Sugar-inducible promoter and application thereof in prevention and treatment of monochamus alternatus

Abstract

The invention discloses a sugar-inducible promoter and application thereof in prevention and treatment of monochamus alternatus, and relates to the technical field of genetic engineering and biological prevention and treatment. Based on the technical background of transgenic symbiotic bacteria in prevention and treatment of monochamus alternatus, the scheme has the advantages that the transgenic symbiotic bacteria and wild strains have a competitive relationship in ecological niche; under high-level expression driven by a constitutive promoter, the transgenic symbiotic bacteria may be too greedy in resource utilization, so that the transgenic symbiotic bacteria conflict with wild strains and even are eliminated by the wild strains; according to the scheme, expression of Cry3Aa-T protein is regulated and controlled by using the sugar-induced promoter so as to reduce the cost of fitness of exogenous genes carried by serratia marcescens, and a hidden and effective method is provided for preventing and controlling propagation of vector insect monochamus alternatus.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering technology and biological control technology, and in particular to sugar-inducible promoters and their application in the control of pine sawyer beetle. Background Technology

[0002] Pine wilt disease (PWD) is a devastating coniferous disease caused by the invasive pine wood nematode (Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle). It severely damages pine forest resources and causes adverse ecological consequences, resulting in significant economic losses. The pine sawyer beetle can carry the pine wood nematode and infect healthy pine trees, causing PWD. The disease spreads rapidly, and pine trees die within weeks of inoculation with the nematode; therefore, by the time it is discovered, it is often in an untreatable, late stage. To achieve effective and long-term prevention, transgenic symbiotic bacteria have been considered a promising biocontrol method to block the spread of vector-borne infectious diseases.

[0003] The convergence of synthetic biology development and insect symbiosis research has led to a novel pest control strategy—paratransgenics (transgenic symbiotic bacteria / fungi / viruses). Through genetic manipulation of insect symbionts, these paratransgenics can act as gene expression vectors to induce transgenic effects in the host, thereby altering host physiology and reducing vector ability. Compared to genetically modified insects, paratransgenic technology is safer, more durable, easier to manipulate, and can spread throughout the host population in a short time. Furthermore, producing large quantities of transformed microorganisms is much easier than generating sufficient numbers of transgenic insects. It shows particular promise in controlling important agricultural and public health pests, and microorganisms from vector insects such as mosquitoes, tsetse flies, and aphids have been successfully recombined. To express toxic proteins in large quantities and effectively control pests, constitutive promoters are used to modify the initial strain. However, researchers hope to achieve safety and specificity for other bacterial strains by constructing inducible promoters. Because the symbiotic bacteria exist in the natural environment and within the insect's gut, transgenic symbiotic bacteria compete with wild-type strains for niches. The production of these constitutive proteins is detrimental to the adaptability of these strains. High-level expression driven by constitutive promoters may lead to overly greedy resource utilization by the transgenic symbiotic bacteria, resulting in conflicts with wild-type strains or even elimination by them. The fitness cost of the symbiotic bacteria to pests is a crucial factor in the success of the paratransgenic strategy. To prevent the introduction of exogenous genes from becoming a burden on the bacteria, this protocol analyzes the environmental factors of pine trees. Since pine trees contain cellulose and hemicellulose, studies have found that they contain various sugars. The engineered bacteria constructed will come into contact with various sugars in the intestine of the pine sawyer beetle. This indicates that using sugars as inducers to induce the expression of insecticidal toxin genes is feasible. Inducible promoters are a class of specific regulatory elements that respond to sugar-related signals and regulate the expression of downstream genes; a small number of inducible promoters also exist in *Serratia marcescens*.

[0004] Serratia marcescens is widely distributed in forests and accumulates extensively in the midgut of the pine sawyer beetle. In this study, the applicant constructed a transgenic symbiotic bacterium using Serratia marcescens modified with a lactose-inducible promoter.

[0005] Therefore, finding a way to regulate gene expression to prevent and control pine wilt disease transmitted by the pine sawyer beetle is a research topic with significant practical implications. Summary of the Invention

[0006] In view of this, the purpose of this invention is to propose a reliable and flexible sugar-inducible promoter and its application in the control of the pine sawyer beetle. This method reduces the fitness cost of carrying foreign genes by modifying the promoter to regulate the expression of Cry3Aa-T protein, and provides a covert and effective method for preventing and controlling the spread of the vector insect, the pine sawyer beetle.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows:

[0008] A sugar-inducible promoter, comprising promoter Para, promoter Pfru, promoter PsacB, or promoter Plac. The nucleotide sequence of promoter Para is shown in SEQ ID No. 1; the nucleotide sequence of promoter Pfru is shown in SEQ ID No. 2; the nucleotide sequence of promoter PsacB is shown in SEQ ID No. 3; and the nucleotide sequence of promoter Plac is shown in SEQ ID No. 4.

[0009] Based on the above, this solution also proposes a recombinant vector containing the sugar-inducible promoter described above.

[0010] As a preferred implementation option, the recombinant vector described in this scheme is obtained by introducing the sugar-inducible promoter and reporter gene into a plasmid.

[0011] As a preferred implementation option, the plasmids described in this scheme preferably include pGHKW4 plasmid and HasA secretion plasmid pCHSW.

[0012] As a preferred implementation option, the reporter gene described in this protocol includes a fluorescent protein gene.

[0013] Based on the above, this solution also proposes a recombinant strain, which is obtained by introducing the aforementioned recombinant vector into a host bacterium.

[0014] As a preferred implementation option, the host bacterium in this scheme is preferably Serratia marcescens; more preferably, the host bacterium is Serratia marcescens.

[0015] Based on the above, this solution also proposes the application of the sugar-inducible promoter, the recombinant vector, and the recombinant strain mentioned above in regulating the expression of Cry3Aa-T protein, wherein the amino acid sequence of the Cry3Aa-T protein is shown in SEQ ID No. 5.

[0016] Based on the above, this solution also proposes a method for controlling the pine sawyer beetle, which includes the applications described above.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art: the present solution reduces the fitness cost of carrying foreign genes by changing the promoter to regulate the expression of Cry3Aa-T protein, which provides a covert and effective method for preventing and controlling the spread of the vector insect, the pine longhorn beetle. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the plasmid map of the inducible promoter of this scheme, where the recombinant vectors are labeled as follows: A: pGHKW4-Plac; B: pGHKW4-Pfru; C: pGHKW4-araP BAD (or pGHKW4-Para); D: pGHKW4-PsacB.

[0020] Figure 2 This is a diagram showing the cloning verification results of the recombinant vector in this scheme. Figure 2 In A: M: 1kb DNA ladder; A: Plasmid extraction results, 1: pGHKW4-Plac; 2: pGHKW4-Pfru; 3: pGHKW4-Para; 4: pGHKW4-PsacB; Figure 2 In B: 1: pGHKW4-Plac double digestion (KpnI, XhoI), 1216bp; Figure 2 C represents double digestion of the plasmid: 1: pGHKW4-Para, 1190bp; 2: pGHKW4-PsacB, 667bp; 3: pGHKW4-Pfru, 1695bp.

[0021] Figure 3 This is a diagram validating the clone of *Serratia marcescens* transfected using this protocol. M: 5000b DNA ladder; A: Results of BRC plasmid extraction via electroporation; 1: BRC-lac; 2: BRC-fru; 3: BRC-araP BAD ; 4: BRC-sac; B: Plasmid digestion results (BamHI), 1: BRC-fru, 6779bp; 2: BRC-araP BAD C: BRC-lac plasmid double digestion (SacI, NheI), 1216bp and 5084bp; D: BRC-sac plasmid double digestion (SacI, NheI), 667bp and 5084bp.

[0022] Figure 4These are fluorescence micrographs of several *Serratia marcescens* BRC strains expressing GFP using different plasmids under different promoters in dark and bright fields. Among them, (a) plasmids containing the nptII promoter, (b) plasmids containing arabinose-inducible promoters, plasmids without arabinose, and plasmids with 0.2% arabinose added, (c) plasmids containing fructose-inducible promoters, plasmids without fructose, and plasmids with 0.5% fructose added, (d) plasmids containing sugar-inducible promoters, plasmids without sugar, and plasmids with 2% sugar added, and (e) plasmids containing lactose-inducible promoters, plasmids without lactose, and plasmids with 0.02% lactose added.

[0023] Figure 5 The fluorescence intensities are those of wild-type and engineered *Serratia marcescens* BRC cells expressing GFP plasmids under different promoters in this scheme. (a) *Serratia marcescens* BRC cells; (b) GFP expression in *Serratia marcescens* BRC cells regulated by the nptII promoter; (c) GFP expression in *Serratia marcescens* BRC cells regulated by the arabinose promoter; (d) GFP expression in *Serratia marcescens* BRC cells regulated by the fructose promoter; (e) GFP expression in *Serratia marcescens* BRC cells regulated by the sucrose promoter; (f) GFP expression in *Serratia marcescens* BRC2 cells regulated by the lactose promoter.

[0024] Figure 6 This is a comparison of the activation ability of the control group of this scheme and strains containing four different sugar-inducible promoters in sugar-supplemented medium.

[0025] Figure 7 Information about the pCHSW vector and schematic diagrams of four promoter modifications are shown.

[0026] Figure 8 It is a schematic diagram and characterization diagram of gene conjugation and transfer.

[0027] Figure 9 This is a characterization diagram of the extracellular protein Cry3Aa-T secreted by the transgenic strain and the control group in this protocol.

[0028] Figure 10This presents comparative experimental data showing that the inducible strain exhibits stronger adaptability compared to the constitutive mean. A represents the measurement of growth curves; B represents the CFUs of the transgenic and wild-type strains after 12 hours of separate culture; C represents the ratio of transgenic CFUs to total CFUs in each strain competing with wild-type *Serratia marcescens* BRC in LB culture (initially 50 / 50 co-culture for 6 hours). The 50:50 ratio indicates the number of transgenic strains not lost during the experiment; D represents the competition between each strain and wild-type *Serratia marcescens* BRC-CXG2 in LB culture (initially 50 / 50 co-culture for 36 generations, with transfer every 12 hours). Statistical significance for each experiment was assessed using one-way ANOVA and Tukey's HSD test. Significance was expressed as *P < 0.05, **P < 0.01, and *** < 0.001. Experiments were replicated (n = 3).

[0029] Figure 11 The curves show the survival rates of pine sawyer beetle larvae treated with different strains in this protocol. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] 1.1 Experimental strains, plasmids, and growth conditions

[0033] The Serratia marcescens (BNCC186186), Escherichia coli S17-1λpir (BNCC357926), and Escherichia coli trans-T1 used in this protocol were all purchased from Beina Biotechnology; among them, Serratia marcescens (BNCC186186) was named Serratia marcescens BRC.

[0034] The plasmid pGL3-Basic-promoter used in this scheme is the pGL3-Basic vector synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.

[0035] The pGHKW plasmid used in this scheme is the same as that in patent CN 116004492A, and the pCHSW plasmid was synthesized by GenScript Biotech Co., Ltd. The pCHSW plasmid was constructed by replacing the AmpR gene in the pBAM1 plasmid with the spectinomycin resistance gene (SmR) for subsequent donor strain screening, and inserting an expression element—the nptII-cry3AaT-hasA gene—composed of the nptII promoter, cry3AaT gene, and hasA gene sequentially spliced ​​upstream of the KanR gene. The nptII promoter (nucleotide sequence number SEQ ID No. 24) and the hasA gene are derived from the pGHKW4 plasmid (Genbank: MK562404).

[0036] Luria-Ber (LB) medium contains 5.0 g / L yeast extract, 10.0 g / L peptone, and 10.0 g / L NaCl.

[0037] Super OptimalBroth with Catabolite repression (SOC) medium contains 20.0 g / L peptone, 5.0 g / L MgCl2, 0.5 g NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose.

[0038] 100 mL of EZ-rich medium containing a 10×MOPS mixture (1.9 M NH4Cl, 0.276 M K2SO4, 0.02 M CaCl2·2H2O, 2.5 M MgCl2, 5 M NaCl), 10 mL of 0.132 M K2HPO4, 100 mL of 10×ACGU (adenine 0.270 g / L, cytosine 0.222 g / L, uracil 0.224 g / L, guanine 0.302 g / L), and 200 mL of 5×EZ were prepared. Carbon source was added at a 1% ratio before use. Yeast extract and peptone were purchased from OXOID (UK), adenine, cytosine, uracil, and guanine were purchased from Macklin (Shanghai, China), and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0039] *Serratia marcescens* BRC was cultured at 30°C and 200 rpm, while *Escherichia coli* was cultured at 37°C and 200 rpm. Strains carrying the recombinant plasmid were cultured in a solution containing 100 μg / mL... -1 Ampicillin or 100 μg / mL -1 Cultured in kanamycin culture medium.

[0040] 1.2 Conditions for raising the pine sawyer beetle

[0041] Second-instar larvae of the pine sawyer beetle can survive under different temperatures and time conditions. The artificially bred population of the pine sawyer beetle used in the experiment was a dedicated population from the Fujian Provincial Key Laboratory of Major Pests in Ecological Public Welfare Forests. Adult beetles collected from beetle traps were placed in rearing boxes for mating, and then laid eggs in fresh production wood. All pine sawyer beetles were reared in a constant temperature chamber at 28℃, 80% relative humidity, and a photoperiod of 16:8h. When the beetles in the production wood reached the second-instar larval stage, they were dissected from the production wood for subsequent experiments.

[0042] Example 2

[0043] 2.1 Plasmid construction method for sugar-induced promoters

[0044] The primers used in this protocol were synthesized by Sunya Biotechnology Co., Ltd. (Fuzhou, China) and are listed in the primer sequence list below.

[0045]

[0046]

[0047] This protocol uses GFP as the reporter gene and plasmid pGHKW4 as the vector. The original nptII constitutive promoter (nucleotide sequence number SEQ ID No. 24) in the plasmid was replaced with the inducible promoters of this protocol (promoter Para (nucleotide sequence number SEQ ID No. 1), promoter Pfru (nucleotide sequence number SEQ ID No. 2), promoter PsacB (nucleotide sequence number SEQ ID No. 3), and promoter Plac (nucleotide sequence number SEQ ID No. 4)). The promoter fragments were then amplified from plasmid pGL3-Basic-promoter (pGL3-Basic vector synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.), and pGHKW4 was digested with SacI and NheI (Beijing Takara) to obtain pGL3-Para plasmid, pGL3-Pfru plasmid, pGL3-PsacB plasmid, and pGL3-Plac plasmid containing promoters Para, promoter Pfru, promoter PsacB, and promoter Plac, respectively. Then follow - The Seamless Cloning and Assembly Kit (Beijing, TransGen) provides instructions for seamlessly connecting two fragments.

[0048] To obtain a pGHKW4-sugar-inducible recombinant vector capable of expressing green fluorescent protein, molecular cloning was performed using a pGHKW4 expression vector carrying GFP and four sugar-inducible promoters (promoter Para, promoter Pfru, promoter PsacB, or promoter Plac).

[0049] In particular, regarding the construction of expression vectors for the promoter Plac, combined with Figure 1 The plasmid map of the inducible promoter shown and Figure 2 The image shows the verification results of pGHKW4-Plac cloning.

[0050] This protocol utilizes pGHKW4-lac-F / R primers to amplify the pGL3-Plac plasmid containing the Plac promoter by PCR. After gel extraction, a 1216 bp Plac fragment was obtained. The original pGHKW4 plasmid was then double-digested with SacI and NheI to obtain a 5129 bp target fragment. This fragment was ligated and transformed into pGHKW4-Plac-DH5α *E. coli*, and plasmid extraction and restriction enzyme digestion verification were performed. The plasmid extraction electrophoresis image showed bands (…). Figure 2 A), and the sizes of the two enzyme digestion bands in the SacI and NheI double digestion electrophoresis results are consistent with the sizes of the 1216bp Plac fragment and the 5129bp pGHKW4 fragment. Figure 2 B) This proves that pGHKW4-Plac was successfully cloned.

[0051] This protocol utilizes pGHKW4-fru-F / R primers to perform PCR amplification of the pGL3-Pfru plasmid containing the Pfru promoter. After gel extraction and recovery, a 1695 bp Pfru fragment was obtained. This fragment was then ligated into the pGHKW4 vector, which had been double-digested with SacI and NheI. The resulting pGHKW4-Pfru-DH5α *E. coli* was transformed, and plasmid extraction and restriction enzyme digestion verification were performed. The electrophoresis results showed that the sizes of the two digested fragment bands corresponded to the 1695 bp Pfru fragment and the 5129 bp pGHKW4 fragment. Figure 2 C) This proves that pGHKW4-Pfru was successfully cloned.

[0052] This protocol utilizes pGHKW4-ara-F / R primers to perform PCR amplification of the pGL3-Para plasmid containing the Para promoter. After gel extraction and recovery, a 1190 bp Para fragment was obtained. This fragment was then ligated into the pGHKW4 vector, which had been double-digested with SacI and NheI. The resulting pGHKW4-Para-DH5α *E. coli* was transformed, and plasmid extraction and restriction enzyme digestion were performed for verification. The electrophoresis results showed that the sizes of the two digested fragment bands corresponded to the 1190 bp Para fragment and the 5129 bp pGHKW4 fragment. Figure 2 C) This proves that pGHKW4-Para cloning was successful.

[0053] This protocol utilizes pGHKW4-sacB-F / R primers to perform PCR amplification of the pGL3-PsacB plasmid containing the PsacB promoter. After gel extraction and recovery, a 667bp PsacB fragment was obtained. This fragment was then ligated into the pGHKW4 vector, which had been double-digested with SacI and NheI. The resulting pGHKW4-PsacB-DH5α *E. coli* was transformed, and plasmid extraction and restriction enzyme digestion verification were performed. The electrophoresis results showed that the sizes of the two digested fragment bands corresponded to the 667bp PsacB fragment and the 5129bp pGHKW4 fragment. Figure 2 C) This proves that pGHKW4-PsacB was successfully cloned.

[0054] Thus, the proposed method has successfully verified through experiments the feasibility and predictability of cloning four sugar-inducible promoters (promoter Para, promoter Pfru, promoter PsacB, and promoter Plac) into the pGHKW4 expression vector. This also yielded four recombinant vectors corresponding to promoters Para, Pfru, PsacB, and Plac: pGHKW4-Para, pGHKW4-Pfru, pGHKW4-PsacB, and pGHKW4-Plac.

[0055] Example 3

[0056] 3.1 Electroconversion and Electrochemical Validation Methods

[0057] Preparation of competent Serratia marcescens cells: 50 μL of Serratia marcescens culture was transferred to 5 mL of liquid LB medium and incubated at 30°C for 12 h. Then, it was transferred to 100 mL of liquid LB medium and incubated at 30°C for another 2-2.5 h to allow the bacterial culture to reach OD500. 600 The concentration was increased to 0.3. After incubating on ice for 20 minutes to stop growth, the cells were collected in 50 mL tubes. Then, the cells were resuspended three times with 10 mL of 10% glycerol. Finally, the cells were gently resuspended with 1 mL of 10% glycerol, and 200 μL was aliquoted into 1.5 mL EP tubes to obtain Serratia marcescens electrocompetent cells.

[0058] Electroporation: 200 μL of competent cells containing 10 μL of desalted recombinant plasmid were added to a 2 mm electrode cup, gently stirred to mix, and immediately placed on ice for 10 minutes. Electroporation was then initiated at 2500 V, 25 μF, and 200 Ω. Immediately after electroporation, 800 μL of SOC medium was added to the electrode cup, and the cells were incubated at 30°C and 200 rpm for 2 hours for recovery. The recovered cell suspension was centrifuged to obtain a cell pellet, and 100 μL of liquid medium was mixed with the pellet and spread onto a solid plate containing kanamycin resistance. The pellet was then incubated at 30°C for 12 hours. Finally, transformed single colonies were picked and incubated in 5 mL of LB liquid medium containing kanamycin resistance for 12 hours at 30°C and 200 rpm. Subsequently, the *Serratia marcescens* clone was validated by plasmid extraction, restriction enzyme digestion, and sequencing.

[0059] 3.2 Electroporation of Serratia marcescens and its validation

[0060] To obtain a modified *Serratia marcescens* strain with the potential to express green fluorescent protein (GFP) via sugar induction, four recombinant vectors corresponding to promoters Para, Pfru, PsacB, or Plac—pGHKW4-Para, pGHKW4-Pfru, pGHKW4-PsacB, and pGHKW4-Plac—were electroporated into *Serratia marcescens* BRC cells. The resulting vector, pGHKW4-araP, was then cloned. BAD Four modified Serratia marcescens strains, pGHKW4-fruP, pGHKW4-PsacB, and pGHKW4-Plac (abbreviated as BRC-araP), were identified. BAD (BRC-fruP, BRC-PsacB, BRC-Plac), and then plasmid extraction and enzyme digestion verification were performed respectively. The results are shown in […]. Figure 3 As shown.

[0061] from Figure 3 The plasmid extraction results shown in the figure indicate that all four modified Serratia marcescens plasmids exhibited bands on electrophoresis. Figure 3 A). The enzyme digestion electrophoresis results show that BRC-fruP and BRC-araP were digested with BamHI. BAD Subsequently, BRC-fruP theory yielded a single fragment of size 6779bp, BRC-araP BAD Theoretically, two fragments of 793bp and 5481bp were obtained, consistent with the results of single enzyme digestion. Figure 3B), after double digestion of BRC-Plac and BRC-PsacB with SacI and NheI, BRC-Plac theoretically yielded two fragments of 1216bp and 5084bp, and BRC-PsacB theoretically yielded two fragments of 667bp and 5084bp, consistent with the experimental double digestion results. Figure 3 C Figure 3 D) This demonstrates that the proposed method successfully transformed four recombinant vectors, pGHKW4-Para, pGHKW4-Pfru, pGHKW4-PsacB, and pGHKW4-Plac, into Serratia marcescens, and correspondingly obtained four transgenic Serratia marcescens pGHKW4-araP BAD , pGHKW4-fruP, pGHKW4-PsacB, pGHKW4-Plac.

[0062] Example 4

[0063] 4.1 Fluorescence observation of recombinant strains

[0064] The successfully cloned Serratia marcescens was validated by sugar-induced expression.

[0065] First, the successfully cloned *Serratia marcescens* culture was placed in 5 mL of Kan-resistant liquid LB and incubated overnight at 30°C. Next, it was transferred to 3 mL of Kan-resistant EZ Rich synthetic medium, and a specific sugar solution was added to achieve a final sugar concentration of 0.02% lactose, 2% sucrose, 0.5% fructose, 0.2% arabinose, and 1% xylose. The medium was then incubated at 30°C with shaking. After 6 hours of incubation, GFP fluorescence was observed under a ZEISS fluorescence microscope at 40x magnification.

[0066] 4.2 Expression of four inducible promoters in Serratia marcescens

[0067] Four strains containing inducible promoters were activated and constructed (Serratia marcescens pGHKW4-araP). BAD Plasmids containing different inducible promoters (pGHKW4-fruP, pGHKW4-PsacB, and pGHKW4-Plac) were cultured in LB medium at 30°C with 50 μL of glycerol. The plasmids were then added to their respective inducers, and fluorescence micrographs of the plasmids without and without inducers were analyzed and compared. The results are shown in [Figure number missing]. Figure 4 As shown.

[0068] Depend on Figure 4 The results showed that, under dark-field microscopy, the pGHKW4 plasmid expression induced by the npt II constitutive promoter exhibited significant green fluorescence. Figure 4a). Without added sugar, all four promoters were able to induce minimal gene expression. When an inducer was added, fluorescence was observed in almost every cell. Plasmids containing arabinose as promoters were induced; after 6 hours of induction with 0.2% arabinose, all cells expressed green fluorescent protein (GFP). Figure 4 b). Plasmids containing a fructose-inducible promoter, plasmids without fructose, and plasmids with 0.5% fructose added expressed more green fluorescent protein than plasmids without fructose. Figure 4 c). In plasmids containing sucrose-inducible promoters, the expression levels of fluorescent proteins differed significantly between plasmids without sucrose and those with 2% sucrose. Figure 4 d). Plasmids containing a lactose-inducible promoter, after 6 h of induction with 0.02% lactose, resulted in all cells expressing green fluorescent protein (GFP). Figure 4 e). This indicates that all four sugar promoters (promoter Para, promoter Pfru, promoter PsacB, or promoter Plac) can be normally stimulated in Serratia marcescens BRC.

[0069] To further confirm the nature of these promoters (the pGHKW4 inducible promoter in Serratia marcescens BRC), all constructed recombinants were cultured in EZ-rich medium at 30°C. Biomass and fluorescence intensity were measured every 2 hours. The EZ-rich medium required the addition of 1% carbon source. 1% glucose was added as a carbon source to the original pGHKW4 plasmid containing the fluorescent gene, and 1% arabinose, fructose, sucrose, or lactose was added to the other four inducible promoters.

[0070] The fluorescence intensity characterization results of Serratia marcescens BRC expressing GFP plasmid under different promoters are shown in the figure. Figure 5 As shown, from Figure 5 As shown in Figure A, the addition of glucose to the empty strain had no effect on fluorescence intensity (approximately 15,000), but due to the availability of carbon source, the growth of the empty strain with added glucose was better than that without added glucose. Therefore, using *Serratia marcescens* BRC strains without recombinant vectors as a blank control, a certain fluorescence intensity could be measured on the SPECTRAMAX instrument without plasmid addition, with a reference value of approximately 15,000 for no fluorescence intensity. Since the addition of carbon source can accelerate cell growth, the fluorescence intensity of *Serratia marcescens* BRC strains containing the constitutive promoter pGHKW4 plasmid increased to some extent after the addition of glucose as cell density increased relative to the absence of carbon source, eventually stabilizing at around 30,000. The fluorescence intensity of the four inducible promoters after sugar induction stabilization was all higher than 50,000, and they can be divided into two categories based on fluorescence intensity. For class I promoters, arabinose promoters, and lactose promoters, the fluorescence intensity increased with increasing cell growth until the stationary phase (…). Figure 5C, D, F), while class II promoters and sucrose promoters only accumulate during the 12-hour stationary period (C, D, F). Figure 5 E). All four promoters were successfully induced by sugar-inducible promoters, laying the foundation for their application in the larvae of the pine sawyer beetle. The fluorescence intensity of the *Serratia marcescens* BRC strain remained unchanged under glucose supplementation. This study compared the fluorescence intensity of *Serratia marcescens* during its logarithmic growth phase (4 hours). All four sugar-inducible promoters (promoter Para, promoter Pfru, promoter PsacB, and promoter Plac) responded to the corresponding sugars for conditional expression in vitro. In contrast, the fluorescence intensity of *Serratia marcescens* BRC strain and pGHKW4-nptII did not show significant differences between EZ-rich medium and the same medium supplemented with 1% sugar. However, all four sugar-inducible promoters were significantly activated in the glucose-supplemented medium. Figure 6 ).

[0071] 4.3 Promoter characterization method based on GFP fluorescence intensity

[0072] Verification of promoter expression intensity in Serratia marcescens.

[0073] First, 30 μL of each glycerol bacterium clone was added to 3 mL of kanamycin-resistant liquid EZ-Rich medium and cultured overnight at 30°C and 200 rpm. Then, they were transferred to 3 mL of kanamycin-resistant EZ-Rich medium, and specific sugars were added to achieve sugar concentrations of 0.2% arabinose, 0.5% fructose, 2% sucrose, and 0.02% lactose, respectively. The medium was then incubated at 30°C and 200 rpm with shaking. Four transgenic *Serratia marcescens* strains BRC-pGHKW4 were used as positive controls, and *Serratia marcescens* BRC as a negative control. Culture was continued for 48 h, with growth curves and fluorescence measured every 2 h. 200 μL of culture medium was taken per well, with each bacterial strain tested in triplicate and each variable measured three times. The samples were transferred to clear and black 96-well plates, respectively. Finally, the clear and opaque 96-well plates were placed in a microplate reader for analysis. OD was measured every 2 hours using a SPECTRAMAX M4. 600 The test was conducted continuously for 48 hours. Data was collected using SoftMax Pro 7 software and analyzed using Origin Pro 2024. 600 And fluorescence data.

[0074] Example 5

[0075] 5.1 Construction and Validation of Engineering Strains with Sugar-Inducible Promoters

[0076] After confirming that four sugar-inducible promoters could be used for Serratia marcescens, this protocol cloned these validated promoters from the GFP reporter plasmid pGHKW4 into the HasA secretory plasmid pCHSW.

[0077] In this embodiment, the insecticidal gene was transferred horizontally from the plasmid to the Serratia marcescens BRC genome via oriT conjugation transfer gene transfer, thus ensuring genetic stability while avoiding the risk of plasmid loss. Fluorescence observation and fluorescence intensity measurements showed that all four promoters could be activated in Serratia marcescens with added sugar inducers. Subsequently, the four sugar-inducible promoters were cloned upstream of the Cry3Aa-T toxin gene targeting the larvae of the pine sawyer beetle. After verifying promoter function with GFP fluorescence, the target promoter fragment was amplified by PCR from the pGHKW4-promoter plasmid and cloned into the pCHSW vector. The resulting recombinant plasmid contained both the inducible promoter and the Cry3Aa-T gene, and was subsequently introduced into Escherichia coli S17-1λpir via heat shock transformation.

[0078] To ensure that the plasmid is retained only in *E. coli* S17-1λpir and not in *Serratia marcescens*, this protocol uses a suicide vector (pCHSW) containing the oriR6Kγ origin of replication. This origin of replication functions only in strains expressing the pir protein (such as *E. coli* S17-1), thus preventing plasmid proliferation in other bacteria. Furthermore, pCHSW carries a transposase gene that enables conjugative transfer, while pGHKW4 cannot replicate in S17-1 and lacks a conjugative transfer mechanism. The replication of this vector depends on the π protein (provided by the pir gene in the donor bacterium), thus ensuring insertional mutations in *Serratia marcescens*. The promoter fragment was amplified from plasmid pGHKW4-promoter. The primer F and R sequences for Para are SEQ ID NO.14 and SEQ ID NO.15, respectively; for Pfru, SEQ ID NO.16 and SEQ ID NO.17, respectively; for Psac, SEQ ID NO.18 and SEQ ID NO.19, respectively; and for Plac, SEQ ID NO.20 and SEQ ID NO.21, respectively. The obtained PCR product was then cloned into the pCHSW vector. Figure 7 The two fragments were seamlessly cloned and ligated, and the resulting plasmid was transformed into E. coli S17-1λpir via heat shock transformation. Figure 7 Information about the pCHSW vector and schematic diagrams of four promoter modifications are shown.

[0079] 5.2 Binding Transfer and Verification Methods

[0080] Schematic diagram of gene conjugation transfer ( Figure 8 A). *Escherichia coli* S17-1λpir, carrying the Cry3Aa-T gene and used as a donor strain, and *Serratia marcescens* BRC, used as a recipient strain, were cultured to the logarithmic growth phase, washed with 10 mM MgSO4 solution, and resuspended at OD. 600 1. Mix them in a 1:1 ratio and spread them on a plate without antibiotics, then incubate at 37°C for 5 hours. Scrape the colonies off the plate and incubate them in ampicillin LB broth at 30°C, 200 rpm for 12 hours, then incubate them on LB plates containing kanamycin at 30°C for 12 hours. Afterwards, pick single colonies of *Serratia marcescens* carrying the Cry3Aa-T gene obtained through conjugation transfer. Inoculate the single colonies with antibiotics containing kanamycin and ampicillin (100 μg / mL). -1 In 5 mL of LB liquid medium, incubate at 37 °C and 150 r / min for 12 h.

[0081] After successfully constructing four transgenic strains with sugar promoters, pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac (corresponding to promoters Para, Pfru, PsacB, or Plac, respectively), bacterial DNA was extracted according to the instructions of the bacterial DNA kit (OMEGABio-Tek, America). Finally, the extracted DNA was used as a template, and the presence of the Cry3Aa-T gene was verified by PCR using Cry primers.

[0082] For ease of comparison, this protocol also simultaneously constructed the original strain (pCHSW) according to the above method. The E. coli S17-1λpir strain carrying the pCHSW plasmid was co-cultured with Serratia marcescens BRC-CXG2 to obtain the engineered Serratia marcescens strain BRC-CXG2-pCHSW carrying the nptII constitutive promoter and capable of exocrine expression of Cry3AaT protein.

[0083] To verify the insertion of the promoter –Cry3Aa-T-hasA gene into the Serratia marcescens genome, this protocol extracted genomic DNA from four transgenic strains carrying sugar promoters (pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac) and performed PCR detection using Cry3Aa-T gene primers. The expected gene fragment size was 1845 bp. Figure 8 As shown in Figure B, a target band of approximately 2000 bp can be observed, indicating that the exogenous gene was successfully inserted into the Serratia marcescens genome. This demonstrates that the transgenic strains pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac were all successfully constructed.

[0084] 5.3 Inducible expression of transgenic strains with inducible promoters

[0085] To ensure the Cry3Aa-T gene was expressed in the inducible promoter plasmid, extracellular and intracellular proteins of these plasmids were extracted using a HasA transport system and analyzed by Western blotting. Cells were then inoculated with 100 μg / mL... -1 Cultured in LB liquid medium containing kanamycin until OD 600 =0.6. Protein overexpression was induced for 24 h using different sugar concentrations mentioned above. Extracellular proteins were concentrated using a 50 kDa Merck Millipore condenser, washed three times with the supernatant (PBS), and then resuspended in 5 mL of PBS. For intracellular protein extraction, harvested cells were washed three times in phosphate-buffered saline (PBS) and then sonicated on ice (50 Hz) for 30 min at 10-second intervals of 5-second. Cell debris was removed by centrifugation at 8000 rpm and 4°C for 8 min, and the resulting supernatant contained intracellular proteins. These strains secrete Cry3Aa-T extracellular protein only in the presence of the corresponding sugars. Figure 9 The expression pattern was consistent with the inducible characteristics of the GFP reporter gene. SDS-PAGE analysis verified the intracellular and extracellular expression of the sugar-induced strains. Notably, all four strains carrying sugar-induced promoters were able to successfully secrete Cry3Aa-T into the extracellular environment for expression under sugar induction, laying a solid foundation for future forestry applications.

[0086] Example 6

[0087] 6.1 Method for determining the fitness cost of transgenic engineered bacteria

[0088] Before testing whether inducible promoters can activate *Acer truncatum* in vivo, it is necessary to verify whether inducible promoters can overcome the fitness cost associated with constitutive promoters. This can be determined using the following three methods:

[0089] First, the growth curves of each strain were determined. The original strain (pCHSW), *Serratia marcescens* BRC, and four transgenic strains with sugar promoters (pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac) were cultured in 100 mL of LB broth at 30°C and 200 rpm for 24 h. Uninoculated LB broth served as a blank control. 3 mL samples were taken every 2 hours, and absorbance was measured at 600 nm using a spectrophotometer. The culture was observed continuously for 24 h, and the experiment was repeated three times. The average value was calculated to determine the growth characteristics, and growth curves were plotted using Graphpad Prism 8.0.2. Data were analyzed using IBM SPSS Statistics 27. Figure 10 -A). To quantify the growth of different strains, the viable cell count of six strains after 12 hours of cultivation was determined using the plate count method. Figure 10 -B).

[0090] In a competitive experiment, the survival ability of four transgenic strains with sugar promoters (pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac) co-cultured with *Serratia marcescens* BRC was tested. Each transgenic strain was cultured to the logarithmic growth phase, with each strain cultured in triplicate, and mixed with *Serratia marcescens* BRC at a 50 / 50 ratio, and cultured until OD (Occurrence Difference). 600 =0.5. The mixed culture was cultured for 6 hours, then serially diluted, and 100 μL of the mixture was taken and diluted with or without 100 μg / mL. -1 Kanamycin was plated on LB agar plates, and the colony count was recorded as 30-300. The ratio of transgenic bacteria to wild-type bacteria was calculated by comparing the CFU on selective and non-selective media. The data was plotted in Chiplot (https: / / www.chiplot.online / ) using box plots. Figure 10 -C).

[0091] The transgenic strains competed with wild-type Serratia marcescens for genetic stability after multiple generations. The transgenic strains (pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac) were mixed with Serratia marcescens BRC at a 50 / 50 ratio and tested at OD... 600 Under conditions of 0.5, culture for 12 h, then inoculate at a 1% concentration. The bacterial solutions obtained after the first, third, and fifth transfers were progressively diluted and plated onto plates containing or without 100 μg / mL. -1On LB solid medium containing kanamycin. The ratio of transgenic bacteria to wild-type was calculated by comparing CFU on selective medium with CFU on non-selective medium to determine the genetic stability of wild-type strains carrying the exogenous gene under non-selective stress. Data were analyzed using the same methods described above.

[0092] 6.2 Construction of strains for controlling *Pinus sylvestris* and results of fitness cost determination

[0093] Cell growth density was monitored for wild-type strain (Serratia marcescens BRC), original strain (pCHSW), and transgenic Serratia marcescens with promoter genes (pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac). CFU was measured for Serratia marcescens BRC and transgenic Serratia marcescens after 12 hours of co-culture, and the retention rates of Serratia marcescens BRC and transgenic Serratia marcescens after multiple passages were also measured.

[0094] By continuously measuring the growth of four transgenic strains with inducible promoters (pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac), the original strain (pCHSW), and the wild-type strain (Serratia marcescens BRC) for 24 hours, the results showed that the overall growth trend of the strains with constitutive promoters was no different from that of the empty strains, while the growth trend of the strains with constitutive promoters was lower than that of the other five strains. Figure 10 A).

[0095] To quantify the growth of different strains, the viable cell counts of six strains after 12 hours of culture were determined using the plate count method. The results showed that the viable cell count of strain nptII, which has a constitutive promoter, was significantly lower than that of *Serratia marcescens* BRC, and also significantly lower than that of the other four sugar-inducible strains (pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac). Figure 10 The growth curves in A are consistent. Furthermore, the CFU of the strain with the constitutive promoter nptII after continuous culture for 12 h were significantly lower than those of the strain with the inducible promoter and Serratia marcescens BRC, meaning that the cell density was significantly lower than that of the original empty strain.

[0096] This preliminarily confirms that changing the promoter can affect bacterial growth. To further determine the survival of the transgenic strain when wild-type (Serratia marcescens BRC) and transgenic strain coexist, wild-type Serratia marcescens (BRC) and transgenic Serratia marcescens were co-cultured for 12 hours without selective pressure. CFU (Cellular Fumes Count) were measured on selective and non-selective media. The percentage of colonies on selective and non-selective media represented the survival rate of the strain carrying the foreign gene. The results showed that the transgenic strain carrying the constitutive promoter PnptII had a survival rate of only 25% in the presence of the wild-type strain (BRC), while the four transgenic strains carrying inducible promoters had average survival rates of 70% for Para and Psac, 80% for Plac, and even 100% for Pfru. Figure 10 B) After 36 generations of continuous subculturing, the survival rate of PnptII strains was only 3%, almost eliminated by (Serratia marcescens BRC), while the survival rate of the four sugar-induced strains (pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac) was around 50% on average. Figure 10 C). This indicates that inducible promoters can be well hidden in wild-type Serratia marcescens without selective pressure and inducers. Inducible promoters can effectively reduce the burden of foreign genes carried by wild-type strains (Serratia marcescens BRC) and reduce a certain fitness cost. This result provides a solid theoretical basis for long-term survival in the wild.

[0097] Example 7

[0098] 7.1 Bioassay methods for *Pseudomonas pineensis*

[0099] First, modified bacterial strains (transgenic strains pCHSW-ara, pCHSW-fru, pCHSW-sac, and pCHSW-lac), the original strain (pCHSW), and the wild-type strain (Serratia marcescens BRC) were cultured in 100 mL Erlenmeyer flasks for 24 h. Then, the supernatant was removed by centrifugation at 8000 rpm for 10 min at 4 °C. The remaining suspension was then resuspended in sterile water and centrifuged under the same conditions. This process was repeated three times, followed by resuspending in 5 mL of sterile water. The resulting bacterial suspension was used for plating and counting at a concentration of 9.5 × 10⁻⁶. 8 CFU mL -1For bioassays, the diluted bacterial suspension was added to 400 μL of 0.4 g paper towel and placed in a 1.5 mL centrifuge tube to feed second-instar larvae of the pine sawyer beetle. The original strain pCHSW and the wild-type strain (Serratia marcescens BRC) were used as a blank control group in the rearing experiment, with ddH2O as the blank control. During the rearing process, the larval susceptibility to the virulence of the strain was observed daily, with 10 larvae in each treatment group, replicated three times. Data were processed and analyzed using Graphpad Prism 8.0.2.

[0100] 7.2 Bioassay of transgenic Serratia marcescens against the longhorn beetle Serratia marcescens

[0101] All strains were treated with 9.5 × 10⁻⁶. 8 CFU mL -1 The concentration was determined by bioassay, and the results were as follows: Figure 11 As shown, it can be seen that the transgenic strains (pCHSW-ara, pCHSW-fru) corresponding to the two inducible promoters Para and Pfru are LT 50 The mortality rate was similar in both strains, reaching 50% on day 9. The transgenic strain (pCHSW-sac) LT corresponding to the PsacB promoter... 50 =10, of which the survival rate of the transgenic strain corresponding to the promoter Pfru gradually decreased during the bioassay, reaching 25% on the last day. The transgenic strain with promoter Plac (pCHSW-lac) LT 50 =4, the survival rate on the last day of the bioassay was 20%. Meanwhile, the original strain LT, carrying the constitutive promoter PnptII... 50 =8, the survival rate only dropped to 50% on the 8th day of the biopsy and remained there until the end of the biopsy.

[0102] pass Figure 11 Survival curve results showed that all four sugar-inducible promoters (promoter Para, promoter Pfru, promoter PsacB, or promoter Plac) could be activated in the intestine of second-instar larvae of the pine sawyer beetle, and the transgenic symbiotic bacteria modified with the lactose promoter Plac showed improved insecticidal activity against pine sawyer beetle larvae compared with the constitutive promoter.

[0103] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A sugar-inducible promoter, characterized in that, It is the promoter Para, promoter Pfru, promoter PsacB, or promoter Plac; The nucleotide sequence of the promoter Para is shown in SEQ ID No. 1; The nucleotide sequence of the promoter Pfru is shown in SEQ ID No. 2; The nucleotide sequence of the promoter PsacB is shown in SEQ ID No. 3; The nucleotide sequence of the promoter Plac is shown in SEQ ID No.

4.

2. A recombinant vector, characterized in that, It contains the sugar-inducible promoter as described in claim 1.

3. The recombinant vector as described in claim 2, characterized in that, The recombinant vector is obtained by introducing the sugar-inducible promoter and reporter gene into a plasmid.

4. The recombinant vector as described in claim 3, characterized in that, The plasmids include pGHKW4 plasmid and HasA secretion plasmid pCHSW.

5. The recombinant vector as described in claim 3, characterized in that, The reporter genes include fluorescent protein genes.

6. A recombinant bacterial strain, characterized in that: The recombinant strain is obtained by introducing the recombinant vector according to any one of claims 2 to 5 into a host bacterium.

7. The recombinant strain according to claim 6, characterized in that: The host bacterium is Serratia marcescens.

8. The application of the sugar-inducible promoter of claim 1, the recombinant vector of any one of claims 2 to 5, and the recombinant strain of any one of claims 6 to 7 in regulating Cry3Aa-T protein expression, characterized in that, The amino acid sequence of the Cry3Aa-T protein is shown in SEQ ID No.

5.

9. A method for controlling the pine sawyer beetle, characterized in that, It includes the application described in claim 8.

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