Laburentz strain

Abstract

The invention provides a strain of the genus laburensis. The provided laburensis can be used for remarkably improving the high-temperature resistance of isochrysis zhanjiangensis. The preservation number of the labrentz's bacteria is CCTCC (China Center for Type Culture Collection) NO: M2025963. The strain of the Labarenaria sp. Obtained by screening can effectively promote the growth of the isochrysis zhanjiangensis under high temperature stress, so that the biomass of the isochrysis zhanjiangensis is improved. Sufficient bait guarantee is provided for the shellfish planktonic larvae in the high-temperature season.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of functional strain screening, and particularly relates to a labrenzia sp. BACKGROUND

[0002] At present, sinonovacula constricta and other beach clam culture has become an important economic source for some breeders. In order to meet the large demand of the shellfish breeding industry for seedlings, it is necessary to artificially cultivate microalgae on a large scale as feed supply. Therefore, the stable cultivation of high-quality feed microalgae, especially the opening feed chlorella, is particularly crucial to the sustainable development of the shellfish breeding industry. Chlorella zhanjiangensis (Chlorella zhanjiangensis) and other chlorella species are currently the most commonly used important opening feed, which has the characteristics of small individual, no cell wall, rich in polyunsaturated fatty acids and is very suitable as a nutritional source for shellfish larvae. In addition, chlorella also shows wide application potential in the fields of biological energy development, synthesis of bioactive substances and water quality regulation. Isochrysis zhangjiangensis

[0003] However, affected by global warming, the water temperature of the culture is often higher than 30℃ in the high-temperature season or in the southern coastal areas, and the growth of chlorella zhanjiangensis and other chlorella species is inhibited, the cell viability decreases, and even large-scale death occurs, which is difficult to meet the feed demand of the large-scale seedling breeding of shellfish. This problem not only directly affects the stability of the feed supply in the shellfish breeding process, but also restricts the healthy development of the entire aquaculture industry. At present, the stress resistance improvement techniques for algae under high temperature mainly include selection breeding, mutagenic breeding and genetic engineering breeding methods, but these techniques still have limitations such as unstable genetic traits, low screening efficiency, lack of chlorella genome information and unstable expression of exogenous genes. Therefore, it is of important practical application value and popularization prospect to explore a safe and effective technical approach to promote the growth of chlorella zhanjiangensis under high temperature conditions.

[0004] It has been found that the interaction between bacteria and plants has an important influence on the resistance of plants to high temperature stress. For example, Brevibacillus linens enhances the heat tolerance of rice by regulating the ethylene release pathway and up-regulating the expression of antioxidant enzymes and heat shock protein-related genes. In recent years, the complex interaction between algicola bacteria and microalgae has attracted increasing attention and has become a hot research field in algae. However, there are still few studies on the interaction between bacteria and microalgae to improve the high-temperature tolerance of microalgae. Only a few reports have been found: for example, when rhizobium is co-cultured with chlamydomonas reinhardtii, the high-temperature tolerance of chlamydomonas reinhardtii can be significantly improved, and further studies have shown that the effect is related to the secretion of vitamin B 12 ​Sanchez-Garcia et al. (2022) found that phyllosphere bacterial community could respond to temperature stress through changes in core bacterial species and specific bacterial abundance. Hünken et al. (2015) found that phyllosphere bacteria of diatoms contributed significantly to the enhancement of their antioxidant defense capacity and growth in special environments. The research results of these plant-bacteria and part of microalgae-bacteria interactions provide enlightenment for exploring the high-temperature tolerance mechanism of chrysophytes. SUMMARY

[0005] The purpose of the present application is to provide a strain of Labrenzia, which can significantly improve the high-temperature tolerance of Isochrysis zhanjiangensis.

[0006] The Labrenzia provided by the present application is Labrenzia sp. Labrenzia aggregata ) NMB-Iz-Ls-0001 strain, the preservation number of which is CCTCC NO: M2025963, the preservation date of which is May 6, 2025, and the preservation unit of which is China Center for Type Culture Collection, located in Wuhan, Wuhan University.

[0007] The present application also provides a use of the Labrenzia, which is applied to promote the culture of bivalve feed algae at high temperature. As a specific description of the embodiment, the bivalve feed algae is Isochrysis zhanjiangensis. The present application also provides a method for culturing Isochrysis zhanjiangensis at high temperature, which adds live bacteria of the Labrenzia to the culture solution of the chrysophyte.

[0008] The Labrenzia strain screened by the present application can effectively promote the growth of Isochrysis zhanjiangensis under high-temperature stress, thereby improving the biomass. This provides sufficient feed guarantee for bivalve plankton larvae in high-temperature seasons. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 is a screening result diagram of symbiotic heat-resistant bacteria of Isochrysis zhanjiangensis; Figure 2 is a colony morphology diagram of the Labrenzia strain Labrenzia aggregata ) NMB-Iz-Ls-0001 strain; Figure 3 is a diagram showing the influence of the Labrenzia in the present application on the high-temperature tolerance of Isochrysis zhanjiangensis. DETAILED DESCRIPTION

[0010] The present application will be described in detail below in combination with specific embodiments and drawings.

[0011] Embodiment 1: Isolation and screening of symbiotic heat-resistant bacteria The strain of this invention was isolated and screened in November 2021 from the culture medium of the thermotolerant mutant strain NMBjih021-10 of *Isochrysis zhanjiangensis*, wherein *Isochrysis zhanjiangensis* (… Isochrysis zhangjiangensis The heat-resistant mutant strain NMBjih021-10 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, on August 14, 2019, with accession number CCTCC NO:M2019629.

[0012] The bacterial strain isolation method is as follows: The culture medium of *Isochrysis galbana* was diluted and spread onto 2216E solid medium. The culture conditions were 35℃, protected from light, and incubated upside down for about one week, yielding monoclonal colonies of different species, morphologies, and sizes. Monoclonal colonies with high separation levels, clear boundaries, and diverse morphologies were selected and transferred to fresh 2216E solid medium. The purified strains were obtained through repeated streak plating (three-zone method) and numbered. The purified strains were then inoculated into 2216E liquid medium and placed in a shaking incubator at 35℃, protected from light, and a rotation speed of 220 r·min⁻¹. -1 Logarithmic bacterial culture can be obtained after 12 hours. Add glycerol and store at -80℃.

[0013] The screening method for symbiotic thermotolerant bacteria is as follows: Aseptically purified *Isochrysis zhanjiangensis* and the isolated bacterial strains were cultured separately to the exponential growth phase. Each culture was cultured at 8000 rpm for 10 min, the supernatant was discarded, and the culture was resuspended in sterile seawater and centrifuged again. After removing the supernatant, different algal bacteria were added to the sterilized *Isochrysis zhanjiangensis* culture medium at an algae-bacteria cell density ratio of 1:50 (cells / cells). After mixing, the control and experimental groups were separately added to NMB3 agar plates using the drop plate method. The left three portions of each plate were the co-culture group, and the right three portions were the control group. Each group was divided into three replicates. The plates were cultured in a light incubator at 4000 Lux and 35℃ (the optimal temperature for *Isochrysis zhanjiangensis* culture is around 25℃). The growth status of the algal strains was photographed and recorded on day 7. The effects of various algal bacteria on the growth of *Isochrysis var. *zhanjiangensis* under high temperature were analyzed, and six strains that significantly promoted the growth of *Isochrysis var. *zhanjiangensis* under high temperature were finally screened. The strain numbers were IM-1, IM-2, IM-3, IM-6, IM-7, and IM-10. Figure 1a). Further compare the promotion effect of the 6 strains by liquid culture method, the liquid culture method is as follows: when the Chrysochromulina zhanjiangensis is cultured to the exponential phase, 50 mL of Chrysochromulina zhanjiangensis algal liquid is inoculated into a 100 mL glass conical flask, and the 6 strains of activated bacteria are inoculated at a ratio of 1:50 (cells / cells), mixed uniformly, and placed in a light incubator at 35°C, light intensity 4000 Lux, light cycle light / dark (12 h / 12 h) for culture, and algal cell counting is performed every two days. The results show that the strain IM-6 has a promoting effect on the growth of Chrysochromulina zhanjiangensis at high temperature from the 2nd day to the 12th day of co-culture, and is significantly stronger than the other 5 strains Figure 1 b).

[0014] Example 2: Identification and physicochemical properties of the screened symbiotic heat-resistant strain The 16S rDNA of the strain IM-6 was amplified, and the specific steps were as follows: 1) PCR amplification: universal primers 27F and 1492R were used for PCR amplification, 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, sterile water 11 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, 32 cycles; 72°C extension for 10 min, 4°C termination reaction.

[0015] 2) Gel electrophoresis and gel recovery of PCR products: the PCR products were identified by 1.5% agarose gel electrophoresis. The target PCR products were recovered and purified by using a fast agarose gel DNA recovery kit (Beijing Baitaike Biotechnology Co., Ltd.).

[0016] 3) PCR product ligation T vector and transformation of competent cells: the recovered DNA fragments were ligated with pMD18-T vector, and the ligation system was as follows: pMD18-T Vector, 1 μL; DNA fragments, 1 μL; ddH2O, 3 μL; Solution 1, 5 μL; total 10 μL. Reaction conditions: 16°C for 30 min. 10 μL of ligation product was mixed with 100 μL of E. coli DH5α competent cells, and placed in ice bath for 30 min. After heating in 42°C water bath for 90 s, it was placed in ice for 2 min. 200 μL of LB medium was added, and cultured at 37°C for 1 h. 100 μL of transformed E. coli was taken and spread on LB solid medium containing ampicillin, and cultured overnight until single colonies grew.

[0017] 4) Positive colony identification and sequencing: 10-20 single colonies were picked and added to fresh LB liquid medium, and placed in a shaking incubator for culture. The culture conditions were 28°C, dark, and the rotation speed was 220 r·min -1 . The bacterial solution was used as a template for PCR amplification with primers M13-F (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and M13-R (5'-CACACAGGAAACAGCTATGAC-3'). The PCR reaction system and amplification program were the same as above. PCR product identification was performed by 1.5% agarose gel electrophoresis, and 2 kb electrophoretic bands were considered as positive colonies, i.e. successful ligation of the vector, and the corresponding bacterial solution of successful ligation was taken for 16S rRNA full-length sequencing.

[0018] The sequence result of the 16S rDNA of the strain is SEQ ID NO: 1: Homology comparison and phylogenetic analysis were performed on the obtained 16S rDNA sequence. Blast alignment was performed on NCBI to identify homologous sequences, and a phylogenetic tree was constructed using MEGA5 software. Based on the Blast alignment and phylogenetic analysis results, this strain belongs to the phylum Proteobacteria, class Alphaproteobacteria, family Rhodobacteraceae, and genus Rabronzella. Labrenzia One of them, named Rabrenzella ( Labrenzia aggregata Strain NMB-Iz-Ls-0001, with accession number CCTCC NO:M2025963, accession date May 6, 2025, is deposited at the China Center for Type Culture Collection (CCCCC), Wuhan University, Wuhan. The biological characteristics of this strain are as follows: rod-shaped, colonies are round, opaque off-white, with a moist and smooth surface, and a viscous consistency when picked up; Gram-negative bacteria. Figure 2 ).

[0019] Example 3: Application of *Labrintzia* strains in promoting the growth of *Isochrysis var. *zhanjiangensis* under high temperature When *Isochrysis zhanjiangensis* reached the exponential growth stage, 50 mL of *Isochrysis zhanjiangensis* algal culture was inoculated into a 100 mL glass conical flask. Activated *Rabronzella* strain NMB-Iz-Ls-0001 was then inoculated at an algae-to-bacterial density ratio of 1:50 (cells / cells). After thorough mixing, the flask was placed in a light incubator at 35℃, 4000 Lux light intensity, and a light / dark cycle (12 h / 12 ​​h). To compare the propagation efficiency of this method, *Isochrysis zhanjiangensis* was cultured in NMB3 medium without added bacteria as a control. The co-culture period was 12 days. The effect of bacteria on algal growth density was assessed by detecting algal cell density every other day and the content of chlorophyll a and carotenoids during the growth cycle. Figure 3 ).

[0020] The cultivation results showed that, under high-temperature stress, the algal density of *Isochrysis zhanjiangensis* was significantly higher when co-cultured with the bacteria described in this invention than when cultured in NMB3 medium. Furthermore, the chlorophyll a and carotenoid contents of the algal cells in the co-culture group were significantly higher than those in the control group at all three stages of algal culture, and the differences were greater with longer culture times. This indicates that the effect of *Rabronzella* strains screened in this invention on promoting the high-temperature propagation of *Isochrysis zhanjiangensis* becomes more significant with prolonged culture time. Figure 3 As can be seen, the culture method provided by this invention has a significantly better effect on expanding microalgae culture than conventional NMB3 culture medium culture. Therefore, the culture method of this invention has a promising application prospect.

Claims

1. A Lachnospira bacterium, characterized in that, The preservation number of the Labrenzia is CCTCC NO:M2025963.

2. The Lactobacillus of claim 1, wherein The sequence of 16S rDNA of the Labrenzia is SEQ ID NO:

1.

3. The Labrenzia for use in promoting the culture of bivalve feed algae at high temperature according to claim 1.

4. The use according to claim 3, wherein the compound is ###0002### The bivalve feed algae is Isochrysis zhanjiangensis.

5. A method for culturing Isochrysis galbana at high temperature, characterized in that, The method is adding the live bacteria of the Labrenzia according to claim 1 into the culture solution of Isochrysis zhanjiangensis.

6. The method of claim 5, wherein, The high temperature is 35℃.

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