Trans-aconitic acid responsive transcription factor TarR, and coding gene and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a trans-aconitic acid (TAA) responsive transcription factor TarR as well as a coding gene and application thereof. The transcription factor TarR provided by the invention is directly combined with TAA to be activated, and then the transcription factor TarR is combined with a promoter region of the TAA assimilation utilization operon to promote transcription translation of related genes in the TAA assimilation utilization operon, so that TAA assimilation utilization of microbial cells is facilitated, and the transcription factor TarR has a good application value in efficient synthesis and utilization of the TAA of the microorganisms and has a wide application prospect. And an excellent gene resource and a new strategy are provided for improving the TAA utilization efficiency and enhancing the carbon source utilization capacity by utilizing a genetic engineering means. Besides, the transcription factor can be specifically bound with TAA, has no binding effect with molecules with similar structures such as citric acid, can be used as a filler to be applied to affinity purification of TAA molecules and improve the purity of the TAA molecules, and has good application potential in the field of TAA affinity purification.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a trans-aconitate responsive transcription factor TarR, a coding gene thereof and an application thereof. Background Art

[0002] Aconitic acid (C6H6O6), also known as propylene-1,2,3-tricarboxylic acid, was first discovered in the plant Aconitum napellus. It is a low molecular weight organic tricarboxylic acid with cis- and trans-isomers, namely cis-aconitic acid (CAA) and trans-aconitic acid (TAA). Among them, CAA is an intermediate product of the isomerization of citric acid to isocitrate catalyzed by aconitase in the tricarboxylic acid cycle, and TAA has a certain inhibitory effect on aconitase. Aconitase is a key enzyme in the tricarboxylic acid cycle. Inhibiting the activity of aconitase can interfere with the tricarboxylic acid cycle, thereby affecting life activities, and shows good application prospects in the field of biological pesticides. Many studies have shown that TAA has good effects in nematode control and can inhibit or kill sweet potato stem nematodes, soybean cyst nematodes and southern root-knot nematodes. In addition, studies have shown that TAA can inhibit the growth of Leishmania, prevent brown planthoppers from feeding, and has anti-edema and anti-inflammatory effects.

[0003] TAA is mainly secreted by plants and microorganisms. It is a widely distributed plant carbon source in the rhizosphere environment. The ability of rhizosphere microorganisms to assimilate and utilize plant root secretions, especially carbon sources, is crucial to their growth and colonization. Therefore, actively exploring the mechanism of microbial assimilation and utilization of new carbon sources TAA, mining and screening genes related to TAA assimilation and utilization at the genome level, and functional analysis of key regulatory genes can not only provide a research basis for the mechanism of microbial carbon source nutrient utilization, but also provide important genetic resources and technical support for expressing TAA assimilation and utilization related genes in probiotics through directed genetic manipulation technologies such as genetic engineering technology to create microbial varieties that efficiently synthesize and utilize TAA. It plays an important role in improving the carbon metabolism ability of probiotics and enhancing their ability to colonize in the rhizosphere. This is also of great theoretical and practical significance for expanding the application of probiotics in the field of bio-agriculture, such as the production and development of biopesticides and bio-fertilizers.

[0004] However, there are few reports on the research of genes related to TAA assimilation and utilization by microorganisms, and there is a lack of effectively utilized gene resources. Therefore, it is urgent to develop and find new genes related to the regulation of TAA carbon source metabolism. Summary of the invention

[0005] In view of the above-mentioned technical problems in the prior art, the present invention provides a TAA-responsive transcription factor TarR and its encoding gene. The transcription factor TarR is activated by direct binding to TAA, and then promotes the transcription and translation of related genes in the TAA assimilation and utilization operon by binding to the promoter region of the TAA assimilation and utilization operon, thereby facilitating the assimilation and utilization of TAA by microbial cells. It has good application value in the efficient synthesis and utilization of microbial TAA, and provides excellent gene resources and new strategies for improving TAA utilization efficiency and enhancing microbial carbon source utilization ability by genetic engineering means. Moreover, the transcription factor of the present invention can specifically bind to TAA and has good application potential in the field of TAA affinity purification. Therefore, the present invention provides the application of the TAA-responsive transcription factor TarR or its encoding gene in regulating the expression of the trans-aconitate assimilation and utilization operon and regulating the trans-aconitate utilization ability of microorganisms, as well as in trans-aconitate affinity purification. The present invention is specifically implemented through the following scheme:

[0006] The first aspect of the present invention provides a trans-aconitate responsive transcription factor TarR, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The second aspect of the present invention provides a trans-aconitate responsive transcription factor TarR encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The third aspect of the present invention provides the use of the trans-aconitate responsive transcription factor TarR or its encoding gene as described above in regulating the expression of a microbial trans-aconitate assimilation and utilization operon, wherein the trans-aconitate assimilation and utilization operon includes a promoter, and the transcription factor promotes the transcription of the trans-aconitate assimilation and utilization operon by binding to the promoter, and the nucleotide sequence of the promoter is shown in bases 862-1034 of SEQ ID NO.3.

[0009] Furthermore, the trans-aconitate assimilation and utilization operon also includes a tarA gene and a tarB gene, the nucleotide sequence of the tarA gene is shown in bases 1035-2165 of SEQ ID NO.3, and the nucleotide sequence of the tarB gene is shown in bases 2209-3585 of SEQ ID NO.3.

[0010] Furthermore, regulating the expression of the trans-aconitate assimilation and utilization operon includes positive regulation and negative regulation, wherein the expression of the trans-aconitate assimilation and utilization operon is promoted by enhancing the expression level of the transcription factor TarR; and the expression of the trans-aconitate assimilation and utilization operon is inhibited by reducing the expression level of the transcription factor TarR.

[0011] Furthermore, promoting the expression of the aconitate assimilation and utilization operon includes the following steps: constructing a transcription factor TarR expression vector, transforming wild-type microorganisms, and culturing transgenic microorganisms expressing TarR coding genes.

[0012] Furthermore, constructing a transcription factor TarR expression vector includes the following steps: using the genome of Bacillus velezensis FZB42 as a template, using the amyE-UF / amyE-UR primer pair, the CtarR-F / CtarR-R primer pair and the amyE-DF / amyE-DR primer pair to perform PCR amplification to obtain amyE-up, tarR and amyE-down fragments respectively; using the kanamycin resistance gene as a template, using the Kan-F / Kan-R primer pair to amplify the Kan fragment; wherein the nucleotide selection sequence of the amyE-UF / amyE-UR primer pair is shown in SEQ ID NO.20-21, the nucleotide selection sequence of the CtarR-F / CtarR-R primer pair is shown in SEQ ID NO.22-23, the nucleotide sequence of the Kan-F / Kan-R primer pair is shown in SEQ ID NO.24-25, and the nucleotide sequence of the amyE-DF / amyE-DR primer pair is shown in SEQ ID NO. As shown in NO.26-27; the amyE-up, tarR, Kan, and amyE-down fragments were connected in sequence by overlapping extension PCR to obtain the fused target fragment, and the target fragment was connected to the T vector by TA cloning technology to obtain the transcription factor TarR expression vector.

[0013] Furthermore, inhibiting the expression of the aconitate assimilation and utilization operon comprises the following steps: constructing a transcription factor TarR knockout vector, transforming a wild-type microorganism, and culturing a transgenic microorganism lacking a TarR coding gene.

[0014] Furthermore, constructing a transcription factor TarR knockout vector includes the following steps: using the Bacillus velezensis FZB42 genome as a template, using the tarR-UF / tarR-UR primer pair and the tarR-DF / tarR-DR primer pair to perform PCR amplification to obtain tarR-up and tarR-down fragments respectively; using the chloramphenicol resistance gene as a template, using the Cm-F / Cm-R primer pair to perform PCR amplification to obtain the Cm gene fragment; wherein the nucleotide selection sequence of the tarR-UF / tarR-UR primer pair is shown in SEQ ID NO.4-5, the nucleotide selection sequence of the tarR-DF / tarR-DR primer pair is shown in SEQ ID NO.8-9, and the nucleotide sequence of the Cm-F / Cm-R primer pair is shown in SEQ ID NO.6-7; the tarR-up, Cm and tarR-down fragments are sequentially connected by overlapping extension PCR to obtain the fused target fragment, and the target fragment is connected to the T vector by TA cloning technology to obtain the transcription factor TarR knockout vector.

[0015] Furthermore, the microorganism is a bacterium; furthermore, it is Bacillus velezensis.

[0016] The fourth aspect of the present invention provides the use of the trans-aconitate responsive transcription factor TarR or its encoding gene as described above in regulating the trans-aconitate utilization ability of microorganisms.

[0017] The fifth aspect of the present invention provides the use of the above trans-aconitate responsive transcription factor TarR or its encoding gene in trans-aconitate affinity purification. The advantages and positive effects of the present invention are:

[0018] 1. The transcription factor TarR provided by the present invention is activated by direct binding with TAA, and then by binding with the promoter region of the TAA assimilation and utilization operon, the transcription and translation of tarA and tarB genes in the TAA assimilation and utilization operon are promoted, and then the extracellular TAA is transported and accumulated into the cell and converted into CAA in the tricarboxylic acid cycle pathway, which is beneficial to the assimilation and utilization of TAA by microbial cells, and has good application value in the efficient synthesis and utilization of microbial TAA, and provides excellent gene resources and new strategies for improving the utilization efficiency of TAA and enhancing the carbon source utilization ability of microorganisms by genetic engineering means. By using the TarR genetic resources provided by the present invention, by expressing its coding gene in probiotics and promoting the expression of TarR transcription factors, the ability of microorganisms to transport, transform, accumulate and utilize TAA can be enhanced, and its carbon source assimilation and utilization ability can be enhanced, thereby improving the rhizosphere colonization ability and stress resistance survival performance of probiotics, which is conducive to the development of more stable and reliable probiotic products. In addition, the transcription factor TarR of the present invention can be used to regulate the expression of the TAA assimilation and utilization operon and control the turning on and off of gene expression. This system (transcription factor TarR-small molecule TAA-target gene) has certain application value in the field of synthetic biology.

[0019] 2. The transcription factor of the present invention can specifically bind to TAA, and has no binding effect with molecules with similar structures such as citric acid. It can be used as a filler in the affinity purification of TAA molecules to improve the purity of TAA molecules, and has good application potential in the field of TAA affinity purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings used in describing the embodiments.

[0021] Figure 1 This is a schematic diagram of the gene structure of the tar gene cluster according to an embodiment of the present invention;

[0022] Figure 2 It is a graph showing the growth of the wild strain FZB42, mutant strain ΔtarR and complemented strain CtarR of Bacillus velezensis according to the present invention on an ACO solid plate with trans-aconitic acid as the sole carbon source;

[0023] Figure 3 It is a graph of the relative expression of tarA and tarB of the wild strain FZB42, mutant strain ΔtarR and complemented strain CtarR of Bacillus velezensis in the embodiment of the present invention in the presence or absence of TAA environment;

[0024] Figure 4 The electrophoresis diagram of the purified TarR protein in the embodiment of the present invention;

[0025] Figure 5The present invention uses EMSA experiments to verify the interaction between TarR protein and P tar Electropherograms of promoter interactions;

[0026] Figure 6 The present invention uses a microscale thermophoresis (MST) experiment to verify the binding curves of TarR protein with trans-aconitic acid and citric acid; wherein, Figures AB are trans-aconitic acid and citric acid, respectively;

[0027] Figure 7 The present invention uses EMSA experiments to verify the effects of TAA and citric acid on the interaction of TarR protein with P tar Influence on promoter binding ability; Figures AB are trans-aconitic acid and citric acid, respectively. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0030] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "approximately" in all cases. The term "approximately" has its usual meaning, which is used to indicate that a value includes the inherent variation of the error of the device or method used to determine the value, or contains a value close to the value, such as within 10% of the value (or range of values). Therefore, unless otherwise specified, the numerical parameters listed in the specification and the attached claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained.

[0031] In addition, it should be noted that the meanings of the terms "include", "comprises", "contains", "has" and the like are non-restrictive, that is, other steps and other components that do not affect the results may be added. The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0033] LysR family transcription factors (LysR-type transcriptional regulator, LTTR) are a type of regulatory factor that has been widely studied and can perform regulatory functions. It is also the largest prokaryotic transcription factor family, present in most prokaryotes, regulating a series of genes with different functions, including genes involved in toxicity, metabolism, quorum sensing and movement, and this family of transcription factors can often bind to small molecules in the regulated path and affect the expression of target genes. The regulatory effect of LTTRs means that they have evolved regulatory mechanisms for different structural genes in the genome, and their products can participate in physiological processes such as metabolism, cell division, quorum sensing, toxicity, movement, nitrogen fixation, oxidative stress response, toxin production, attachment and secretion. Although LysR family transcription factors have a wide range of biological functions, there are still few studies on LysR family transcription factors in regulating TAA synthesis, utilization and accumulation. Therefore, this application uses LysR family transcription factors as research objects, aiming to dig out new TAA regulatory genes.

[0034] Trans-aconitic acid (TAA) is a widely existing plant carbon source that can be used by bacteria, and its metabolic pathway has a high research value for the colonization of plant probiotics. This paper takes the plant rhizosphere growth-promoting microorganism Bacillus velez FZB42, which can efficiently assimilate and utilize TAA, as the object, and explores the regulatory effect of the LysR family transcription factor tarR (TAAassimilation-related gene R) gene and its protein TarR on the assimilation of TAA carbon source by Bacillus velez.

[0035] First, by measuring and comparing the growth of wild Bacillus velezensis FZB42 and tarR gene knockout mutant ΔtarR in the presence of TAA carbon source, it was found that the ΔtarR gene deletion mutant could not grow in the culture medium with TAA as the only carbon source, which was manifested as an inability to utilize TAA. However, after complementing the tarR gene, it was able to use TAA as a nutrient carbon source to maintain growth and reproduction; furthermore, the positive regulatory effect of the tarR gene on the assimilation of TAA carbon source by FZB42 was clarified. At the molecular level, qRT-PCR was used to verify the regulatory mode of tarR gene on TAA assimilation function. It was found that TAA signal was transmitted in the cell through TarR transcription factor to induce the expression of TAA assimilation and utilization operon (including tarA and tarB). EMSA experiments were further used to confirm that TarR transcription factor promoted the transcription of tarA and tarB genes by directly interacting with the promoter region of TAA assimilation and utilization operon. Among them, TarA protein encoded by tarA has the function of aconitate isomerase that mediates the conversion of TAA to CAA, and TarB protein encoded by tarB has the function of TAA transporter that mediates the transport of extracellular TAA to the cell. The high expression of the above genes is conducive to promoting the accumulation of intracellular TAA and its conversion to the tricarboxylic acid cycle pathway, which is conducive to the assimilation and utilization of TAA carbon source by cells. Finally, MST and EMSA experiments clarified that TAA directly binds to TarR and enhances its binding to the promoter region of TAA assimilation operon. The above results reveal the positive regulatory effect of tarR on the assimilation and utilization of TAA carbon sources by microorganisms and its mechanism of action, enrich the factors and mechanisms regulating TAA carbon source assimilation in bacteria, and also provide new genetic resources and scientific guidance for the development of more stable and effective carbon metabolism probiotic products and the enhancement of microbial colonization ability.

[0036] Based on the above findings, an embodiment of the present invention provides a trans-aconitate-responsive transcription factor TarR, the amino acid sequence of the transcription factor TarR is shown in SEQ ID NO.2.

[0037] Another embodiment of the present invention provides a trans-aconitate responsive transcription factor TarR encoding gene, the gene encoding the trans-aconitate responsive transcription factor TarR as described above, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0038] The present invention discloses for the first time the positive regulation mechanism of the transcription factor TarR on the accumulation and utilization of trans-aconitic acid (TAA) in microbial cells, and has good application value in the efficient synthesis and utilization of microbial TAA, and provides excellent gene resources and new strategies for improving the utilization efficiency of TAA and enhancing the carbon source utilization ability of microorganisms by genetic engineering means. It is well known that the stronger the ability of microorganisms to utilize various carbon sources in the environment, the more conducive to their colonization in the natural environment. TAA, as a widely existing secretory plant carbon source, can be assimilated and utilized by many plant probiotic bacteria, and the TAA metabolic pathway plays an important role in participating in the rhizosphere colonization of probiotics. Using the TarR genetic resources provided by the present invention, by expressing its coding gene in probiotics and promoting the expression of TarR transcription factors, the ability of microorganisms to transport, transform, accumulate and utilize TAA can be enhanced, and its carbon source assimilation and utilization ability can be enhanced, thereby improving the rhizosphere colonization ability of probiotics and the stress resistance survival performance in the rhizosphere environment, which is conducive to the development of more stable and reliable probiotic products. In addition, the transcription factor TarR of the present invention can be used to regulate the expression of the TAA assimilation and utilization operon, and control the opening and closing of gene expression. This system (transcription factor TarR-small molecule TAA-target gene) has certain application value in the field of synthetic biology. In addition, the transcription factor of the present invention can specifically bind to TAA, and has no binding effect with molecules with similar structures such as citric acid. It can be used as a filler for affinity purification of TAA molecules to improve the purity of TAA molecules, and has good application potential in the field of TAA affinity purification.

[0039] Another embodiment of the present invention provides the use of the transcription factor TarR or its encoding gene as described above in regulating the expression of a trans-aconitate assimilation and utilization operon of a microorganism, wherein the trans-aconitate assimilation and utilization operon includes a promoter, and the transcription factor promotes the transcription of the trans-aconitate assimilation and utilization operon by binding to the promoter, and the nucleotide sequence of the promoter is shown in bases 862-1034 of SEQ ID NO.3.

[0040] Furthermore, the trans-aconitate assimilation and utilization operon also includes a tarA gene and a tarB gene, the nucleotide sequence of the tarA gene is shown in bases 1035-2165 of SEQ ID NO.3, and the nucleotide sequence of the tarB gene is shown in bases 2209-3585 of SEQ ID NO.3.

[0041] The transcription factor TarR provided by the present invention can promote the transcription of tarA and tarB genes in the TAA assimilation and utilization operon by binding to the promoter region of the TAA assimilation and utilization operon. The expression of these genes is conducive to promoting the accumulation of intracellular TAA and its conversion to the tricarboxylic acid cycle pathway, thereby promoting the assimilation and utilization of TAA by microbial cells, and plays an important role in the microbial utilization of TAA metabolic pathways. The elucidation of the mechanism by which the transcription factor TarR and its encoding gene regulate the utilization and metabolism of TAA carbon sources by plant probiotics provides a reference for the regulatory mechanisms of many other plant probiotics that can also utilize TAA carbon sources as nutrients. The application of the transcription factor TarR of the present invention or its encoding gene to the study of the molecular regulatory mechanism for genetic improvement of probiotic TAA assimilation performance and the creation of probiotics that efficiently utilize TAA has good application prospects.

[0042] Optionally, regulating the expression of the trans-aconitate assimilation and utilization operon includes positive regulation and negative regulation, and the expression of the aconitate assimilation and utilization operon is promoted by enhancing the expression level of the transcription factor TarR, and the expression of the aconitate assimilation and utilization operon is inhibited by reducing the expression level of the transcription factor TarR.

[0043] Specifically, promoting the expression of the aconitate assimilation and utilization operon includes the following steps: constructing a transcription factor TarR expression vector, transforming a target microorganism, and culturing a transgenic microorganism expressing the TarR coding gene.

[0044] More specifically, constructing the transcription factor TarR expression vector comprises the following steps:

[0045] Using the genome of Bacillus velezensis FZB42 as a template, PCR amplification was performed using the amyE-UF / amyE-UR primer pair, the CtarR-F / CtarR-R primer pair, and the amyE-DF / amyE-DR primer pair to obtain amyE-up, tarR, and amyE-down fragments, respectively; using the kanamycin resistance gene as a template, the Kan fragment was amplified using the Kan-F / Kan-R primer pair; wherein the nucleotide sequence of the amyE-UF / amyE-UR primer pair is shown in SEQ ID NOs.20-21, the nucleotide sequence of the CtarR-F / CtarR-R primer pair is shown in SEQ ID NOs.22-23, the nucleotide sequence of the Kan-F / Kan-R primer pair is shown in SEQ ID NOs.24-25, and the nucleotide sequence of the amyE-DF / amyE-DR primer pair is shown in SEQ ID NOs.26-27;

[0046] The amyE-up, tarR, Kan, and amyE-down fragments were sequentially connected by overlap extension PCR to obtain the fused target fragment, and the target fragment was connected to the T vector by TA cloning technology to obtain the transcription factor TarR expression vector.

[0047] Specifically, inhibiting the expression of the aconitate assimilation and utilization operon comprises the following steps: constructing a transcription factor TarR knockout vector, transforming a wild-type microorganism, and culturing a transgenic microorganism with a TarR coding gene deleted.

[0048] More specifically, constructing the transcription factor TarR knockout vector comprises the following steps:

[0049] Using the genome of Bacillus velez FZB42 as a template, PCR amplification was performed using the tarR-UF / tarR-UR primer pair and the tarR-DF / tarR-DR primer pair to obtain tarR-up and tarR-down gene fragments respectively; using the chloramphenicol resistance gene as a template, PCR amplification was performed using the Cm-F / Cm-R primer pair to obtain the Cm fragment; wherein the nucleotide sequence of the tarR-UF / tarR-UR primer pair is shown in SEQ ID NO.4-5, the nucleotide sequence of the tarR-DF / tarR-DR primer pair is shown in SEQ ID NO.8-9, and the nucleotide sequence of the Cm-F / Cm-R primer pair is shown in SEQ ID NO.6-7;

[0050] The tarR-up, Cm and tarR-down fragments were connected in sequence by overlapping extension PCR to obtain the fused target fragment, and the target fragment was connected to the T vector by TA cloning technology to obtain the transcription factor TarR knockout vector.

[0051] Optionally, the microorganism or probiotic mentioned above is preferably a bacterium, such as Bacillus Velezii.

[0052] Based on the same inventive concept as above, another embodiment of the present invention provides the use of the trans-aconitate responsive transcription factor TarR or its encoding gene as described above in regulating the trans-aconitate utilization ability of microorganisms.

[0053] The embodiment of the present invention also provides the use of the trans-aconitate responsive transcription factor TarR or its encoding gene as described above in trans-aconitate affinity purification.

[0054] The present invention uses microthermophoresis (MST) experiment to detect the binding characteristics of transcription factor TarR with citric acid and TAA, showing that transcription factor TarR only binds to TAA molecules, and the dissociation equilibrium constant K D=11.08±4.35μM, with strong specificity, suitable as a filler for affinity purification of TAA molecules.

[0055] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Manual (Fourth Edition) published by Cold Spring Harbor Laboratory, or the conditions recommended by the manufacturer.

[0056] Main reagents: Molecular biology enzyme reagents were purchased from Takara Biotechnology Co., Ltd. TAA standard was purchased from TCI (Shanghai). DNA gel recovery kit and PCR clean up kit were purchased from Beijing Biotech Biotechnology Co., Ltd. Protein concentration determination kit was purchased from White Shark Biotechnology Co., Ltd. RNA extraction kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0057] Main culture medium: 1) SPI Medium includes: 0.19% (NH4)2SO4, 1.36% K2HPO4·3H2O, 0.58% KH2PO4, 0.10% Trisodium citrate dihydrate, 202μL 5% MgSO4·7H2O, 202μL 50% glucose and 202μL 1% CAYE, the above percentages are weight volume ratio (w / v); 2) SPII Medium includes: 19.60mL SPI, 200μL 50mM CaCl2 and 200μL 250mM MgCl2, the final concentration of CaCl2 in the culture medium is 0.5mM, and the final concentration of MgCl2 is 2.5mM; 3) Liquid ACO medium includes the following concentration components: 7.5g / L TAA, 2 g / L (NH4)2SO4, 1 g / L K2HPO4, 0.5 g / LMgSO4 and 0.1 g / L FeCl3·6H2O, pH 7.0. Solid ACO medium supplemented with 1.6%-1.8% agar.

[0058] Primers: The primer sequences used in the present invention are shown in Table 1, and the primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0059] Table 1 Primer sequences used in the examples of the present invention

[0060]

[0061]

[0062] 1. Transcription factor tarR gene, protein and structure analysis

[0063] The experimental material used in this study is Bacillus velezensis (B. velezensis) FZB42. FZB42 is a classic plant rhizosphere growth-promoting microorganism with advantages such as clear genetic background and mature operating system. Therefore, FZB42 is also a model strain in the study of bacteria-plant interactions. The genome sequence can be found in NCBI accession number NC_009725.2.

[0064] FZB42 can efficiently assimilate and utilize TAA carbon source. The present invention analyzes its genome sequence and successfully locates the target gene cluster for TAA assimilation and utilization, named TAAassimilation-related gene (tar). The gene cluster contains two genes, tarA (TAAassimilation-related gene A) and tarB (TAAassimilation-related gene B), which exist in the form of operons. It is confirmed at the genetic and biochemical levels that tarA plays the role of aconitate isomerase when mediating TAA carbon source assimilation, and tarB plays the role of TAA intracellular transporter.

[0065] There is a hypothetical protein coding gene 173bp upstream of tarA, which is opposite to the transcription direction of the tar gene, i.e., shares a promoter with it. The conservative domain analysis was performed using NCBI BLAST comparison analysis (Protein BLAST: search protein databases using a protein query (nih.gov)), which showed that the gene belongs to the LysR family transcription factor, and it was named tarR (TAAassimilation-related gene R), and the encoded protein was named TarR. The present invention reveals for the first time from the genetic and biochemical levels the positive regulatory effect of tarR from the LysR family on the assimilation of TAA carbon source by the plant probiotic FZB42, and explains the specific regulatory mechanism. The TarR protein is activated by direct binding to TAA, and then directly interacts with the promoter of the TAA assimilation and utilization operon to promote the transcription and translation of the tar gene, thereby promoting the process of TAA carbon source assimilation and utilization by cells.

[0066] Figure 1The physical position relationship between the tarR gene and the tar gene in the tar operon in the FZB42 genome is shown. The arrow indicates the transcription direction of the gene. The blue region is the tarR gene, which is located on the negative strand of the chromosome; the yellow region is the tarA gene, and the red region is the tarB gene. Both are located on the positive strand of the chromosome and are the same operon; the 173bp sequence between the tarR and tarA genes is the promoter sequence regulated by the TarR protein. The corresponding sequence information is shown in Table 2.

[0067] Table 2 tar operon gene and protein sequence of the present invention embodiment

[0068]

[0069]

[0070]

[0071] 2. Construction and phenotypic detection of the tarR gene deletion mutant of Bacillus velezinis ΔtarR

[0072] 2.1. Construction of tarR-up-Cm-tarR-down fusion fragment

[0073] Using the genome of Bacillus velez FZB42 and the chloramphenicol resistance gene (synthesized artificially using the chloramphenicol resistance gene information on the cloning vector pAgR-gCBE4, the GenBank accession number of the cloning vector is MW344269.1) as templates, tarR-UF / tarR-UR primer pair, Cm-F / Cm-R primer pair, tarR-DF / tarR-DR primer pair were used to obtain three DNA target fragments of the upstream arm homology fragment of the tarR gene (tarR-up), the chloramphenicol resistance gene fragment (Cm) and the downstream arm homology fragment of the tarB gene (tarR-down) by PCR technology. The PCR configuration system is: 1μL DNA template, 2μL upstream primer, 2μL downstream primer, 1μL high-fidelity PCR polymerase, 20μL 5× buffer, 2μL dNTP, and ddH2O supplemented to 100μL. PCR program: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 50℃ annealing for 40s, 72℃ extension for 1min, 30 cycles. The PCR products were subjected to agarose gel electrophoresis, and the target bands were cut and recovered to obtain tarR-up, Cm and tarR-down fragments respectively.

[0074] There are overlapping regions between the above three DNA fragments, which can be used as primers and templates for each other, and the corresponding fragments can be connected by overlap extension PCR (SOE-PCR). First, tarR-up and Cm fragments are used as templates, 0.5 μL each, and tarR-UF and Cm-R primer pairs are used for PCR amplification. The system and program settings are the same as above; the PCR products are subjected to agarose gel electrophoresis and the gel is cut and recovered to purify the tarR-up-Cm fusion fragment. Then, the tarR-up-Cm and tarR-down fragments are used as templates, 0.5 μL each, and the tarR-UF and tarR-DR primer pairs are used for the second round of PCR amplification. The PCR products are subjected to agarose gel electrophoresis and the gel is cut and recovered to purify the fused tarR-up-Cm-tarR-down fusion fragment.

[0075] 2.2. Construction and transformation of tarR gene knockout vector

[0076] The tarR-up-Cm-tarR-down gene fragment was connected to the T vector by TA cloning technology, and the vector construction was successfully verified by sequencing by Qingke Biotechnology. The linearized recombinant plasmid tarR-up-Cm-tarR-down-T-simple was obtained by BamHI digestion, and the digestion product was recovered using a PCR clean up kit. The linearized recombinant plasmid was then transformed into Bacillus Velez FZB42 using a chemical transformation method. The operation is as follows: Inoculate the overnight activated Bacillus Velez FZB42 in 20mL SPI Medium and culture at 200rpm and 37℃ until the OD 600 When the p-value reaches 1.4-1.5, take 2.5 mL of culture solution and inoculate it into 20 mL of SPII Medium, and culture it at 37°C and 100 rpm for 1.5 h; add 250 μL of 10 mM EGTA (pH 8.0) solution, and continue to culture it at 37°C and 100 rpm for 10 min to obtain FZB42 competent cells; then divide the FZB42 competent cells into 500 μL tubes; add 15 μL of linearized recombinant plasmid to each tube, mix well, and culture it at 37°C and 100 rpm for 50 min; add 800 μL of LB culture medium to each tube, and continue to culture it at 200 rpm and 37°C for 2 h; spread on LB plates containing chloramphenicol and culture them at 37°C for 24 h. The transformed product was the tarB gene deletion mutant strain ΔtarR. Sequencing verification showed that the tarR-up-Cm-tarR-down gene fragment was successfully integrated into the tarR gene site of the FZB42 chromosome. The sequence was complete, correct and without mutation, and the target mutant strain ΔtarR was obtained.

[0077] 2.3. Detection of the phenotype of Bacillus velezensis ΔtarR mutant using TAA carbon source

[0078] The wild-type strain of Bacillus velezensis FZB42 and the ΔtarR mutant were inoculated into liquid LB medium and cultured overnight at 37°C and 200 rpm. The cells were collected by centrifugation at 6000 rpm, washed twice with sterile water, and then streaked onto solid ACO medium and cultured at 37°C for 16 h. The growth of the strains was observed. The results are shown in Table 1. Figure 2 .

[0079] from Figure 2 It can be seen that the wild-type strain grew well in ACO medium, while the ΔtarR mutant strain could not grow in ACO medium, indicating that the ΔtarR mutant strain could not utilize TAA, indicating the positive regulatory effect of transcription factor tarR on genes related to TAA utilization.

[0080] 3. tarR gene complementation and phenotypic detection of the ΔtarR mutant of Bacillus velezini

[0081] 3.1. Construction of amyE-up-tarR-Kan-amyE-down fusion fragment

[0082] Using the genome of Bacillus velez FZB42 and the kanamycin resistance gene (synthesized artificially using the kanamycin resistance gene information on Bacillus velez HNU24, the GenBank accession number of the strain is CP092412.1) as templates, the primer pairs amyE-UF / amyE-UR, CtarR-F / CtarR-R, Kan-F / Kan-R, and amyE-DF / amyE-DR were used to amplify and recover four DNA target fragments, amyE-up, tarR, kanamycin resistance gene fragment (Kan), and amyE-down, using PCR technology. The four fragments were connected using overlap extension PCR (SOE-PCR). First, amyE-up and tarR, Kan and amyE-down were connected respectively to obtain two gene fragments, amyE-up-tarR and Kan-amyE-down, and then amyE-up-tarR and Kan-amyE-down were connected to obtain the fused amyE-up-tarR-Kan-amyE-down gene fragment. The PCR configuration, procedure, and rubber tapping recovery operation of this embodiment are the same as those in the above-mentioned Part 2.

[0083] 3.2. Construction and transformation of tarR gene complementation vector

[0084] The amyE-up-tarR-Kan-amyE-down fusion fragment was connected to the T vector by TA cloning technology, and the vector construction was successfully verified by sequencing by Qingke Biotechnology. The linearized complementation plasmid amyE-up-tarR-Kan-amyE-down-T-simple was obtained by BamHI digestion, and the digestion product was recovered using a PCR clean up kit. The linearized recombinant plasmid was then transformed into the ΔtarR mutant strain of Bacillus velezinus by chemical transformation. The amyE-up-tarR-Kan-amyE-down fusion gene was integrated into the amyE gene site of the FZB42 chromosome by the principle of homologous double exchange, thereby constructing the CtarR complementation strain. The genetic transformation and strain verification operations of this embodiment are the same as those in Part 2 above.

[0085] 3.3. Detection of the phenotype of Bacillus velezensis CtarR complemented strain using TAA carbon source

[0086] The CtarR complemented strain of Bacillus velez was inoculated into liquid LB medium and cultured overnight at 37°C and 200 rpm. The cells were collected by centrifugation at 6000 rpm, washed twice with sterile water, and then streaked onto solid ACO medium and cultured at 37°C for 16 h. The growth of the strain was observed. The results are shown in Figure 2 .

[0087] Figure 2 The growth status of the CtarR complemented strain in ACO medium is shown. It can be seen that the CtarR complemented strain grows well in the ACO solid plate, that is, the TAA utilization phenotype is restored, further suggesting that the tarR gene has a positive regulatory effect on the process of TAA assimilation and utilization.

[0088] 4. Verification of the regulatory effect of tarR gene on TAA function of strain assimilation and utilization

[0089] The wild strain, ΔtarR mutant and CtarR complemented strain of Bacillus velezinsis FZB42 were inoculated into liquid LB medium and cultured overnight at 37°C and 200 rpm. Subsequently, the three strains were transferred to half LB medium without TAA and containing TAA (7.5 g / L) respectively, and cultured at 37°C and 200 rpm for 20 h. The bacteria were collected and RNA was extracted using the TransZol UP kit (Cat. No.: ER501-01-V2) of Beijing Quanshijin Biotechnology Co., Ltd., and the RNA was reverse transcribed into cDNA using the reverse transcription kit (Cat. No.: RR047A) of Baoriyi Biotechnology (Beijing) Co., Ltd. The expression level of each gene of the tar operon was detected by real-time fluorescence quantitative PCR (qRT-PCR), with the 23S rRNA gene as the internal reference gene.

[0090] The SYBR Green I method was used for quantitative detection. The qRT-PCR reaction system was: 1 μL cDNA, 1 μL upstream primer, 1 μL downstream primer, 10 μL qPCR SYBR Green Master Mix (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 11184ES08), and ddH2O was added to 20 μL. PCR reaction and fluorescence signal collection were performed. The number of cycles required for each reaction system to reach the fluorescence threshold was recorded, and the fluorescence signal was calculated according to the 2 -ΔΔCt The expression levels of each gene in the tar operon under different treatments were calculated by this method.

[0091] Figure 3 The expression levels of tarA and tarB genes in the FZB42 wild strain, ΔtarR mutant and CtarR complemented strain in the environment without TAA (indicated by "-") and with TAA (indicated by "+") are shown. In the FZB42 strain, compared with the environment without TAA, the expression levels of tarA and tbrB genes in the environment with TAA were upregulated by 60 times and 8 times, respectively, indicating that the transcription of the TAA assimilation and utilization operon requires the induction of TAA; when the tarR gene was knocked out, the expression levels of tarA and tbrB genes remained unchanged in the same TAA environment, indicating that the transcription of the operon induced by TAA requires the participation of TarR protein, and also indicating that tarR positively regulates the transcription of the TAA assimilation and utilization operon. In the CtarR complemented strain, the expression levels of tarA and tbrB genes were consistent with those in the FZB42 wild strain.

[0092] 5. TarR transcription factor directly binds to the promoter region of the tar operon to regulate the assimilation and utilization of TAA

[0093] 5.1. Expression and purification of TarR protein

[0094] Using the genome of Bacillus velezinis FZB42 as a template, the tarR-F-28b / tarR-R-28b primer pair was used to amplify the open reading frame (ORF) gene fragment of the heterologous expression of the TarR protein. The PCR configuration system was: 1 μL DNA template, 2 μL upstream primer and downstream primer, 1 μL high-fidelity PCR polymerase, 20 μL 5× buffer, 2 μL dNTP, and ddH2O supplemented to 100 μL. PCR program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 50℃ annealing for 40 s, 72℃ extension for 1 min, and 30 cycles. The PCR product was digested overnight with NcoI and XhoI, and the digested product was recovered with the Axygen PCR Clean up kit to obtain the tarR ORF fragment with sticky ends. The fragment was then ligated in vitro with the pET28b vector with the same sticky ends. The ligation product was transformed into E. coli BL21 (DE3) competent cells, and the cells were spread on LB plates containing 50 μg / mL kanamycin resistance and cultured overnight at 37° C. The transformation was successfully constructed by colony PCR and sequencing to obtain the TarR recombinant expression strain.

[0095] The TarR recombinant expression strain activated overnight was inoculated into 200 mL LB liquid medium containing 50 μg / mL kanamycin and cultured at 200 rpm and 37°C until OD 600 The p-value was about 1, and 100 μL of 0.5 M IPTG solution was added, and the culture was induced at 16°C and 200 rpm for 18 h.

[0096] After induction, the cultured bacterial solution was centrifuged, the bacterial precipitate was collected, and 30 mL of binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, pH 8.0) was added to resuspend the bacterial cells. The bacterial cells were disrupted by ultrasound, with the amplitude set to 6, the power set to 200 W, and the time set to 30 min. The bacterial solution after ultrasonic disruption was centrifuged at 4 ° C and 12000 rpm for 10 min, and the supernatant was collected for later use.

[0097] According to the instruction manual, the supernatant was affinity purified using Ni-NTA purification medium (purchased from Nanjing GenScript Biotechnology Co., Ltd., catalog number L00250) to obtain the TarR protein eluate. The TarR protein eluate was placed in a dialysate (20 mM Tris-HCl, 10% glycerol (v / v), 1 mM EDTA, 0.1 mM DTT, pH 7.5) and dialyzed in an ice-water bath for 6 h to obtain the purified TarR protein.

[0098] The purified protein was subjected to SDS-PAGE electrophoresis. Figure 4It can be seen that the molecular weight of the purified protein is between 20-35 kDa, which is consistent with its theoretical size of 32.26 kDa, indicating that TarR protein has been expressed in large quantities in pET28b-tarR / Rostta bacteria, providing a material basis for subsequent experiments.

[0099] 5.2. TarR protein and P tar Promoter interaction validation

[0100] The gel retardation mobility shift assay (EMSA) was used to determine whether TarR protein exerts its regulatory function by interacting with the promoter region of the TAA assimilation and utilization operon.

[0101] The genomic DNA of the starting strain FZB42 was used as a template and P tar -EMSA-F / P tar -EMSA-R primer pair (where P tar -EMSA-R primer 5' end with 6-FAM fluorescent labeling) to amplify the promoter fragment of the tar operon P tar PCR products were recovered using the PCR clean up kit to obtain pure P tar Fragment.

[0102] EMSA experiments were used to detect the interaction between TarR protein and promoter. Different incubation systems were prepared, and P tar The fragment concentration remained constant (all 50 nM), the TarR protein concentration showed a gradient change (0-2 μM), and was incubated at room temperature for 2 hours. Polyacrylamide gel electrophoresis was performed at 150 V for 1 hour. After the electrophoresis, the gel was taken out and placed in a chemiluminescence imager to observe the experimental results.

[0103] Figure 5 The TarR protein is shown to be tar The results of the promoter-mixed reaction system after polyacrylamide gel electrophoresis. When TarR protein is 0, P tar The fragment migration rate was the fastest. When the concentration of TarR protein gradually increased, P tar The fragment showed an obvious migration band with a slower migration rate, and the migration band gradually became denser, indicating that the TarR protein can directly bind to the promoter sequence of the tar operon.

[0104] 5.3. Verification of the interaction between TarR protein and TAA molecules

[0105] The interaction between TarR protein and TAA molecules was analyzed by microthermophoresis (MST) technique, with TAA analog citric acid as control molecule. The principle of MST is: one of the interacting molecules (such as protein) is labeled with fluorescent dye, and the other interacting molecule (such as protein ligand) is mixed with the labeled protein in a capillary according to a specific concentration gradient. During detection, the infrared laser beam in the instrument heats the center of the detection area of ​​the capillary to generate a microscopic temperature gradient field. Changes in the size, hydration layer and charge of the labeled protein and ligand complex can cause the thermophoresis velocity of the complex molecule to change relative to the thermophoresis velocity of the labeled protein molecule. Therefore, it is easy to detect the MST signal change caused by the binding of the labeled protein and the ligand molecule. Specifically, 800nM purified TarR protein and 100nM NTA dye (manufacturer: Nano temper Technologies; item number: SKU: MO-L018) were mixed evenly, incubated for 30min, and centrifuged to obtain the supernatant. A total of 16 concentration gradient compounds were prepared with 1 micromole TAA or citric acid as the highest concentration. Then, 10 μL of TarR protein labeled with NTA dye was added to each test tube and loaded onto the detection device using a standard capillary. The scanning parameters were MST power of 40% and LED power of 80%. MonolithNT.115 was used for fluorescence measurement to record the change of fluorescence intensity in each capillary over time in the temperature gradient. The fluorescence intensity value was then used as the ordinate and the ligand concentration as the abscissa for fitting. The affinity constant K for the binding of TarR protein and TAA was calculated. D Data were analyzed using MO.AffinityAnalysis V2.3* (Nano temper Technologies, Germany).

[0106] Figure 6 The binding curves between the labeled purified TarR protein and TAA (Figure A) and citric acid (Figure B) molecules are shown. The results show that the TarR protein binds to the TAA substrate molecule and exhibits a ligand dose-dependent change in fluorescence intensity, indicating that the TarR protein can directly bind to the TAA molecule. The dissociation equilibrium constant between the two is K D =11.08±4.35μM, while TarR had no obvious interaction with citric acid, indicating that the binding of TarR protein to substrate TAA has high affinity and specificity.

[0107] 5.4. TAA molecules can enhance the interaction between TarR and P tar The ability to combine

[0108] EMSA experiments detected the effects of TAA molecules on TarR protein and P tarDifferent incubation systems were prepared, with the concentration of TarR protein (all 1 μM) and P tar The fragment concentration remained constant (all 50 nM), and the citric acid concentration of TAA molecules or control molecules showed a gradient change (0-80 mM), and was incubated at room temperature for 2 hours. Polyacrylamide gel electrophoresis was performed at 150 V for 1 hour. After the electrophoresis, the gel was taken out and placed in a chemiluminescence imager to observe the experimental results.

[0109] Figure 7 TarR protein, P tar The result of polyacrylamide gel electrophoresis of the reaction system mixed with promoter fragment and TAA molecule or citric acid molecule. When the concentration of TAA in the reaction system gradually increases, P tar The migration band of the fragment gradually became denser, while when the concentration of the control molecule citric acid gradually increased, the concentration of the migration band remained unchanged, indicating that the TAA molecule can enhance the binding of TarR protein to the promoter of the tar operon and has good specificity.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A trans-aconitate responsive transcription factor TarR, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. A trans-aconitate responsive transcription factor TarR encoding gene, characterized in that: Its nucleotide sequence is shown as SEQ ID NO.

1.

3. The use of the trans-aconitate responsive transcription factor TarR or its encoding gene as claimed in claim 1 in regulating the expression of the trans-aconitate assimilation and utilization operon of microorganisms, characterized in that: The transaconitate assimilation and utilization operon includes a promoter, and the transcription factor promotes the transcription of the transaconitate assimilation and utilization operon by binding to the promoter. The nucleotide sequence of the promoter is shown in bases 862-1034 of SEQ ID NO.

3.

4. The use of the trans-aconitate responsive transcription factor TarR or its encoding gene according to claim 3 in regulating the expression of the trans-aconitate assimilation and utilization operon of microorganisms, characterized in that: The trans-aconitate assimilation and utilization operon also includes a tarA gene and a tarB gene. The nucleotide sequence of the tarA gene is shown in bases 1035-2165 of SEQ ID NO.3, and the nucleotide sequence of the tarB gene is shown in bases 2209-3585 of SEQ ID NO.

3.

5. The use of the trans-aconitate responsive transcription factor TarR or its encoding gene according to claim 3 in regulating the expression of the trans-aconitate assimilation and utilization operon of microorganisms, characterized in that: Regulating the expression of the trans-aconitate assimilation and utilization operon includes positive regulation and negative regulation, wherein the expression of the trans-aconitate assimilation and utilization operon is promoted by enhancing the expression level of the transcription factor TarR; and the expression of the trans-aconitate assimilation and utilization operon is inhibited by reducing the expression level of the transcription factor TarR.

6. The use of the trans-aconitate responsive transcription factor TarR or its encoding gene according to claim 5 in regulating the expression of the trans-aconitate assimilation and utilization operon of microorganisms, characterized in that: Promoting the expression of the aconitate assimilation and utilization operon comprises the following steps: Constructing a transcription factor TarR expression vector, transforming a target microorganism, and culturing a transgenic microorganism expressing a TarR encoding gene; wherein constructing a transcription factor TarR expression vector comprises the following steps: Using the genome of Bacillus velezensis FZB42 as a template, PCR amplification was performed using the amyE-UF / amyE-UR primer pair, the CtarR-F / CtarR-R primer pair and the amyE-DF / amyE-DR primer pair to obtain amyE-up, tarR and amyE-down fragments respectively; using the kanamycin resistance gene as a template, the Kan fragment was amplified using the Kan-F / Kan-R primer pair; wherein the nucleotide sequence of the amyE-UF / amyE-UR primer pair is shown in SEQ ID NO.20-21, the nucleotide sequence of the CtarR-F / CtarR-R primer pair is shown in SEQ ID NO.22-23, the nucleotide sequence of the Kan-F / Kan-R primer pair is shown in SEQ ID NO.24-25, and the nucleotide sequence of the amyE-DF / amyE-DR primer pair is shown in SEQ ID NO.26-27; The amyE-up, tarR, Kan, and amyE-down fragments were sequentially connected by overlapping extension PCR to obtain the fused target fragment, and the target fragment was connected to the T vector by TA cloning technology to obtain the transcription factor TarR expression vector.

7. The use of the trans-aconitate responsive transcription factor TarR or its encoding gene according to claim 5 in regulating the expression of the trans-aconitate assimilation and utilization operon of microorganisms, characterized in that: Inhibiting the expression of the aconitate assimilation and utilization operon comprises the following steps: Constructing a transcription factor TarR knockout vector, transforming a target microorganism, and culturing to obtain a transgenic microorganism with a TarR coding gene deleted; wherein constructing a transcription factor TarR knockout vector comprises the following steps: Using the genome of Bacillus velez FZB42 as a template, PCR amplification was performed using the tarR-UF / tarR-UR primer pair and the tarR-DF / tarR-DR primer pair to obtain tarR-up and tarR-down fragments respectively; using the chloramphenicol resistance gene as a template, PCR amplification was performed using the Cm-F / Cm-R primer pair to obtain the Cm gene fragment; wherein the nucleotide sequence of the tarR-UF / tarR-UR primer pair is shown in SEQ ID NO.4-5, the nucleotide sequence of the tarR-DF / tarR-DR primer pair is shown in SEQ ID NO.8-9, and the nucleotide sequence of the Cm-F / Cm-R primer pair is shown in SEQ ID NO.6-7; The tarR-up, Cm and tarR-down fragments were connected in sequence by overlapping extension PCR to obtain the fused target fragment, and the target fragment was connected to the T vector by TA cloning technology to obtain the transcription factor TarR knockout vector.

8. The use of the trans-aconitate responsive transcription factor TarR or its encoding gene according to claim 3 in regulating the expression of the trans-aconitate assimilation and utilization operon of microorganisms, characterized in that: The microorganism is Bacillus Velezii.

9. Use of the trans-aconitate responsive transcription factor TarR or its encoding gene as claimed in claim 1 in regulating the trans-aconitate utilization ability of microorganisms.

10. Use of the trans-aconitate responsive transcription factor TarR or its encoding gene as claimed in claim 1 in trans-aconitate affinity purification.

Images