogen fixation activity and can secrete and produce 3-indoleacetic acid, so that the lean sphingomyelinia sp scsio T-2 has higher value and wide application prospect in the aspect of marine microbial fertility preparation.
Leptolyngbya sp.scsio T-2 was deposited at the chinese culture collection center (CCTCC) on 1 month and 22 days 2015, address: wuhan university, the preservation number: CCTCC NO: m2015061.
Description of the drawings:
FIG. 1 is a phylogenetic tree of 16S rDNA of Sphingomonas leptocyania (Leptolyngbya sp.) scsio T-2, where scsio T-2 represents Sphingomonas leptocyania (Leptolyngbya sp.) scsio T-2;
FIG. 2 is a phylogenetic tree of nifH of Sphingomonas leptocystis (Leptolyngbya sp.) scsio T-2, where scsio T-2 represents Sphingomonas leptocystis (Leptolyngbya sp.) scsio T-2.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: isolation, purification and characterization of Sphingomonas leptospira (Leptolyngbya sp.) scsio T-2
1. Isolation and purification of Sphingomonas leptospira (Leptolyngbya sp.) scsio T-2
Collecting Thalassia sp collected from the New village of Lingshui, south China, adding seawater, scrubbing, and filtering to obtain blue algae solution. Selecting blue algae mycelium, and culturing in marine blue algae culture Medium ATCC Medium 957, blue algae culture Medium ATCC 819, and marine blue algae culture Medium ATCC Medium 1077 and BG-11Medium respectively with continuous illumination intensity of 150 μ E/m2S, lightThe illumination period is 12:12h (light: dark period), the temperature is 23-27 deg.C, the illumination incubator (model is LRH-250-GH microcomputer control illumination incubator) is transferred into fresh culture medium every two weeks until microscopic examination shows single mycelium. Thus obtaining the blue algae scsio T-2 by separation and purification.
2. Identification of Sphingomonas leptospira (Leptolyngbya sp.) scsio T-2
1) Morphological identification: under an optical microscope, the blue algae scsio T-2 cultured for two weeks is picked and placed on a glass slide, and is fixed through a cover glass for observation. The cyanobacteria scsio T-2 obtained in the embodiment is filamentous non-heteromorphic cytocyanobacteria, is not branched, and has different variable sheaths with different thicknesses.
2) Determination of blue algae nitrogen fixation activity
The azotobacter Activity of blue-green algae in this example was measured by gas chromatography for the Acetylene-reducing Activity (ARA) of azotobacter (refer to Capone 1993, Capone DG (1993) Determination of nitrifying Activity in aqueous sampling using the azonylene reduction process. aqueous microbiological Ecology, 621-631).
This example was divided into an experimental group and a blank control group.
Experimental groups: separating and purifying to obtain blue algae scsio T-2, inoculating the blue algae scsio T-2 into 100mL of selective liquid nitrogen-free liquid improved culture medium, and continuously irradiating at 150 muE/m2The light period is 12:12h (light: dark period), the temperature is 23-27 ℃, after 2 weeks of culture, 5mL of the solution is taken out of a sterilized 20mL small bottle, a rubber plug is covered, the edge of the bottle cover is sealed by paraffin, 1mL of acetylene is injected into the 20mL small bottle by an injector, the solution reacts for 12h at the temperature of 30 ℃, then 40 mu L of reacted gas is taken out to be measured; three replicates were set for each experimental group.
The formula of the selective liquid nitrogen-free liquid improved culture medium is as follows: MgSO (MgSO)4·7H2O,0.08g；CaCl20.04g；NaNO3 1.5g；K2HPO4 0.04g；NaHCO30.04 g; 2ml of Fe-EDTA; TA 52 ml; VB120.32ml, 500ml of distilled water and 1500ml of sterilized seawater are sterilized for later use.
Trace metal mix A5(TA5):H3BO3 2.86g；MnCl24H2O 1.81g；ZnSO4 7H2O 0.222g；NaMoO42H2O 0.39g；CuSO4 5H2O 0.079g；Co(NO3)26H2O49.4 mg; distilled water 1.0L, sterilized for use.
Blank control group: acetylene gas was added to the control vessel but no blue algae was added to the control vessel, for the same experimental group.
All samples were checked for ethylene production by gas chromatography.
40 μ L of reacted gas was used to measure the peak value of ethylene on the Shanghainegaku apparatus electric chromatography apparatus (GC-112A), and the ethylene concentration of the standard gas was 138 ppm. The operating conditions of the gas chromatograph are set as follows: the detection chamber temperature is 200 ℃, the column box temperature is 60 ℃, and the injection port is 120 ℃. The gauge head carrier gas pressure of nitrogen is 0.95kg cm-2Hydrogen gas of 0.8kg · cm-2Air of 0.6kg cm-2. The acetylene reduction activity calculation method comprises the following steps:
ARA(nmol C H·H-1·Culture-1)＝Vst×Cst×Asa×Vtu/Vsa/Ast/H/22.4
where Vst is the volume (ml) of injected standard gas, Cst is the concentration of standard gas, Vtu is the volume (ml) of the test tube used, Asa is the area (cm) of the ethylene peak of the sample, Vsa is the sample injection volume (ml), Ast is the area (cm) of the standard gas peak, and H is the incubation time.
The detection result shows that the pure bacterial strain scsio T-2 obtained by separation and purification of the embodiment can utilize N2As nitrogen source, has high biological nitrogen fixation activity, and the average nitrogen fixation activity under pure culture condition is 45 +/-12.9 nmol N2μg-1Chla。
Example 2 measurement of amount of produced indole acetic acid in cyanobacteria
The salkowski colorimetric method is adopted to determine the capability of the isolate to produce the plant growth hormone substances, and a standard curve is made by pure 3-IAA (3-indoleacetic acid). Inoculating blue algae scsio T-2 into 100mL of selective liquid nitrogen-free liquid modified culture medium (formula same as example 1), and continuously irradiating at 150 muE/m2The light period is 12:12h (light: dark period), the temperature is 25-28 deg.C, and culturing is carried outCentrifuging 5mL of culture supernatant of cyanobacteria at 10000r/min for 10min every 24h for 120h, adding colorimetric solution into the supernatant, standing in the dark for 0.5h, taking out, and immediately measuring with spectrophotometer at wavelength of 530nm (Glickmann and Dessux Y.A).
The indoleacetic acid content (μ g/mg) in the sample was (a × V1)/(W × V2) × 1000
In the formula: A-IAA amount (μ g) found on the standard curve
V l sample volume (mL)
W-sample weight (g)
V2-volume of sample reaction solution (mL)
The IAA production amount of the blue algae scsio T-2 obtained by the invention is about 9.5 +/-0.71 mu g/mg.
Example 3 16SRNA and Nitrogen fixation Gene amplification of Strain
The pure bacterial strain scsio T-2 obtained by separation and purification in example 1 was selected for DNA extraction and purification, and the operation method of the DNA extraction and purification in this example refers to Dongxiu bead "Manual of identification of common bacteria System" P409. PCR amplification was carried out using 1SF/16SR (Weisenburg et al, 1991, 16S ribosol DNA amplification for the same, Journal of bacteriology, 1991173 (2): 697. sup. -) 703 and nifH Gene general primer PolF/PolR (refer to Poly et al, 2001, company of nifH Gene in Soils and Soil microorganisms with control primers, Applied and Environmental Microbiology,2001, 67(5) 2255. sup. -. 5) of nitrogen-fixing bacteria, and the PCR reaction system is shown in Table 1.
TABLE 1PCR reaction System
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s for 30 cycles; post extension at 72 ℃ for 10 min.
Taking 2 mu l of PCR reaction products, detecting by using 1.5% agarose gel electrophoresis, if a target band appears, directly delivering the rest PCR products to Invitrogen biotechnology limited company for sequencing by using an ABI Prism 3730XL DNA analysis system, and comparing 16S rDNA (the nucleotide sequence of the 16S rDNA is shown as SEQ ID NO. 1) and a nitrogen-fixing gene sequence nifH (the nucleotide sequence of the nitrogen-fixing gene sequence is shown as SEQ ID NO. 2) obtained by sequencing analysis with sequences in a GenBank database through BLAST in the GenBank, and preliminarily determining the species identification. The phylogenetic tree is established, as shown in fig. 1 and fig. 2, it can be seen from the phylogenetic tree that the sequences having close relativity with the 16S rDNA sequence of the blue algae scsio T-2 are all from the genus coleus, and have 93.79% similarity with the 16S rDNA sequence of the Leptolyngbya nodulosa of the known strain, the similarity of the nitrogen-fixing gene and the sphingomyelina lysimachiae of the genus coleus is the highest, and is only 91%, which indicates that the blue algae scsio T-2 is a potential new species, which belongs to the leptocystis, and is named as leptocystis (Leptolyngbya sp) scsio T-2, which is preserved in the typical culture center (CCTCC) in china 22 days 1/2015, address: wuhan university, the preservation number: CCTCC NO: m2015061.
Example 4 effectiveness test of growth promoting effect of cyanobacteria culture solution in mangrove seed germination and rooting process
The supernatant of Sphingomonas leptocyclina (Leptolyngbya sp.) scio T-2 bacterial liquid cultured for 2 weeks in a selective liquid nitrogen-free liquid modified culture medium (the formula is the same as that in example 1) is respectively taken out by 5mL and added into a culture bottle containing mangrove plant Brugia gymnorrhiza seeds, wherein only 5mL of ultrapure water is added into a control group, the three steps are repeated, and then the control group is placed into an incubator and is placed for 12h: dark cycle (200. mu.E/m)2S), cultivation at 27 ℃ and 20 days later, the number of roots was counted and the length of the roots was measured, and the results are shown in Table 2.
Table 2: the number and average root length of the red sea olive of different treatment groups
As shown in table 2, the comparison of the experimental results of the experimental group and the control group after 20 days of growth shows that the root number and the average root length of the experimental group are both significantly higher than those of the control group, the root number is about 2 times that of the control group, and the average root length is 1.37 times that of the control group, which shows that the scsio T-2 (leptolengbya sp.) has a good growth promoting effect and has a great potential for application in the repair and protection of marine ecosystems such as mangrove forest.
Claims (3)
1. Sphaeria leptocyani (Leptolyngbya sp.) scsio T-2, accession number: CCTCC NO: m2015061.
2. The use of the sphingomonas leptin scsio T-2 as claimed in claim 1 for the preparation of a growth promoting azotobacter fertilizer.
3. Use of the sphingomonas leptin T-2 of claim 1 for the preparation of 3-indoleacetic acid.
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| CN111642166A (en) * | 2020-06-04 | 2020-09-11 | 浙江大学 | Preparation method of algae powder and application of algae powder in improving desert soil |
| CN111996135B (en) * | 2020-07-22 | 2022-04-08 | 中国科学院青岛生物能源与过程研究所 | High-temperature-resistant high-carbohydrate-yield hot spring cyanobacteria and application thereof |
| CN111876350B (en) * | 2020-07-22 | 2022-04-05 | 中国科学院青岛生物能源与过程研究所 | Sphingomyelina gracilis for high yield of carbohydrate and application thereof |
| CN111925958B (en) * | 2020-07-23 | 2022-02-22 | 大连理工大学 | Coleomyces tenuis strain tolerant to high-concentration sodium bicarbonate and culture method and application thereof |
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| The morphology and bioactivity of the rice field cyanobacterium Leptolyngbya;Mehboob Ahmed等;《Rev. Biol. Trop.》;20140901;第62卷(第3期);1251-1260页 * |
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