CN114539424A

CN114539424A - Fusion protein combined with glucan, recombinant microorganism, preparation method of recombinant microorganism and application of recombinant microorganism in remodeling rhizosphere microbial community

Info

Publication number: CN114539424A
Application number: CN202210135742.XA
Authority: CN
Inventors: 喻其林; 尹宏达; 冯丽安; 陈煜樵; 冯禹铭
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2022-02-15
Filing date: 2022-02-15
Publication date: 2022-05-27
Anticipated expiration: 2042-02-15
Also published as: CN114539424B

Abstract

The invention provides a fusion protein combined with glucan, a recombinant microorganism, a preparation method of the recombinant microorganism and application of the recombinant microorganism in remodeling rhizosphere microbial community, and belongs to the technical field of rhizosphere bacteria. The invention designs a novel fusion protein with high-efficiency glucan binding through simulating the binding relationship between glucan binding protein and a glucan substrate. The fusion protein is displayed and expressed on the surface of a microbial cell, the obtained recombinant microorganism is combined into a plant rhizosphere through glucan secreted by the rhizosphere, and meanwhile, the recombinant microorganism has the capability of recruiting natural glucan-producing microorganisms, so that the microbial community of the plant rhizosphere is remodeled, the plant growth is promoted, the stress resistance of the plant is improved, and the plant repair is effectively carried out in the sewage treatment and soil pollutant removal processes.

Description

Fusion protein combined with glucan, recombinant microorganism, preparation method of recombinant microorganism and application of recombinant microorganism in remodeling rhizosphere microbial community

Technical Field

The invention belongs to the technical field of rhizosphere bacteria, and particularly relates to glucan-combined fusion protein, recombinant microorganism, a preparation method of the fusion protein and the recombinant microorganism and application of the fusion protein and the recombinant microorganism in remodeling of rhizosphere microbial communities.

Background

Microbial communities are ubiquitous in nature and play an important role in a large number of biological processes, such as degradation of environmental pollutants (e.g., persistent organic pollutants, micro-plastics), production of biofuels (e.g., methane, ethanol, and petroleum), tolerance to various stresses (e.g., heat, cold, dry, hypertonic, oxidative, antibiotic, and pathogen invasion conditions), and maintenance of intestinal ecosystem homeostasis. The modification of natural microbial communities to improve the synergistic efficiency among microbial individuals is becoming a hot spot in synthetic biology and biotechnology.

Plant rhizosphere microflora is a narrow area of plant roots tightly surrounded by microflora (such as bacteria, fungi, protozoa, and algae) and dynamically influenced by root metabolism. These microbial communities interact strongly with the plant root system to improve plant growth by releasing inorganic nutrients, producing phytohormones and alleviating environmental stresses. In addition, benign microbial communities form a physical protective layer, produce antimicrobial agents, activate the immune response of plants, and protect plants from pathogen infection. Therefore, optimizing the rhizosphere microbial community structure is undoubtedly an effective way to promote plant growth, enhance the plant's resistance to physical, chemical and biological stresses.

Environmental pollution, especially multiple pollution caused by emerging pollutants (such as heavy metals, nutrient components associated with eutrophication, persistent organic pollutants, etc.), is becoming a significant problem for water and soil systems and seriously threatening people's health and global ecosystem. Phytoremediation is a popular environmental pollution treatment strategy, and has been successfully applied to the removal of volatile organic compounds and PM 2.5, the removal of heavy metals in soil and water, and the degradation of organic pollutants. It is not certain that the rhizosphere microflora, an important interacting factor for plants, plays a crucial role in promoting plant growth, metabolism and contaminant removal during phytoremediation. However, although a lot of research has focused on the influence of contaminants (such as heavy metals, persistent organic contaminants, and petroleum hydrocarbons) on rhizosphere microbial communities, artificially modifying rhizosphere microbial communities, removing various contaminants through microbial community-plant symbionts, and relieving various stresses of contaminated sites, there has been no report yet.

Microbial cells and associated Extracellular Polymeric Substrates (EPSs) constitute the natural physical contact between organisms. However, these contacts are unstable and are often subject to damage by complex environmental factors (e.g., temperature, pH, C/N ratio, metal ions, etc.). Synthetic biology, a powerful strategy for large-scale remodeling of cell surface components, would be a promising approach to enhance the physical link between plants and organisms.

Disclosure of Invention

In view of the above, the present invention aims to provide a glucan-binding fusion protein having a more desirable binding property to a glucan-containing substance.

The invention also aims to provide a recombinant microorganism, a preparation method thereof and application thereof in remodeling the microorganism community at the rhizosphere, wherein the recombinant microorganism enhances physical contact with the root system of a plant through fusion protein, and forms the microorganism community with the plant repairing function at the rhizosphere.

The invention provides a fusion protein combined with glucan, which is obtained by sequentially connecting the following structures: a clostridium cellulolyticum CipC protein glucan binding domain, a marker protein domain, and a clostridium cellulolyticum CipC protein glucan binding domain.

Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:1 is shown.

The invention provides a fusion gene for coding the fusion protein, which is obtained by connecting a microbial secretion signal peptide coding sequence and a coding sequence of the fusion protein in sequence.

Preferably, the nucleotide sequence of the fusion gene is shown as SEQ ID NO:2, respectively.

The invention provides a recombinant expression vector containing the fusion gene.

The invention provides a recombinant microorganism which binds glucan and displays the fusion protein on the cell surface of the recombinant microorganism.

The invention provides application of the recombinant microorganism in remodeling of plant rhizosphere microbial communities.

Preferably, the microorganisms in the plant rhizosphere microflora include glucan-producing bacteria or fungi;

the bacteria comprise one or more of the following: cuprinus taiwanensis, pseudomonas fluorescens, bacillus subtilis and acetobacter xylinum;

the fungi comprise one or more of the following: trichoderma harzianum, Saccharomyces cerevisiae, Paecilomyces varioti and Aspergillus niger.

The invention provides application of the recombinant microorganism in promoting plant growth, improving plant stress resistance or repairing plants.

Preferably, said improving plant stress resistance is said recombinant microorganism reducing oxidative stress;

the phytoremediation is to remove heavy metal pollution and eutrophication in the planting matrix; the heavy metal comprises one or more of the following components: cd. Pb, Hg, Cu, Cr, and Co.

The invention provides a fusion protein combined with glucan, which is obtained by sequentially connecting the following structures: a clostridium cellulolyticum CipC protein glucan binding domain, a marker protein domain, and a clostridium cellulolyticum CipC protein glucan binding domain. The invention designs a novel fusion protein by simulating the combination relationship between glucan binding protein and glucan substrate. Experiments prove that the fusion protein can be well combined with glucan by taking microorganisms as carriers. Experiments show that compared with a control microorganism which does not express the fusion protein, the content of glucan on the surface of the microorganism which expresses the fusion protein on the surface is obviously increased, which indicates that the fusion protein has stronger glucan binding capacity.

The invention provides a recombinant microorganism binding to dextran, the cell surface of which displays the fusion protein. The recombinant microorganism has the ability to recruit naturally occurring glucan-producing microorganisms while also being able to bind to plant roots by binding rhizosphere secreted glucans, thereby enhancing physical contact of the natural microorganism with the plant roots. The enhancement of physical contact further remodels the rhizosphere microbial communities of the model repair plants such as eichhornia crassipes, duckweed, solanum nigrum and the like, and promotes the accumulation of various pollutants in the plant body. The recombinant microorganism provided by the invention provides a new idea for remodeling rhizosphere microbiota, so that phytoremediation can be effectively carried out in the processes of sewage treatment and soil pollutant removal. Meanwhile, the combination of the recombinant microorganism and the plant rhizosphere also has the effects of promoting the plant growth and improving the stress resistance of the plant.

Drawings

FIG. 1 is a graph showing the effect of EcCMC on the diversity of the rhizosphere bacteria of Eichhornia crassipes; wherein FIG. 1(a) is Shannon index of rhizosphere bacteria of each treatment group, FIG. 1(b) is Simpson index of rhizosphere bacteria of each treatment group, and FIG. 1(c) is relative abundance distribution of bacteria at each sample boundary level;

FIG. 2 is a graph showing the effect of EcCMC on the growth of Eichhornia crassipes plants and roots, wherein FIG. 2(a) is a photograph of Eichhornia crassipes plants in each treatment group, FIG. 2(b) is the wet weight of the plants in each treatment group, and FIG. 2(c) is the length of the roots in each treatment group;

FIG. 3 shows the effect of EcCMC on the ability of Eichhornia crassipes to remove pollutants, where FIG. 3(a) shows the change of cadmium content in root systems of each treatment group, FIG. 3(b) shows the change of cadmium content in wastewater, FIG. 3(c) shows the change of COD content in wastewater, FIG. 3(d) shows the change of total nitrogen content in wastewater, FIG. 3(e) shows the change of total potassium content in wastewater, and FIG. 3(f) shows the change of total phosphorus content in wastewater.

Detailed Description

The invention provides a fusion protein combined with glucan, which is obtained by connecting the following structures in sequence: a clostridium cellulolyticum CipC protein glucan binding domain, a marker protein domain, and a clostridium cellulolyticum CipC protein glucan binding domain.

In the present invention, the marker protein in the fusion protein includes a fluorescent protein or phycoerythrin. The fluorescent protein comprises a red fluorescent protein mCherry or a green fluorescent protein. In the present example, the red fluorescent protein mCherry domain is used as the marker protein domain to represent, which illustrates the biological function of the fusion protein. The labeled protein plays a role in protein identification and does not influence the performance of glucan binding. The amino acid sequence of the fusion protein is preferably as shown in SEQ ID NO:1 (MKKTAIAIAVALAGFATVAQAAGTGVVSVQFNNGSSPASSNSIYARFKVTNTSGSPINLADLKLRYYYTQDADKPLTFWCDHAGYMSGSNYIDATSKVTGSFKAVSPAVTNADHYLEVALNSDAGSLPAGGSIEIQTRFARNDWSNFDQSNDWSYTAAGSYMDWQKISAFVGGTLAYGSTPDGGNPPPQDPTINPTSISAKAGSFADTKITLTPNGNTFNGISELQSSQYTKGTPGGGGVSKGEDDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSASAGASASAGASASAGASASAAGTGVVSVQFNNGSSPASSNSIYARFKVTNTSGSPINLADLKLRYYYTQDADKPLTFWCDHAGYMSGSNYIDATSKVTGSFKAVSPAVTNADHYLEVALNSDAGSLPAGGSIEIQTRFARNDWSNFDQSNDWSYTAAGSYMDWQKISAFVGGTLAYGSTPHHHHHH).

The invention provides a fusion gene for coding the fusion protein, which is obtained by connecting a microbial secretion signal peptide coding sequence and a coding sequence of the fusion protein in sequence. When the fusion protein is expressed by taking a microorganism as a vector, the fusion protein needs to be expressed on the surface of a cell, so that the fusion protein needs to be positioned on the surface of the cell wall for expression by secreting a signal peptide by the microorganism. The microbial secretion signal peptide varies according to the kind of microbe, and includes a bacterial secretion signal peptide and a fungal secretion signal peptide. The present invention is not particularly limited in the kind of the bacterial secretion signal peptide and the fungal secretion signal peptide, and a microbial secretion signal peptide well known in the art may be used. In the present invention, the recombinant expression of the fusion protein in bacteria is illustrated by taking the secretion signal peptide OmpA of Escherichia coli as an example. The nucleotide sequence of the fusion gene is preferably as shown in SEQ ID NO:2 (ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCAGGTTTCGCTACCGTCGCTCAGGCTGCGGGCACCGGCGTGGTGTCCGTGCAGTTCAACAACGGCTCCAGCCCTGCAAGCTCCAACTCGATCTACGCTCGTTTCAAGGTCACCAACACCAGCGGCAGCCCGATCAACCTGGCGGATCTGAAATTGCGCTATTACTACACCCAAGATGCTGATAAACCGTTGACCTTCTGGTGTGATCACGCGGGTTACATGAGCGGCTCTAACTATATCGATGCGACCTCGAAGGTTACCGGTTCCTTTAAGGCCGTGAGCCCAGCGGTTACGAACGCGGATCACTATCTGGAAGTCGCACTTAATTCTGATGCCGGGTCGCTGCCGGCTGGCGGTTCTATCGAGATCCAGACTCGCTTCGCACGTAATGACTGGTCAAACTTCGACCAGTCGAACGATTGGAGCTACACCGCTGCAGGCTCCTACATGGATTGGCAAAAGATTAGCGCATTCGTTGGTGGCACGTTGGCGTATGGTTCCACCCCGGACGGTGGCAATCCGCCGCCGCAGGACCCGACCATTAACCCGACCTCCATTAGTGCTAAAGCCGGTTCGTTTGCGGACACCAAGATCACGCTGACCCCAAATGGTAACACGTTCAATGGCATAAGCGAACTGCAGTCTAGCCAGTACACCAAAGGTACCCCGGGTGGTGGAGGTGTTAGCAAGGGTGAAGATGATAACATGGCAATTATCAAGGAGTTCATGCGTTTCAAAGTTCACATGGAAGGTAGCGTCAATGGTCACGAGTTCGAGATTGAGGGCGAGGGCGAGGGCCGTCCGTATGAAGGCACCCAAACCGCTAAATTGAAGGTCACCAAGGGCGGACCGCTCCCATTTGCCTGGGATATTCTGAGCCCACAGTTTATGTACGGTTCGAAAGCGTACGTGAAACATCCGGCTGACATCCCGGATTACCTGAAGCTTAGCTTTCCGGAGGGTTTTAAGTGGGAACGTGTTATGAATTTTGAGGACGGTGGGGTGGTGACGGTGACCCAAGACAGCAGCCTGCAAGACGGTGAGTTCATTTACAAGGTAAAATTGAGAGGTACGAACTTCCCGAGCGACGGCCCGGTTATGCAGAAAAAGACCATGGGTTGGGAAGCAAGTAGCGAGCGCATGTACCCGGAGGATGGTGCTCTGAAGGGTGAGATCAAGCAGCGTCTAAAGCTCAAAGATGGTGGCCACTACGATGCTGAAGTTAAAACTACCTATAAAGCGAAAAAGCCGGTTCAACTGCCGGGTGCGTATAACGTGAACATTAAATTGGACATTACCTCCCATAATGAGGATTATACGATCGTGGAACAATATGAACGTGCGGAAGGCCGTCATAGCACCGGTGGCATGGACGAACTGTACAAAAGCGCGTCCGCGGGTGCGAGCGCTAGCGCGGGCGCTTCTGCTTCCGCCGGGGCGAGCGCCAGCGCCGCAGGCACCGGCGTTGTGAGCGTTCAGTTTAACAACGGCAGCTCTCCCGCGAGCTCTAACAGCATCTACGCGCGTTTCAAAGTCACCAACACCTCCGGCAGCCCGATCAATCTGGCGGACTTGAAGTTACGCTATTACTACACCCAGGACGCGGACAAACCGCTCACCTTCTGGTGCGATCATGCCGGTTACATGTCCGGCAGCAATTATATTGACGCGACATCCAAGGTGACTGGCAGCTTTAAGGCGGTGTCACCAGCGGTTACCAATGCGGATCATTATCTGGAAGTTGCTCTGAACAGCGACGCAGGTAGCTTGCCGGCCGGCGGGTCCATCGAGATCCAAACCCGCTTCGCCCGTAATGACTGGTCAAACTTCGATCAATCAAACGACTGGAGCTATACTGCGGCAGGTAGCTATATGGATTGGCAAAAAATCAGTGCGTTTGTAGGTGGCACCTTGGCATACGGTTCAACCCCTCATCACCACCACCACCACTAA).

The backbone vector of the recombinant expression vector of the present invention is not particularly limited, and any backbone vector known in the art, such as pET-28a plasmid or pVLT33 plasmid, may be used. When the backbone vector is pET-28a plasmid, the insertion site of the fusion gene is preferably BamH I and Xho I to form pET-28 a-CMC. When the backbone vector is pVLT33 plasmid, the insertion site of the fusion gene is preferably EcoRI and SacI, forming pVLT 33-CMC. The method for inserting is not particularly limited, and the method for inserting can be realized by adopting an inserting method known in the field, for example, a primer with an enzyme cutting site is utilized to carry out PCR amplification on a fusion gene, the obtained PCR product and a skeleton vector are subjected to double enzyme cutting treatment by adopting BamH I and Xho I, an enzyme cutting fragment is connected with the obtained linear vector, and a recombinant expression vector is obtained by screening. Wherein the primer with the enzyme cutting site comprises a nucleotide sequence shown as SEQ ID NO: 3 (CGC)GGATCCATGAAAAAGACAGCTATCGCGAT) and SEQ ID NO: 4 (CCG)CTCGAGTTAGTGGTGGTGGTGGTGATG) is shown. The reaction condition of PCR amplification is preferably 94 ℃ for 5min, (94 ℃ for 30s, 50 ℃ for 2min10s, 72 ℃ for 30s) multiplied by 30 cycles, 72 ℃ for 7min, and the temperature is reduced to 4 ℃; the reaction system for PCR amplification is preferably 5 μ l of Long Taq enzyme buffer solution, 4 μ l of 2.5mM dNTP, 2 μ l of upstream primer, 2 μ l of downstream primer, 2 μ l of template, 1 μ l of Long Taq enzyme and ddH₂O was supplemented to 50. mu.l.

In the present invention, the method for constructing the recombinant microorganism preferably comprises the steps of:

and (3) transforming the recombinant expression vector in the scheme into a microorganism competence, and culturing and screening to obtain the recombinant microorganism.

The method of transformation is not particularly limited in the present invention, and may be performed by a method well known in the art, for example, CaCl₂And (3) a conversion method. The screening utilizes the resistance gene carried in the recombinant expression vector to carry out resistance screening.

In the present invention, when the recombinant expression vector is pET-28a-CMC, the microorganism competence is preferably Escherichia coli competence. When the recombinant expression vector is pVLT33-CMC, the microorganism competence is preferably Pseudomonas putida KT2440 competence.

The cell surface expression fusion protein of the recombinant microorganism has strong binding capacity to glucan, so that the recombinant microorganism can be bound with a plant root system secreting glucan by using the fusion protein, meanwhile, the recombinant microorganism can recruit and bind a large number of microorganisms producing glucan naturally based on the fusion protein, the abundance and the number of the microorganisms at the plant rhizosphere are greatly improved, a new microbial community is formed at the plant rhizosphere, and the microorganisms producing glucan naturally have the effects of wide growth promotion and stress resistance and removing heavy metal pollutants, so that the growth speed and the stress resistance of the plant are improved, and the plant restoration effect is exerted.

In the present invention, the microorganism in the plant rhizosphere microbial community preferably comprises a glucan-producing bacterium or fungus. The bacteria comprise one or more of the following: cuprinus taiwanensis, pseudomonas fluorescens, bacillus subtilis and acetobacter xylinum. The fungi comprise one or more of the following: trichoderma harzianum, Saccharomyces cerevisiae, Paecilomyces varioti and Aspergillus niger. The plants preferably include aquatic plants and terrestrial plants. The present invention is not particularly limited in kind of the plant, and plants well known in the art may be used. Taking the aquatic model plants Eichhornia crassipes and duckweed as examplesThe recombinant microorganism has the function of remodeling the rhizosphere microbial community of the plant. Meanwhile, the black nightshade is taken as a representative of terrestrial plants, so that the efficacy of the recombinant microorganisms in remodeling the rhizosphere microbial communities of the plants is demonstrated. Experimental results show that the recombinant microorganism has a reconstruction effect on plant rhizosphere microbial communities. The recombinant microorganism was administered at a concentration of 10⁷～10⁸Individual cell/L or 10⁷～10⁸Individual cells/kg.

In the present invention, the promotion of plant growth is expressed by increasing the plant height, wet weight and root length of a plant.

In the present invention, it is preferred that the plant stress resistance is improved by the recombinant microorganism that the oxidative stress response is reduced. The reduction of oxidative stress is manifested by increasing activities of superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD), and reducing MDA content.

In the present invention, the phytoremediation is preferably the removal of heavy metal contamination and eutrophication in the planting substrate. The heavy metal preferably comprises one or more of the following: cd. Pb, Hg, Cu, Cr, and Co. Reducing COD value in the water body and the content of total nitrogen, total phosphorus and total potassium. Experiments prove that the plant growth promotion and pollutant treatment capacity is realized by remolding rhizosphere microbial communities by using the recombinant microorganisms.

The glucan-binding fusion protein, the recombinant microorganism, the preparation method thereof and the application thereof in remodeling rhizosphere microbial community provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example 1

Effect of recombinant microorganism EcCMC on remodeling and repairing and enhancing rhizosphere microbial community of eichhornia crassipes

1. Test method

(1) Preparation method of recombinant microorganism EcCMC

Firstly, an artificially synthesized protein Cmc with high efficiency of combining glucan is designed, and the artificially synthesized protein Cmc can be combined with glucanCombining plant root system with natural microbe for producing glucan. The fusion Cmc protein (SEQ ID NO:1) consists of three domains: clostridium cellulolyticum CipC protein glucan binding Domain (CBM)_CipC) Red fluorescent protein mCherry structural domain, cellulolytic clostridium CipC protein glucan binding structural domain (CBM)_CipC). A coding gene CMC (SEQ ID NO:2) of the protein Cmc is prepared by a de novo synthesis method, and the N end of the gene is provided with an escherichia coli secretion signal peptide OmpA, so that the fusion protein Cmc can be efficiently displayed on the cell surface of a recombinant microorganism.

The coding gene CMC is cloned to pET-28a plasmid, and the multiple cloning sites are BamH I and Xho I, so as to obtain expression plasmid pET-28 a-CMC. Wherein the amplification primer is shown as SEQ ID NO: 3(CGCGGATCCATGAAAAAGACAGCTATCGCGAT, wherein the underlined portions are the cleavage sites) and SEQ ID NO: 4 (CCG)CTCGAGTTAGTGGTGGTGGTGGTGATG, wherein the underlined sections are the cleavage sites).

The expression plasmid pET-28a-CMC utilizes CaCl₂The method is transformed into BL21 competence to obtain the synthetic strain EcCMC. At the same time, a control strain EcM was prepared, which only expressed the control protein mCherry.

(2) Evaluation of polysaccharide binding ability:

to evaluate the polysaccharide binding ability of the recombinant microorganisms, several polysaccharides, including FITC-labeled dextran, mannan, and hyaluronic acid, were dissolved in PBS buffer at a concentration of 100 mg/L. The synthesized cells were cultured at 1X 10⁷cells/mL were added to the culture. The mixture was incubated at 37 ℃ for 30 minutes, followed by centrifugation at 12000rpm for 2 minutes. The cells were washed twice with PBS and the fluorescence intensity of the cells FITC was detected using flow cytometric analysis or a fluorescence microplate reader.

(3) Evaluation of bacterial binding capacity:

to investigate the ability of recombinant microorganisms to recruit natural microbial cells, native glucan-producing cuprum taiwan, pseudomonas fluorescens, bacillus subtilis, acetobacter xylinum were cultured in LB medium containing 2% glucose (W/V) for 12h, fungal strains trichoderma harzianum, saccharomyces cerevisiae, paecilomyces varioti, and aspergillus niger were cultured in PDA medium for 36h, centrifuged to obtain native strains, and 4', 6-diamino-2-was usedPhenyl indole (5mg/L) stained bacteria, fungal cells with fluorescent white (10mg/L) and resuspended in PBS. Newly cultured recombinant microbial cells at 1X 10⁷The concentration of cells/mL is added to the medium. After 30 minutes of co-incubation, the cultures were collected for confocal microscopy.

(4) And (3) analyzing the binding capacity of EcCMC and the water hyacinth root system:

fresh and young roots were taken from healthy Eichhornia crassipes plants and washed twice with PBS. Soaking the roots in PBS, adding the newly cultured recombinant microbial cells to the root suspension to a final concentration of 1X 10⁷Individual cells/L. After incubation at 30 ℃ for 30min, the roots were gently rinsed twice with PBS and observed with a confocal microscope. The bacterial cells remaining in solution were used for Colony Forming Units (CFU) determination.

(5) The remodeling action of EcCMC on the rhizosphere microbial community of eichhornia crassipes is analyzed:

the eichhornia crassipes is cultured in simulated sewage, and the influence of the artificially recombinant microorganisms on the rhizosphere microbial community of the eichhornia crassipes is researched. The sewage is composed of peptone 80mg/L, yeast extract 30mg/L, (NH)₄)₂SO₄28mg/L、NH₄Cl 25mg/L、KH₂PO₄70 mg/L、CdCl₂3.3 mg/L and pH6.0-6.5. COD of sewage is 40mg/L, total nitrogen is 28mg/L, total potassium is 20mg/L, total phosphorus is 16mg/L, Cd²⁺Is 3 mg/L. The 3 rhizosphere microbial communities were re-modeled as a control group, EcM group and EcCMC group. In most experiments, synthetic bacterial cells EcM (EcM group) or EcCMC (EcCMC group) were added to wastewater at a concentration of 1X 10⁸cells/L. The sewage containing recombinant microorganism cells is added into 2L of fish tanks, 1L of the sewage is added into each fish tank, and then 1 plant of Eichhornia crassipes (25 +/-2 g) is added into each fish tank for culture. In the control group, plants were cultured in a fish tank in the presence of wastewater without any recombinant microbial cells. Each group had 3 replicates. After 14 days of culture, plant roots were harvested for further microbiology, metabolomics, biochemistry and contaminant removal analysis. The sampled plant roots were fixed with 2.5% glutaraldehyde for 24h and then lyophilized with a vacuum lyophilizer for 12 h. The dried roots were then observed by scanning electron microscopy. Day 14, harvestingExtracting the total DNA of the eichhornia crassipes rhizosphere microorganism by using a TGuide S96 magnetic bead extraction kit. Bacterial 16S V3+ V4 primer (338F, 5'-ACTCCTACGGGAGGCAGCA-3', SEQ ID NO: 5; 806R, 5' -GGACTACHVGGGTWTCTAAT-3', SEQ ID NO:6) and fungal primers (ITS1F, 5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO: 7; ITS25 "-GCTGCGTTCTTCATCGATGC-3 ', SEQ ID NO:8) were used. The constructed library was sequenced using Illumina novaseq6000 sequencing system (Biomarker ltd., China). Data were analyzed using the SILVA reference database. The Alpha diversity was evaluated using QIIME2 software, and Shannon index and Simpson index were calculated for each group.

(6) The effect of EcCMC on the growth, stress resistance and pollutant removal capacity of Eichhornia crassipes:

on the 14 th day of growth, each treated Eichhornia crassipes was weighed and root length was measured. And (3) crushing and cracking the sampled root system cells by adopting a liquid nitrogen grinding method. The lysates were centrifuged at 12000rpm for 10 minutes and the total protein content of the supernatants was assayed using Coomassie Brilliant blue reagent. The SOD, CAT and POD activities of the supernatant were measured using superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD) detection kits. MDA level was detected using a Malondialdehyde (MDA) detection kit.

The roots, leaves and sewage of the plants are sampled within a specified time of artificially synthesized bacteria for treating the eichhornia crassipes simulated sewage. Drying root and leaf, weighing, and adding 30% HNO₃Digesting, and detecting Cd in digestion solution by using an inductively coupled plasma mass spectrometer²⁺The content of (a). Determination of Cd in wastewater by similar method²⁺Total potassium and total phosphorus content. And (3) measuring the COD value of the sewage sample by using a COD measuring kit. The samples were preheated in potassium persulfate solution and then stained with ammonium molybdate, antimony potassium tartrate and sodium l-ascorbate to quantify total phosphorus.

2 results of the test

(1) Binding ability of EcCMC to dextran

After incubation of EcCMC or EcM cells with FITC-dextran for 10min, EcM cells had almost no FITC fluorescence, whereas EcCMC cells had significant FITC fluorescence. Thus indicating that EcCMC has strong glucan binding capacity.

Confocal microscope magnified images and fluorescence quantification further showed that EcCMC cells had a bilateral distribution of mCherry. These results indicate that the expression of glucan-binding protein is high in the surface of EcCMC cells.

(2) EcCMC adsorption capacity to natural microorganisms

After the EcCMC is incubated with natural cells of the cuprinus taiwan, the pseudomonas fluorescens, the bacillus subtilis and the acetobacter xylinum for 30min, the EcM cells in the control group can not absorb the natural bacterial cells. In contrast, EcCMC cells recruit strongly in conjunction with native bacterial cells, forming a co-aggregate of EcCMC with native bacterial cells. Thus, EcCMC strongly absorbs bacterial cells that naturally produce glucan-containing EPS.

All tested fungal cells were able to bind strongly to EcCMC cells but not to control EcM cells when incubated with EcCMC. In particular, yeast cells dispersed well in the medium when incubated with EcM cells, but yeast cells formed significant aggregates with EcCMC cells, indicating that EcCMC acts as a cross-linking agent to induce fungal cell aggregation.

In addition, EcCMC cells also strongly bound Aspergillus niger hyphae and conidia, whereas EcM cells failed, suggesting that EcCMC may be recruited to fungal cells at different stages of the fungal life cycle. In summary, the above results indicate that EcCMC cells have a strong ability to bind and recruit native bacterial and fungal cells to produce extracellular glucan.

(3) Binding capacity of EcCMC and Eichhornia crassipes root system

Culturing Eichhornia crassipes (lour.) hand-Mazz in water containing recombinant microorganism for 1 day, and observing the soaked roots with confocal microscope. The root system only absorbed very low levels of EcM control cells, but adsorbed very high levels of EcCMC cells. Colony forming unit assays further indicated that the roots adhered EcCMC 12-fold more than EcM. Thus, EcCMC cells have higher root binding capacity than EcM.

(4) Reconstitution effect of EcCMC on eichhornia crassipes root system microbial community

The 16S rDNA sequencing results showed an increase in Shannon index and Simpson index for the EcCMC-treated rhizosphere bacterial diversity compared to the rhizosphere bacterial diversity of the control and EcM-treated groups. Bacterial colony abundance analysis further indicated that there were multiple gates in EcCMC-treated roots with higher relative abundance than the control and EcM-treated roots. These gates include Acidobacterium, Actinobacterium, Gemmatimonadetes, Chloroflexi, Armatimamadeades and Nitrospirae (FIG. 1).

Meanwhile, ITS analysis sequencing results showed that EcM and EcCMC had no significant effect on the composition of the mycogate. However, EcCMC, while not significantly altering fungal diversity, significantly increases fungal biomass on the root system. Thus, EcCMC highly remodels the eichhornia rhizosphere microbial community in simulated sewage. In addition, the addition of 100mg/L penicillin, 50mg/L streptomycin and 100mg/L fluconazole to the culture system can result in the significant reduction of the biodiversity and microorganisms of the Eccmc group iris rhizosphere microbial community, thereby eliminating the microbial community remodeling action of Eccmc.

(5) EcCMC enhances the growth, stress resistance and pollutant treatment of Eichhornia crassipes

After the eichhornia crassipes is cultured in simulated sewage for 14 days, the plant height, wet weight (40g to 30-32 g) and root length (18cm to 12-13 cm) of the eichhornia crassipes plant treated by EcCMC are all higher than those of the other two groups. This suggests that EcCMC has a positive role in plant growth (see FIG. 2).

The enzyme activity determination result shows that after the EcCMC is treated for 3 days, the activity of 3 enzymes is higher than that of the root systems treated by the control and EcM, which indicates that the root system treated by the EcCMC has stronger tolerance to heavy metals. After 14 days of treatment, the activity of these enzymes in EcCMC treated roots was lower than in the other two groups, which may be related to the reduced oxidative stress in this group. After 3 and 14 consecutive days of EcCMC treatment, Malondialdehyde (MDA) levels were significantly reduced. The results show that EcCMC significantly up-regulates the level of antioxidant enzymes in roots under heavy metal stress, and further relieves heavy metal-induced oxidative stress.

The control and EcM groups had lower Cd content on the root surface, while the EcCMC treated root surface had a significant Cd distribution. ICP analysis further shows that Cd of roots treated by EcCMC are in 3-14 days²⁺The content is higher than the other two groups. Meanwhile, Cd in plant leaves of EcCMC group²⁺Is also higher than in the other groups,indicating that EcCMC also promotes Cd²⁺Transport from root system to leaf. Further measuring Cd in sewage by ICP method²⁺The content of (a). After 14 days of treatment, the control and EcM-treated groups removed only a portion of the Cd in the water²⁺Cd in sewage²⁺In an amount of>0.5 mg/L. In contrast, EcCMC treatment group plants almost completely removed Cd from wastewater²⁺The heavy metal content in the sewage is reduced to<0.05mg/L (FIG. 3), which indicates that the heavy metal removal capacity of EcCMC group plants is much higher than that of the other two groups (>98% by weight<80％)。

In addition, compared with the control group and the EcM group, the COD value of the EcCMC group after 14d treatment is much lower than that of the other two groups (the ratio of <7mg/L >25mg/L), and the total nitrogen, the total potassium and the total phosphorus are all lower (the ratio of <1.5mg/L >6mg/L), which indicates that the EcCMC can also obviously promote the treatment capability of plants on eutrophic pollutants.

In addition, 100mg/L penicillin, 50mg/L streptomycin and 100mg/L fluconazole are added into the culture system to disorder the microbial community of the root system, so that the promotion effect of EcCMC on the growth, heavy metal adsorption and eutrophication pollutant removal of the eichhornia crassipes plant can be eliminated, and the promotion effect of EcCMC on the plant growth metabolism and the pollutant treatment capacity is realized by remolding the microbial community of the root system.

Example 2

Effect of recombinant microorganism EcCMC on reconstruction and repair enhancement of microbial community of duckweed rhizosphere

Test method 1

(1) The remodeling action of EcCMC on the microbial community of the duckweed rhizosphere is analyzed:

duckweed is cultured in simulated sewage, and the influence of the artificially recombinant microorganisms on the microbial community of the duckweed rhizosphere is researched. The sewage is composed of peptone 80mg/L, yeast extract 30mg/L, (NH)₄)₂SO₄28mg/L、NH₄Cl 25mg/L、KH₂PO₄70 mg/L、CdCl₂3.3 mg/L, pH 6.0.0-6.5. COD of the sewage is 40mg/L, total nitrogen TN is 28mg/L, total potassium TK is 20mg/L, total phosphorus TP is 16mg/L, Cd²⁺Is 3 mg/L. The 3 rhizosphere microbial communities were re-modeled as a control group, EcM group and EcCMC group. In most experiments, the synthesizedBacterial cells EcM (EcM group) or EcCMC (EcCMC group) were added to the wastewater at a concentration of 1X 10⁸cells/L. The sewage containing the recombinant microorganism cells is added into fish tanks with the volume of 1L, 0.5L is added into each fish tank, and then 1 duckweed (25 +/-2 g) is respectively added into each fish tank for culture. In the control group, plants were cultured in a fish tank in the presence of wastewater without any recombinant microbial cells. Each group had 3 replicates. After 14 days of culture, total DNA of the duckweed rhizosphere microorganisms is extracted by using a TGuide S96 magnetic bead extraction kit. The primers for bacterium 16S V3+ V4 and its fungus were used. The constructed library was sequenced using Illumina novaseq6000 sequencing system (Biomarker ltd., China). Data were analyzed using the SILVA reference database. The Alpha diversity was evaluated using QIIME2 software, and Shannon index and Simpson index were calculated for each group.

(6) Effect of EcCMC on duckweed growth and contaminant removal capacity:

on day 14 of growth, the treated duckweeds were weighed and root length measured. And (3) crushing and cracking the sampled root system cells by adopting a liquid nitrogen grinding method. The lysates were centrifuged at 12000rpm for 10 minutes and the total protein content of the supernatants was assayed using Coomassie Brilliant blue reagent. The SOD, CAT and POD activities of the supernatant were measured using superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD) detection kits. MDA level was detected using a Malondialdehyde (MDA) detection kit.

The roots, leaves and sewage of the plants were sampled at the designated time of artificial synthetic bacteria for treating duckweed-simulated sewage. Drying root and leaf, weighing, and adding 30% HNO₃Digesting, and detecting Cd in digestion solution by using an inductively coupled plasma mass spectrometer²⁺The content of (b). Determination of Cd in wastewater by similar method²⁺Total potassium and total phosphorus content. And (3) measuring the COD value of the sewage sample by using a COD measuring kit. The samples were preheated in potassium persulfate solution and then stained with ammonium molybdate, antimony potassium tartrate and sodium l-ascorbate to quantify total phosphorus.

2 results of the test

(1) Reconstruction effect of EcCMC on duckweed root system microbial community

The 16S rDNA sequencing results showed an increase in Shannon index and Simpson index for the EcCMC-treated rhizosphere bacterial diversity compared to the rhizosphere bacterial diversity of the control and EcM-treated groups. Bacterial colony abundance analysis further indicated that there were multiple gates in EcCMC-treated roots with higher relative abundance than the control and EcM-treated roots. These gates include Acidobacteria, Actinobacteria, Gemmatimonadetes, and the like. ITS analysis sequencing result shows that EcCMC obviously increases abundance of fungi of rhizomorpha and chytrium. Thus, EcCMC highly remodels the duckweed rhizosphere microbial community in simulated wastewater.

(2) EcCMC enhancement of duckweed growth and pollutant treatment

After being cultured in simulated sewage for 14 days, the plant height, the wet weight and the root length of the duckweed treated by the EcCMC are all higher than those of a control group and a EcM group, and the EcCMC has a positive effect on the growth of plants.

The enzyme activity determination result shows that after the EcCMC is treated for 3 days, the activity of 3 enzymes is higher than that of the roots treated by the control and EcM, and the MDA content is remarkably lower, which indicates that the roots treated by the EcCMC have stronger oxidation pressure tolerance to heavy metals.

ICP analysis shows that Cd of roots treated by EcCMC is in 3-14 days²⁺The content is higher than the other two groups. After 14 days of treatment, EcCMC treatment group plants almost completely removed Cd from the wastewater²⁺The heavy metal content in the sewage is reduced to<0.07mg/L, the COD value is far lower than that of the other two groups (<10mg/L ratio>28mg/L), total nitrogen, total potassium and total phosphorus are all low (<2mg/L ratio>8mg/L), which shows that EcCMC can also obviously promote the treatment capacity of duckweed on eutrophic pollutants.

In addition, 100mg/L penicillin, 50mg/L streptomycin and 100mg/L fluconazole are added into the culture system to disorder the microbial community of the root system, and the promotion effect of EcCMC on the growth of duckweed plants, heavy metal adsorption and removal of eutrophic pollutants can also be eliminated, so that the promotion effect of EcCMC on the plant growth and the pollutant treatment capacity is realized by remolding the microbial community of the root system.

Example 3

Effect of recombinant microorganism PpCMC on reconstruction and repair enhancement of rhizosphere microbial community of solanum nigrum

Test method 1

(1) Construction method of recombinant microorganism PpCMC

The CMC gene fragment is cloned to pVLT33 plasmid, and cloning sites are EcoRI and SacI, so that plasmid pVLT33-CMC is obtained. The plasmid is transferred into pseudomonas putida KT2440 to obtain a synthetic strain PpCMC. At the same time, a control strain PpM was prepared, which only expresses the control protein mCherry.

(2) Analysis of the remodeling action of PpCMC on the rhizosphere microbial community of solanum nigrum:

culturing the solanum nigrum in simulated heavy metal contaminated soil, and researching the influence of the artificial recombinant microorganism PpCMC on the rhizosphere microbial community of the solanum nigrum. Simulating Cd in heavy metal contaminated soil²⁺、Pb²⁺、Hg²⁺、Cu²⁺、Cr³⁺And Co²⁺The contents are all 1mg/kg soil. The 3 groups of rhizosphere microbial communities were re-modeled as a control group, a PpM group and a PpCMC group, respectively, with a cell concentration of 10⁷～10⁸Individual cells/kg soil. After culturing for 30 days, shearing the root system of the black nightshade, and extracting the total DNA of rhizosphere microorganisms by adopting a TGuide S96 magnetic bead extraction kit. Libraries constructed with bacterial 16SV3+ V4 primers and fungal primers (ITS1F and ITS2) were sequenced using the Illumina novaseq6000 sequencing System. Data were analyzed using the SILV A reference database. The Alpha diversity was evaluated using QIIME2 software, and Shannon index and Simpson index were calculated for each group.

(3) The effect of EcCMC on the growth, stress resistance and pollutant removal ability of Solanum nigrum:

on day 30 of growth, each treated nightshade was weighed and root length was determined. And (3) crushing and cracking the sampled root system cells by adopting a liquid nitrogen grinding method. The lysates were centrifuged at 12000rpm for 10 minutes and the total protein content of the supernatants was assayed using Coomassie Brilliant blue reagent. SOD, CAT and POD activities of the supernatant were determined using SOD, CAT and POD detection kits. MDA level was detected using a malondialdehyde detection kit.

During the designated time of artificial bacterial treatment of the solanum nigrum simulated sewage, the roots, leaves and sewage of the plants are sampled. Drying the roots and leaves, weighing,with 30% HNO₃Digesting, and detecting Cd in digestion solution by using an inductively coupled plasma mass spectrometer²⁺、Pb²⁺、Hg²⁺、Cu²⁺、Cr³⁺And Co²⁺The content of (a). The content of various heavy metal ions in the soil is determined by the same method.

2 results of the test

(1) Reconstruction effect of PpCMC on microbial community of root system of solanum nigrum

The 16S rDNA sequencing results showed an increase in Shannon index and Simpson index for PpCMC treated rhizospheric bacterial diversity compared to control and PpM treated rhizospheric bacterial diversity. Bacterial colony abundance analysis further showed that: the PpCMC treated roots had multiple gates in relative abundance that were higher than the control and PpM treated roots. These include the phyla Actinomyceta, Bacteroides, etc. ITS analysis sequencing result shows that PpCMC obviously increases the abundance of fungi of rhizomorpha neoflagellata and basidiomycota. Therefore, the PpCMC is highly remodeled for simulating the rhizosphere microbial community of the heavy metal contaminated soil solanum nigrum.

(2) Enhancement effect of PpCMC on growth of black nightshade and pollutant treatment

After the solanum nigrum plants are cultured in simulated heavy metal contaminated soil for 30 days, the plant height, the wet weight and the root length of the PpCMC-treated solanum nigrum plants are higher than those of a control group and a PpM group, and the PpCMC has a positive effect in plant growth.

The enzyme activity measurement result shows that the activity of 3 enzymes is higher than that of the control and PpM treated roots after the PpCMC is treated for 30d, and the MDA content is remarkably lower, which indicates that the PpCMC treated roots have stronger oxidation pressure tolerance to heavy metal. ICP analysis showed Cd in PpCMC-treated roots on day 30²⁺、Pb²⁺、Hg²⁺、Cu²⁺、Cr³⁺And Co²⁺The content of the heavy metal ions in the soil is higher than that of the other two groups, the content of the six heavy metal ions in the soil is reduced by more than 50%, and the reduction rate is far higher than that of the control group and the PpM group by 20-30%, which shows that the PpCMC can also obviously promote the treatment capability of the black nightshade on eutrophic pollutants.

In addition, 100mg/L penicillin, 50mg/L streptomycin and 100mg/L fluconazole are added into the system to disorder the microbial community of the root system, and the promotion effect of PpCMC on the growth and pollutant removal of the solanum nigrum plants can also be eliminated, so that the promotion effect of PpCMC on the plant growth metabolism and the pollutant treatment capacity is realized by remodeling the microbial community of the root system.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of southern kayak

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Val Thr Asn Thr Ser Gly Ser Pro Ile Asn Leu Ala Asp Leu Lys Leu

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Arg Tyr Tyr Tyr Thr Gln Asp Ala Asp Lys Pro Leu Thr Phe Trp Cys

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Asp His Ala Gly Tyr Met Ser Gly Ser Asn Tyr Ile Asp Ala Thr Ser

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Lys Val Thr Gly Ser Phe Lys Ala Val Ser Pro Ala Val Thr Asn Ala

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Asp His Tyr Leu Glu Val Ala Leu Asn Ser Asp Ala Gly Ser Leu Pro

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Ala Gly Gly Ser Ile Glu Ile Gln Thr Arg Phe Ala Arg Asn Asp Trp

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Ser Asn Phe Asp Gln Ser Asn Asp Trp Ser Tyr Thr Ala Ala Gly Ser

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Tyr Met Asp Trp Gln Lys Ile Ser Ala Phe Val Gly Gly Thr Leu Ala

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Tyr Gly Ser Thr Pro Asp Gly Gly Asn Pro Pro Pro Gln Asp Pro Thr

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Ile Asn Pro Thr Ser Ile Ser Ala Lys Ala Gly Ser Phe Ala Asp Thr

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Lys Ile Thr Leu Thr Pro Asn Gly Asn Thr Phe Asn Gly Ile Ser Glu

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Leu Gln Ser Ser Gln Tyr Thr Lys Gly Thr Pro Gly Gly Gly Gly Val

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Ser Lys Gly Glu Asp Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg

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Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile

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Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys

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Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu

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Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala

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Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp

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Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln

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Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg

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Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met

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Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu

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Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His Tyr

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Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu

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atgaaaaaga cagctatcgc gattgcagtg gcactggcag gtttcgctac cgtcgctcag 60

gctgcgggca ccggcgtggt gtccgtgcag ttcaacaacg gctccagccc tgcaagctcc 120

aactcgatct acgctcgttt caaggtcacc aacaccagcg gcagcccgat caacctggcg 180

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aaagccggtt cgtttgcgga caccaagatc acgctgaccc caaatggtaa cacgttcaat 660

ggcataagcg aactgcagtc tagccagtac accaaaggta ccccgggtgg tggaggtgtt 720

agcaagggtg aagatgataa catggcaatt atcaaggagt tcatgcgttt caaagttcac 780

atggaaggta gcgtcaatgg tcacgagttc gagattgagg gcgagggcga gggccgtccg 840

tatgaaggca cccaaaccgc taaattgaag gtcaccaagg gcggaccgct cccatttgcc 900

tgggatattc tgagcccaca gtttatgtac ggttcgaaag cgtacgtgaa acatccggct 960

gacatcccgg attacctgaa gcttagcttt ccggagggtt ttaagtggga acgtgttatg 1020

aattttgagg acggtggggt ggtgacggtg acccaagaca gcagcctgca agacggtgag 1080

ttcatttaca aggtaaaatt gagaggtacg aacttcccga gcgacggccc ggttatgcag 1140

aaaaagacca tgggttggga agcaagtagc gagcgcatgt acccggagga tggtgctctg 1200

aagggtgaga tcaagcagcg tctaaagctc aaagatggtg gccactacga tgctgaagtt 1260

aaaactacct ataaagcgaa aaagccggtt caactgccgg gtgcgtataa cgtgaacatt 1320

aaattggaca ttacctccca taatgaggat tatacgatcg tggaacaata tgaacgtgcg 1380

gaaggccgtc atagcaccgg tggcatggac gaactgtaca aaagcgcgtc cgcgggtgcg 1440

agcgctagcg cgggcgcttc tgcttccgcc ggggcgagcg ccagcgccgc aggcaccggc 1500

gttgtgagcg ttcagtttaa caacggcagc tctcccgcga gctctaacag catctacgcg 1560

cgtttcaaag tcaccaacac ctccggcagc ccgatcaatc tggcggactt gaagttacgc 1620

tattactaca cccaggacgc ggacaaaccg ctcaccttct ggtgcgatca tgccggttac 1680

atgtccggca gcaattatat tgacgcgaca tccaaggtga ctggcagctt taaggcggtg 1740

tcaccagcgg ttaccaatgc ggatcattat ctggaagttg ctctgaacag cgacgcaggt 1800

agcttgccgg ccggcgggtc catcgagatc caaacccgct tcgcccgtaa tgactggtca 1860

aacttcgatc aatcaaacga ctggagctat actgcggcag gtagctatat ggattggcaa 1920

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<213> Artificial Sequence (Artificial Sequence)

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actcctacgg gaggcagca 19

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<213> Artificial Sequence (Artificial Sequence)

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ggactachvg ggtwtctaat 20

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cttggtcatt tagaggaagt aa 22

<210> 8

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gctgcgttct tcatcgatgc 20

Claims

1. A glucan-binding fusion protein comprising the following structures linked in sequence: a clostridium cellulolyticum CipC protein glucan binding domain, a marker protein domain, and a clostridium cellulolyticum CipC protein glucan binding domain.

2. The glucan-binding fusion protein according to claim 1, wherein the amino acid sequence of said fusion protein is as set forth in SEQ ID NO:1 is shown.

3. A fusion gene encoding the fusion protein of claim 1 or 2, wherein the sequence of the coding sequence for the secretion signal peptide of the microorganism and the coding sequence for the fusion protein of claim 1 are linked.

4. The fusion gene of claim 3, wherein the nucleotide sequence of the fusion gene is as set forth in SEQ ID NO:2, respectively.

5. A recombinant expression vector comprising the fusion gene of claim 3 or 4.

6. A recombinant microorganism that binds to glucan, wherein the fusion protein of claim 1 or 2 is displayed on the cell surface of the recombinant microorganism.

7. Use of a recombinant microorganism according to claim 6 for the purpose of remodelling the rhizosphere microflora of a plant.

8. The use according to claim 7, wherein the microorganisms in the plant rhizosphere microflora comprise glucan-producing bacteria or fungi;

9. Use of the recombinant microorganism of claim 6 for promoting plant growth, improving plant stress resistance or phytoremediation.

10. The use according to claim 9, wherein the improvement of plant stress resistance is that the recombinant microorganism reduces oxidative stress;