CN112899187B - Serratia marcescens strain and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a serratia marcescens strain and application thereof. A Serratia marcescens strain, which is characterized in that: the strain is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 23 days in 2020, and the preservation number is as follows: CGMCC NO. 21530. The serratia marcescens strain provided by the invention can promote the synthesis of auxin by a plant root system and promote the development of a plant lateral root. The promotion of the development of the plant root system is specifically shown in the promotion of the generation and the elongation of the lateral root of the plant; the promotion of plant growth and development is embodied in the promotion of fresh weight and yield increase of plants.

Description

Serratia marcescens strain and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a serratia marcescens strain and application thereof.

Background

The root system of the plant is an important organ of the terrestrial plant and plays a role in fixing the plant and promoting the absorption of water and mineral nutrients by the plant. The growth and development of the root system is closely related to the growth of the overground part of the plant, and the 'deep-rooted luxuriant leaves' and 'instinct branches and honor' are the concrete reflection of the growth correlation of the overground part and the underground part of the plant. The lateral roots are important components of the plant root system, the contact area of the plant and the soil is greatly increased, the absorption of water and mineral nutrition by the plant is promoted, and therefore the number and the length of the lateral roots are also important indexes for judging the development condition of the plant root system. The plant hormone-auxin plays an important regulation and control role in the growth of main roots and the development of lateral roots of plants.

The root system of the plant goes deep into the soil, and besides absorbing moisture and mineral nutrition, the plant also secretes various organic matters such as sugar, amino acid, organic acid and the like to the surrounding soil, so that the physicochemical property of the soil is changed and optimized. The soil attached to the surface of the plant root system in the micro-area affected by the plant root system is the rhizosphere of the plant. As the plant rhizosphere is rich in organic matters, a large number of microorganisms grow and reproduce in the plant rhizosphere, and directly or indirectly influence the root system development of the plant, the absorption of water and mineral elements by the plant, the stress tolerance and disease resistance of the plant and the like. The Plant Growth Promoting Rhizobacteria (PGPR) is a type of bacteria that can freely grow in soil or attach to Plant roots, and promote Plant Growth, water and mineral absorption, disease resistance and stress resistance of plants, and the like, by direct or indirect means.

PGPR can promote the growth and development of plants, enhance the stress tolerance of plants by various means: for example, the PGPR promotes the absorption of nutrients in soil by plants by transforming nutrients which cannot be absorbed by the plants in the soil into nutrients which can be absorbed by the plants; the PGPR can generate some plant hormones and volatile gases to promote the root system development of plants and regulate and control the absorption of the plants to water and nutrients in soil; the PGPR can also activate the immune mechanism of the plant, improve the disease resistance of the plant and promote the adaptation of the plant to the external stress environment.

Disclosure of Invention

The invention aims to find rhizosphere microorganisms capable of promoting plant growth and develop an excellent microbial fertilizer.

In order to achieve the purpose, the invention provides the technical scheme that: a serratia marcescens strain is preserved in China general microbiological culture Collection center (CGMCC) on 12-23 days of 2020, with the preservation numbers as follows: CGMCC NO. 21530.

As a preferred mode of the invention, the 16S rDNA sequence of the serratia marcescens strain is shown as SEQ ID No. 1.

The invention also provides the application of the serratia marcescens strain, and the serratia marcescens strain can be used for preparing a microbial agent for promoting the development of plant root systems or a growth promoter for promoting the growth and development of plants.

The invention further provides a microbial agent for promoting the development of plant roots, and the microbial agent comprises the serratia marcescens strain.

The invention further provides a plant growth promoter, which comprises the serratia marcescens strain.

The invention further provides the serratia marcescens with the fluorescent marker, and GFP fluorescence is marked on the serratia marcescens strain.

The invention carries out fluorescence labeling on the serratia marcescens strain in an electric shock conversion mode.

The serratia marcescens strain provided by the invention can promote the synthesis of auxin by a plant root system and promote the development of a plant lateral root. The promotion of the development of the plant root system is specifically shown in the promotion of the generation and the elongation of the lateral root of the plant; the promotion of plant growth and development is embodied in the promotion of fresh weight and yield increase of plants.

Drawings

FIG. 1 is a diagram showing the results of an experiment in which Serratia marcescens 8C-3 promotes lateral root formation in Arabidopsis;

wherein, A: length of main root of arabidopsis; b: lateral root primordial density (LP), lateral root density of the prominent epidermis (LE), and Total lateral root density (Total);

FIG. 2 is a graph showing the results of experiments in which Serratia marcescens 8C-3 activates the auxin signaling pathway in plants; wherein, A: marker genes expressed in response to auxin upregulation in root systems of Arabidopsis thaliana after treatment of control and strain 8C-3 by real-time PCRIAA2、IAA5、IAA31、GH3.5And the downstream transcription factor gene involved in coding auxin to regulate lateral root developmentLBD18、LBD29Detecting the expression level of (a); b and C transgenic plants responding to auxin down-regulation expression and treated by the control and the strain 8C-3 by using a laser confocal microscope respectivelyDⅡ-VINUSAnd transgenic plants that respond to up-regulated expression of auxinDR5:GFPDetecting the fluorescence signal of the root tip;

FIG. 3 shows the experimental results of Serratia marcescens 8C-3 promoting the synthesis of auxin by plants; wherein, A: detecting the expression quantity of key genes participating in auxin synthesis in roots after control and strain 8C-3 treatment by real-time PCR respectively;

b: the promoter of key genes YUC5, YUC7, YUC8 and YUC9 in the auxin synthetic pathway is used for driving GUS expression transgenic plants to be dyed and observed after the treatment of the strain 8C-3;

FIG. 4 is a diagram showing the experimental results of the enrichment of Serratia marcescens 8C-3 strains on the surface of plant roots;

FIG. 5 is a diagram showing the experimental results of Serratia marcescens 8C-3 promoting the growth of Arabidopsis and increasing the yield; wherein A: transferring the wild arabidopsis seedlings on an 1/2MS plate to vermiculite, inoculating a strain 8C-3, and continuously co-culturing for 20 days to obtain a plant phenotype with a scale of 1 cm;

b: counting the fresh weight of the arabidopsis seedlings under the same conditions as the graph A;

c: statistics of seed dry weight harvested from Arabidopsis thaliana individuals under the same conditions as in Panel A.

Detailed Description

In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Example 1 origin and isolation of strains, identification

The inventor of the application separates and obtains the strain 8C-3 from the rhizosphere soil microbial pool of the east-Yingshan saline land, and identifies the strain as serratia marcescens (Serratia marcescens) by observing the morphological characteristics of the strain 8C-3 and combining the 16S rDNA sequence and homology analysisSerratia marcescens) (ii) a Meanwhile, the functions of the plant growth promoter in promoting the development of plant root systems and promoting the growth and development of plants are researched.

(1) Morphological Observation of Strain 8C-3

8C-3 streaking is carried out on an LB solid culture medium, the culture is carried out for 12-24 hours at 30 ℃, the colony morphology is observed, the strain 8C-3 in the logarithmic growth phase is stained by a gram staining method, and then the morphological characteristics of the serratia marcescens strain 8C-3 are observed by an optical microscope.

The observation shows that the single bacterial colony of the strain 8C-3 is round, the center of the bacterial colony is convex, orange red, opaque, moist, neat in edge, easy to pick and not sticky. In addition, microscopic observation revealed that the cells of strain 8C-3 were short rod-shaped, gram-negative, and free of spores.

(2) 16S rDNA sequence homology analysis of Strain 8C-3

Extracting the genome DNA of the strain 8C-3 by adopting a bacterial genome DNA extraction kit, and performing 16S rDNA amplification by using the extracted genome DNA as a template and a bacterial 16S rDNA universal primer.

27F 5’- AGAGTTTGATCCTGGCTCAG-3’，SEQ ID No.2；

1492R 5’- GGTTACCTTGTTACGACTT-3’，SEQ ID No.3。

The high Fidelity DNA Polymerase used was Phanta Max Super-Fidelity DNA Polymerase from vayme. The PCR system was 50. mu.L: ddH₂O is 20 mu L; 25 μ L of 2X Phanta Max buffer; dNTP Mix (10 mM): 1 mu L of the solution; 27F (10 mM): 1 mu L of the solution; 1492R (10 mM): 1 mu L of the solution; phanta Max Super-Fidelity DNA Polymerase: 1 mu L of the solution; 8C-3 genomic DNA: 1 μ L. The PCR conditions were: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 15 sec, annealing at 57 ℃ for 15 sec, extension at 72 ℃ for 1 min, denaturation to extension for 35 cycles, and complete extension at 72 ℃ for 5 min.

The target fragment obtained by PCR amplification is sent to Protecidae sequencing company for sequencing analysis, and the doublet with C, T at 444 th and 1266 th bases of the obtained fragment is represented by Y, the doublet with A, G at 1217 th bases is represented by R, so that a plurality of single clones are obtained by repeated scribing, the possibility of pollution is eliminated, and the 16S rDNA is supposed to have a plurality of copies in the strain and slightly different sequences, wherein the specific sequence is shown as SEQ ID No. 1. The 16S rDNA gene sequence of the strain 8C-3 of the present invention was compared with the 16S rDNA gene sequence of the relevant strain by referring to the International related GenBank at the National Center for Biotechnology Information (NCBI) to analyze homology. The results show that the strain and Serratia marcescens (A), (B), (C) and (C)Serratia marcescens) Has 99.8% similarity, the 16S rDNA sequence is shown in SEQ ID No.1, the strain is named as visciditySerratia marcescens.

The strain is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 23 days in 2020. The preservation number is CGMCC number 21530. The address of the depository: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

Example 2 Experimental study of the Effect of Strain 8C-3 on the promotion of plant lateral root initiation

The method for sterilizing and plating arabidopsis seeds comprises the following steps: taking a certain amount of arabidopsis thaliana seeds in a 1.5 mL EP tube, adding a 1% sodium hypochlorite solution, reversing, uniformly mixing, resuspending for 15 minutes, discarding the sodium hypochlorite waste liquid, and resuspending and rinsing for 3 times by using sterile water. Uniformly scattering the seeds to 1/2MS solid (containing 1% of sucrose and 0.8% of agar powder) culture medium, treating at 4 ℃ for 24-48 hours, taking out the plate, vertically placing the plate to a long-day (16-hour light/8-hour dark) illumination incubator at 22 ℃, and culturing for 3-8 days.

The specific culture and inoculation method of the strain 8C-3 comprises the following steps: streaking the strain on LB solid culture medium at 8C-3, culturing at 30 deg.C for 12-24 hr, selecting the thallus, blowing and resuspending in sterile water to make the thallus fully resuspended in sterile water, measuring OD value of the thallus, and diluting with sterile water to OD value of 0.01-0.1. Transferring the Arabidopsis seedlings vertically cultured on 1/2 solid medium to a new 1/2MS solid medium (without sucrose), inoculating diluted bacterial liquid to the roots of the Arabidopsis seedlings, wherein 1 mu L of the bacteria is inoculated to the roots of each Arabidopsis, culturing for 4-6 days, and observing the plant phenotype or observing the fluorescence signal of the root tip.

Inoculating 8C-3 strain to root system of Arabidopsis seedling growing for 5 days, culturing for 4 days, observing and counting main root length and lateral root number, and counting lateral root density (lateral root number/main root length). Compared with the control group, the results show that the effect on the length of the main root of the arabidopsis seedlings after inoculation of the strain 8C-3 bacterial liquid is not obvious, as shown in A in figure 1, but the density increase of the arabidopsis lateral root primordium (LP), the lateral root (LE) protruding the epidermis and the total lateral root (total = LP + LE) can be obviously promoted, as shown in B in figure 1. The strain 8C-3 can remarkably promote the development of lateral roots of arabidopsis thaliana.

Example 3 Experimental study of Strain 8C-3 activation of auxin signalling pathway

Transferring Arabidopsis thaliana seedling growing for 5 days at 1/2MS (containing 1% sucrose and 0.8% agar powder) to a new 1/2MS solid culture medium (containing 0.8% agar powder), inoculating strain 8C-3 to plant root system, continuously culturing for 4 days, taking root system of seedling, extracting RNA, detecting auxin-induced gene by RT-PCRIAA3、IAA5、IAA31 AndGH3.5expression level of (B), as a result, it was found thatIAA3、IAA5、IAA31AndGH3.5gene expression was up-regulated by induction of strain 8C-3, as shown in FIG. 2A, suggesting auxin signaling pathway activation. At the same time, transgenic Arabidopsis thaliana grown for 3 days on 1/2MS medium in response to up-regulated expression of auxin was usedDR5:GFP After 3 days of treatment with transgenic Arabidopsis thaliana DII-VENUS responding to the down regulation of auxin, by Serratia marcescens 8C-3, a fluorescent signal is observed and found,DR5:GFPthe ability to be up-regulated by strain 8C-3, while the ability to be down-regulated by strain 8C-3 of DIII-VENUS is shown by B, C in FIG. 2, indicating that the Serratia marcescens strain is able to activate the auxin signaling pathway in plants.

Example 4 Experimental study of Strain 8C-3 promoting plant Synthesis of auxin

Transferring an arabidopsis wild type seedling Col-0 growing in 1/2MS solid medium (containing 1% of sucrose and 0.8% of agar powder) for 5 days to a new 1/2MS solid medium (containing 0.8% of agar powder), inoculating a strain 8C-3 on the root system of the arabidopsis wild type seedling Col-0, culturing for 5 days, extracting RNA of the plant root system, and detecting key genes participating in the synthesis process of auxin by RT-PCR (reverse transcription-polymerase chain reaction) such as:CYPB2、 CYPB3、AMI1、AOO1、WEI8、TAR1、TAR2、YUC3、YUC5、YUC7、YUC8、YUC9etc. are detected. As a result, it was found that the above genes were all induced by Serratia marcescens strain and the expression level was up-regulated, as shown in A in FIG. 3.

Transgenic Arabidopsis seedlings treated for 5 days with strain 8C-3YUC5:GUS、YUC7:GUS、YUC8:GUS、YUC9: GUSGUS staining observation shows that the GUS expression level, especially the expression level in roots, of the transgenic plants is induced by Serratia marcescens strain 8C-3, and the expression is up-regulated, as shown in B in figure 3As shown. The experimental result further shows that the serratia marcescens strain 8C-3 can induce the expression of auxin synthetic gene in plants and promote the plants to synthesize auxin.

Example 5 Experimental study of electroporation-transformed Strain 8C-3 for fluorescence labeling and Observation of its distribution on root System

In order to further determine whether the strain 8C-3 can enter the interior of the plant root system, the inventor transfers a vector pPROBE-GTkan which expresses a GFP fluorescent marker in bacteria into strain 8C-3 competent cells by an electric shock transformation method, so that the strain 8C-3 competent cells are provided with the GFP fluorescent marker, and inoculates the roots of wild type Arabidopsis seedlings which grow for 5 days, PI staining is carried out on the root systems of the Arabidopsis seedlings after two days, and the GFP fluorescent marker is observed by a laser confocal scanning microscope, so that the strain 8C-3 is mainly in a short rod shape and is gathered on the surface and the rhizosphere of the plant root, as shown in figure 4.

The strain 8C-3 competent cells were prepared and transformed as follows:

(1) streaking, streaking the strain 8C-3 to LB solid medium, 30 ℃ overnight culture until single colonies are grown.

(2) Small shake, pick the single clone to 1 mL LB liquid medium, 30 ℃, shake bacteria for 8-12 hours.

(3) And then, largely shaking, inoculating 50 mu L of the small-shaking bacterium liquid into 5 mL of LB culture medium, shaking at 30 ℃ for 2-4 hours until the OD value is 0.5-1, standing on ice for 15 minutes, and precooling on ice with sterile deionized water.

(4) Centrifuging, taking 2 mL of bacterial liquid into a centrifuge tube, centrifuging at 8000g and 4 ℃ for 15 seconds, discarding supernatant, and collecting thalli.

(5) Washing, adding 2 mL of sterile precooled deionized water, after resuspending the thalli, centrifuging at 8000g and 4 ℃ for 15 seconds, discarding the supernatant, collecting the thalli, and repeating for 3 times.

(6) Resuspending, adding 50. mu.L of pre-cooled sterile deionized water to resuspend the cells, which is competent.

(7) Electric shock transformation, adding 1 μ g of pPROBE-GTkan plasmid into competent cells, gently mixing uniformly, adding into a precooled electric shock cup, 1250V, shocking for 5 seconds, rapidly adding 800 μ L of LB culture medium, incubating for 1-2 hours with slow shaking at 30 ℃, spreading on a Kanna resistant LB solid culture medium, inverting in an incubator at 30 ℃, and culturing overnight until a monoclonal antibody grows out. The successfully transformed strain 8C-3 was yellow in color, and strong fluorescence of GFP was observed under a fluorescence microscope.

Example 6 study of growth promotion of plants by Strain 8C-3 in vermiculite

The method for culturing arabidopsis thaliana in vermiculite comprises the following steps: adding vermiculite into a culture hole to absorb water, transferring arabidopsis thaliana seedlings growing on a sterile 1/2MS culture medium for 6-7 days into the vermiculite, adding 2-3 mL of a strain 8C-3 (OD value is 1.5-2.0) which is shaken in an LB liquid culture medium into the vermiculite, culturing in a long-day (16-hour light/8-hour dark) culture room at 22 ℃ for 3 weeks to observe phenotype, harvesting single arabidopsis thaliana seeds after culturing for 70 days, placing in an oven at 37 ℃ for one week, and drying the seeds.

In order to further determine whether the serratia marcescens 8C-3 can promote the growth of overground parts of plants and increase the yield of the plants, the wild-type arabidopsis thaliana seedling Col-0 growing for 7 days is transferred to vermiculite and inoculated with the serratia marcescens strain 8C-3, the phenotype of the plants is observed after 20 days, and the fresh weight of the overground parts of the plants is weighed statistically, and as a result, after the serratia marcescens strain 8C-3 is applied, the overground rosette leaves of arabidopsis thaliana are obviously increased and the fresh weight is increased compared with a control, as shown in A, B in fig. 5. Statistics of the dry weight of the seeds obtained from the individual plants show that the application of the serratia marcescens strain 8C-3 can significantly improve the yield of the seeds of the plants, as shown in C in FIG. 5. The serratia marcescens strain 8C-3 can promote the growth of overground parts of plants and increase the yield of the plants, and the application of the serratia marcescens strain as a plant bacterial fertilizer is suggested.

Sequence listing

<120> Serratia marcescens strain and application thereof

<141> 2021-01-28

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<170> SIPOSequenceListing 1.0

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tccccgggtg acgagcggcg gacgggtgag taatgtctgg gaaactgcct gatggagggg 120

gataactact ggaaacggta gctaataccg cataacgtcg caagaccaaa gagggggacc 180

ttcgggcctc ttgccatcag atgtgcccag atgggattag ctagtaggtg gggtaatggc 240

tcacctaggc gacgatccct agctggtctg agaggatgac cagccacact ggaactgaga 300

cacggtccag actcctacgg gaggcagcag tggggaatat tgcacaatgg gcgcaagcct 360

gatgcagcca tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact ttcagcgagg 420

aggaaggtgg tgagcttaat acgytcatca attgacgtta ctcgcagaag aagcaccggc 480

taactccgtg ccagcagccg cggtaatacg gagggtgcaa gcgttaatcg gaattactgg 540

gcgtaaagcg cacgcaggcg gtttgttaag tcagatgtga aatccccggg ctcaacctgg 600

gaactgcatt tgaaactggc aagctagagt ctcgtagagg ggggtagaat tccaggtgta 660

gcggtgaaat gcgtagagat ctggaggaat accggtggcg aaggcggccc cctggacgaa 720

gactgacgct caggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780

cgctgtaaac gatgtcgatt tggaggttgt gcccttgagg cgtggcttcc ggagctaacg 840

cgttaaatcg accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg 900

ggcccgcaca agcggtggag catgtggttt aattcgatgc aacgcgaaga accttaccta 960

ctcttgacat ccagagaact ttccagagat ggattggtgc cttcgggaac tctgagacag 1020

gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg ggttaagtcc cgcaacgagc 1080

gcaaccctta tcctttgttg ccagcggttc ggccgggaac tcaaaggaga ctgccagtga 1140

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cacgtgctac aatggcrtat acaaagagaa gcgacctcgc gagagcaagc ggacctcata 1260

aagtaygtcg tagtccggat tggagtctgc aactcgactc catgaagtcg gaatcgctag 1320

taatcgtaga tcagaatgct acggtgaata cgttcccggg ccttgtacac accgcccgtc 1380

acaccatggg agtgggttgc aaaagaagta ggtagcttaa ccttcgggag ggcgcttacc 1440

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Claims (5)

1. Serratia marcescens strain (A)Serratia marcescens) The method is characterized in that: the strain is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 23 days in 2020, and the preservation number is as follows: CGMCC number 21530.

2. Use of a serratia marcescens strain according to claim 1, wherein: the strain is used for preparing a microbial agent for promoting the development of plant root systems or a growth promoter for promoting the growth and development of plants.

3. A microbial agent for promoting the development of plant roots is characterized in that: the microbial agent comprises the serratia marcescens strain of claim 1.

4. A plant growth promoter characterized by: comprising the serratia marcescens strain of claim 1.

5. A serratia marcescens with a fluorescent label is characterized in that: the Serratia marcescens strain of claim 1, which is labeled with GFP fluorescence; performing fluorescence labeling on the serratia marcescens in an electric shock conversion mode; electric shock conversion: adding 1 mu g of pPROBE-GTkan plasmid into the competent cells of the Serratia marcescens, gently mixing uniformly, adding into a precooled electric shock cup, carrying out electric shock for 5 seconds at 1250V, quickly adding 800 mu L of LB culture medium, and incubating for 1-2 hours at the temperature of 30 ℃ with slow shaking.

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