CN111440737A - Alteromonas strain - Google Patents

Abstract

The invention provides an alteromonas strain and application thereof, wherein the strain is obtained by screening from a dinoflagellate solution such as a dinoflagellate and can promote the growth of the dinoflagellate such as the dinoflagellate. The Alteromonas strain provided by the invention is named Alteromonas (Alteromonas sp.), the strain number is NBU-A-0001, the preservation number is CCTCC M2020010, the preservation date is 2020, 1, 3 days, the preservation unit is China center for type culture preservation, the address is Wuhan, Wuhan university. The alteromonas strain obtained by screening can promote the growth of the dinoflagellates such as the cocci, thereby effectively enlarging the yield of the dinoflagellates such as the cocci and meeting the requirement of the dinoflagellates such as the cocci as feed.

Description

A strain of Alteromonas

technical field

The invention belongs to the technical field of symbiotic bacteria screening, and in particular relates to an Altomonas strain.

Background technique

Isochrysis galbana is small in size, rich in polysaccharides, carotene and other high-energy lipids, especially rich in unsaturated fatty acids DHA, EPA, etc., which are required for the growth and development of shellfish. It is an indispensable opening bait in the breeding process of seedlings.

At present, the research on the factors affecting the cultivation of Isochrysis sphaericus at home and abroad focuses on the influence of environmental factors such as light, salinity, and temperature on the growth of Isochrysis sphaericus. However, even if the suitable environmental conditions and nutritional conditions are controlled as much as possible, in the actual large-scale open-air breeding process, the maximum reproduction density of microalgae is still not high enough, and it is very easy to decay, which cannot meet the needs of large-scale seedling breeding of shellfish.

Studies have shown that interalgal bacteria have an important impact on the proliferation of microalgae. For example, Cytophagasp. can inhibit the proliferation of diatoms and dinoflagellates by direct attack (Imai et al., 1993), while Sulfitobacter sp. can promote the proliferation of diatoms and dinoflagellates by secreting indoleacetic acid (Imai et al., 1993). Reproduction of diatoms (Amin et al., 2015). However, the current actual microalgae propagation technology does not consider the influence of interalgal bacteria.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a strain of Alteromonas and its application, the strain is obtained by screening from Isochrysis sphaericus algal fluid, and can promote the growth of Isochrysis sphaericus.

The strain of Alteromonas sp. provided by the present invention is named as Alteromonas sp., the strain number is NBU-A-0001, the preservation number is CCTCC M2020010, the preservation date is January 3, 2020, and the preservation The unit is the Chinese Collection of Typical Cultures, and the address is Wuhan, Wuhan University.

The Alternomonas strain provided by the present invention is obtained by screening from the algal liquid of Isochrysis globosa;

The Alternomonas strains screened and obtained by the present invention can promote the growth of Isochrysis sphaericus, thereby effectively expanding the output of Isochrysis sphaericus and meeting the requirement of Isochrysis sphaericus as feed.

Description of drawings

Fig. 1 is the co-cultivation result figure of each algal bacterium and Isochrysis globosa isolated by the present invention;

Fig. 2 is the bacterial colony morphology diagram of Alteromonas sp. of the present invention;

Figure 3 is a graph showing the influence of different culture methods on the growth of Isochrysis globosa in the present invention.

Detailed ways

The present invention will be described in detail below with reference to the embodiments and accompanying drawings.

Example 1: Screening of symbiotic strains

The strain of the present invention was isolated and screened from the third-level culture solution of Isochrysis globosa in the aquaculture base of Fujian Baozhi Aquaculture Co., Ltd. in June 2018.

The strain isolation method is as follows: Dilute the Isochrysis sphaericus culture solution and spread it on 2216E solid medium. The culture condition is 28°C, protected from light, and cultivated upside down for about 1 week. Monoclonal colonies of different types, shapes and sizes can be obtained. . Pick monoclonal colonies with a high degree of separation, clear boundaries and diverse shapes, transfer them to a new 2216E solid medium, and obtain purified strains by multiple bacterial three-zone streaking methods. The purified strains were inoculated into 2216E liquid medium respectively, and placed in a shaking incubator for cultivation. The culture conditions were 28°C, protected from light, and the rotation speed was 220r·min ^-1 . After 12h, the log phase bacterial liquid was obtained, and glycerol was added. Store in -80°C refrigerator.

Specifically, the following steps are included: culturing the aseptically purified Isochrysis globosa and each algal inter-algal bacteria obtained by separation to exponential growth phase respectively. Dispense the Isochrysis globosa algal liquid into 100mL sterile conical flasks in 50mL portions. Each strain was collected by centrifugation at 13,000 rpm at low temperature for 10 min, and washed three times with Ningda No. 3 seawater medium to remove 2216E liquid medium. According to the ratio of the algal-bacterial cell density ratio of 1:50, different algal bacteria were added to the Isochrysis globosa culture solution, three parallels were set in each group, and Ningda No. 3 seawater medium was added as the control group.

Using the cell density of Isochrysis sphaericus as an index, the effects of purified interalgal bacteria on the growth of Isochrys sphaericus were analyzed. A total of 5 strains of interalgal bacteria were isolated, and the co-culture results of each interalgal bacterial and Isochrys sphaericus showed that one strain was finally determined to have a significant promoting effect on the growth of Isochrysis sphaericus (Figure 1).

The strain was identified by PCR amplification of 16S rDNA, and the specific steps were as follows:

(1) PCR amplification: PCR amplification was performed with universal primers 27F and 1492R, the sequence of 27F was 5'-AGAGTTTGATCCTGGCTCAG-3', and the sequence of 1492R was 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction system was: Mix (dNTP, enzyme, Mg ²⁺ , 10×buffer) 12.5 μL, 27F primer 0.5 μL, 1492R primer 0.5 μL, bacterial liquid template 0.5 μL, and sterile water 11 μL. The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 90s, 32 cycles; extension at 72°C for 10 min, termination of the reaction at 4°C. (2) Gel electrophoresis and gel-tapping recovery of PCR products: PCR products were identified by 1.5% agarose gel electrophoresis. The target PCR product was recovered and purified by a quick agarose gel DNA recovery kit (Beijing Biotech Biotechnology Co., Ltd.). (3) Transformation of PCR product ligated with T vector and competent cells: The recovered DNA fragment was ligated with pMD18-T vector, and the ligation system was as follows: pMD18-T Vector, 1 μL; DNA fragment, 1 μL; ddH ₂ O, 3 μL; Solution 1, 5 μL; 10 μL total. Reaction conditions: ligation at 16°C for 30min. Mix 10 μL of the ligation product with 100 μL of E. coli DH5α competent cells, and place in an ice bath for 30 min. After heating in a 42°C water bath for 90s, it was placed in ice for 2min. Add 200 μL of LB medium, and shake at 37°C for 1 h. Take 100 μL of transformed Escherichia coli and spread it with LB solid medium containing ampicillin, and cultivate overnight until a single colony grows. (4) Identification and sequencing of positive colonies: Pick 10-20 single colonies, add fresh LB liquid medium, and place them in a shaking incubator for culture. The culture conditions were 28°C, protected from light, and the rotational speed was 220 r·min ^-1 . Using the bacterial solution as a template, primers M13-F (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and M13-R (5'-CACACAGGAAACAGCTATGAC-3') were used for PCR amplification. The PCR reaction system and amplification procedure were the same as above. The PCR product was identified by 1.5% agarose gel electrophoresis detection. The electrophoresis band of about 2kb was regarded as a positive colony, that is, the vector was successfully connected. ID NO: 1.

Homology comparison and phylogenetic analysis were performed on the measured 16S rDNA sequences, and Blast alignment was performed on NCBI to find homology sequences and use MEGA5 software to build a phylogenetic tree. According to the results of Blast comparison and phylogenetic analysis, the strains screened by the present invention can significantly promote the growth of Isochrysis globosa, and are closely related to Proteobacteria, Gammaproteobacteria, Alternomonas Order (Alteromonadales), Alteromonadaceae (Alteromonadaceae), Alteromonas (Alteromonas) have the closest genetic distance, so the classification is attributed to Alteromonas strains, named as Alteromonas sp., strains No. NBU-A-0001, its deposit number is CCTCC M2020010, the deposit date is January 3, 2020, and the deposit unit is China Center for Type Culture Collection, whose address is Wuhan, Wuhan University.

The biological characteristics of strain NBU-A-0001 are as follows: rod-shaped, round colonies, opaque off-white, moist and smooth surface, and sticky when provoked (Figure 2). Marine bacteria, the cells were Gram-negative.

Example 2: Application of Alternomonas strain in promoting the reproduction of Isochrysis globosa

First, prepare the algal culture solution, the composition is as follows: 100mg/L KNO ₃ , 10mg/L KH ₂ PO ₄ , 3mg/L Fe-citrate·5H ₂ O, 0.06g/L brown sugar, 6μg/L VB ₁ , 0.05μg / _LVB12 .

In a 100mL glass conical flask, add 50mL of the above-mentioned sterilized new algal culture solution, add 100μL of activated Alteromonas NBU-A-0001 with OD ₆₀₀ =0.4-0.6, and add 1mL of algal cell density to 10 ⁶ cells/mL of Isochrysis globosa algal species, mixed evenly, placed in a light incubator at 25°C, light intensity of 4000Lux, and light cycle light/dark (12h/12h) for cultivation.

In order to compare the effect of this propagation method, in a 100mL glass conical flask, the culture solution added with the alternamonas NBU-A-0001 of the present invention, the conventional microalgae culture solution (NMB3) and the non- The algal bacteria expansion culture solution of the existing Alteromonas sp. (Alteromonas sp., preservation number 1D00009, China Marine Microbiology Culture Collection and Management Center, Xiamen, Third Institute of Oceanography, Ministry of Natural Resources, China) isolated from the algae was cultivated, Three parallels were set for each culture medium, the seeding density was 2×10 ⁴ cells/mL, and the culture density was counted with a hemocytometer every day. The culture results showed (Fig. 3) that the algal density of Isochrysis globosa was much higher than that of NMB3 culture solution culture and the addition of non-algal-separated Alternaria algae when cultured with the new culture method, and the longer the culture time was. , the effect of promoting multiplication is more obvious, and high-density culture is always maintained.

Therefore, the NBU-A-0001 strain provided by the present invention can effectively promote the culture efficiency of Isochrysis globosa.

sequence listing

<110> Ningbo University

<120> A strain of Alteromonas

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1328

<212> DNA

<213> Artificial Sequence

<400> 1

tcccactccc atggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcagt 60

atgctgacct gcgattacta gcgattccga cttcatggag tcgagttgca gactccaatc 120

cggactacga tatgctttaa ggggtccgct tactctcgca cgttcgcttc cctctgtaca 180

taccattgta gcacgtgtgt agccctactc gtaagggcca tgatgacttg acgtcgtccc 240

caccttcctc cggtttgtca ccggcagtct ccttaaagtg cccaactaaa ggctggcaac 300

taaggacaag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 360

gacagccatg cagcacctgt attccagttc ccgaaggcac taattcatct ctgaaaaatc 420

ctggatatgt caagagtagg taaggttctt cgcgttgcat cgaattaaac cacatgttcc 480

accgcttgtg cgggcccccg tcaattcatt tgagttttaa ccttgcggcc gtactcccca 540

ggcggtctac ttagcgcgtt agcttcgcta cgcacgcatt aaatgcacac acagctagta 600

gacagcgttt acggtgtgga ctaccagggt atctaatcct gttcgctacc cacactttcg 660

cacatgagcg tcagtctttg gccagagagt cgccttcgcc actgttgttc ctccagatat 720

ctacgcattt caccgctaca cctggaattc cactcccctc tccaaaactc tagtctgcca 780

gttctaaaag cccttcccac gttgagcgcg gggctttcac ttctagctta acagaccgcc 840

tgcgtgcgct ttacgaccag taattccgat taacgctcgc accctccgta ttaccgcggc 900

tgctggcacg gagttagccg gtgcttcttc tgttgctaac gtcacagatt gcgggtatta 960

accacaaccc tttcctcaca actgaaagtg ctttacaacc cgaaggcctt cttcacacac 1020

gcggcatggc tgcatcaggg tttcccccat tgtgcaatat tccccactgc tgcctcccgt 1080

aggagtctgg gccgtgtctc agtcccagtg tggctgatct tcctctcaga acagctagag 1140

atcgtcgcct tggtgagcct ttacctcacc aactagctaa tctcgcttag gttcatcctt 1200

tggcgggagg taaacacccc ttttgcccgt aggctatatg cggtattagc catcgtttcc 1260

aatggttatc cccctccaaa gggcagatcc ctaagtatta ctcacccgtc cgccactcgt 1320

cagcaact 1328

Claims (3)

1. The alteromonas strain is characterized in that the preservation number of the strain is CCTCC M2020010.

2. The alteromonas strain of claim 1, wherein said strain is selected from the algal solution of isochrysis galbana.

3. Use of the alteromonas strain of claim 1 for culturing isochrysis galbana.

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