Promoter PinhA1 and application thereof in guiding efficient expression of functional genes

Abstract

The invention relates to a promoter PinhA1 and application thereof in guiding efficient expression of functional genes. The nucleotide sequence of the promoter PinhA1 is as shown in SEQ ID NO: 1. The strong promoter PinhA1 can be used for efficiently guiding the expression of the cry1Ac gene in the bacillus thuringiensis delta cdsR mutant. According to the invention, a strain delta cdsR (PinhA1-1Ac) which is used for guiding cry1Ac expression by PinhA1 and generating a large number of regular bipyramid crystal proteins is constructed, and the Cry1Ac yield of the strain delta cdsR (PinhA1-1Ac) and the insecticidal activity of the strain delta cdsR (PinhA1-1Ac) on plutella xylostella larvae are equivalent to those of a wild strain HD73. Results prove that the strong promoter PinhA1 has a good application prospect in the aspect of constructing a bacillus thuringiensis engineering strain, and a new strategy is provided for improving crystal protein or other insecticides in future agricultural production.

Description

Technical Field

[0001] The invention relates to a novel strong promoter PinhA1, which can guide the expression of functional genes in Bacillus thuringiensis, and in particular, the expression efficiency of cry genes is comparable to that of HD73 wild strain. Background Art

[0002] Pest control is an important means to ensure the health and yield of crops and a guarantee for the sustainable development of agriculture. At present, the main pest control measures in the world are physical, chemical and biological control methods, such as: physical trapping based on the principle of insect phototaxis (Zhang Jinfang, Zhang Yang, Xu Wenping, et al. Research progress on insect phototaxis and its application in pest control [J]. 2020, (011): 042.); spraying chemical pesticides such as imidacloprid (Yang Siyao, Gao Suhong, Qi Huixia. Safety evaluation of five commonly used insecticides in chestnut orchards against Harmonia axyridis (J. Agricultural Disaster Research [J]. 2024, 14 (06): 22-4.); using biological natural enemies to control pests (Yan Yanchun, Qiao Chuanling, Insect Knowledge Qian J. Research progress on insecticide resistance of diamondback moth [J]. 1997, (05): 310-4.), etc. However, physical trapping methods rely on the night environment, chemical pesticides pose the risk of polluting the ecological environment, and biological natural enemy control is slow to take effect. Therefore, the development of green, safe and efficient prevention and control methods is an urgent need for the development of my country's agriculture.

[0003] Bacillus thuringiensis (Bt) is a Gram-positive bacterium belonging to the Bc family. Unlike other Bacillus species, it produces insecticidal crystal proteins (ICPs) encoded by cry or cyt genes during the spore formation period. The expression level of ICPs is very high, accounting for 20% to 30% of the cell dry weight (BRAVO A, GILL SS, SOBERóN M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control [J]. Toxicon: official journal of the International Society on Toxinology, 2007, 49 (4): 423-35.). As of early September 2024, researchers have discovered and recorded 858 cry and cyt genes (http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / ). Different types of cry and cyt toxin proteins have specific insecticidal activity against different types of pests. When the crystal protein is eaten by the target pest and enters the digestive tract, it dissolves and activates the midgut protease in the alkaline environment of the midgut, binds to specific receptors, and causes the insect to die of septicemia (Yan Yanchun, Qiao Chuanling, Insect Knowledge Qian J. Research Progress on Plutella xylostella Resistance [J]. 1997, (05): 310-4.). Bt is widely distributed in nature and has the advantages of good insecticidal activity, environmental friendliness, simple formulation production and high yield. It is currently the most successfully applied insect pathogen and the main biological insecticide produced worldwide, with sales accounting for 90% of all biological pesticides (TORRES MANNO MA, REPIZO GD, MAGNI C, et al. The assessment of leading traits in the taxonomy of the Bacillus cereus group[J]. Antonie van Leeuwenhoek, 2020, 113(12): 2223-42.).

[0004] Although Bt biopesticides have the advantages of high insecticidal activity and safety to non-target organisms, they still have problems such as short duration, easy decomposition of crystals and insect resistance in field applications, which limit their application in long-term pest management. Therefore, strategies for improving insecticides have emerged, including adding protective agents to prevent UV damage and encapsulating crystal proteins to prevent them from becoming ineffective; slowing down the development of pest resistance by rotating different types of Bt preparations or using them in combination with other biopesticides and chemical pesticides; and using genetic engineering technology to transform Bt strains to improve insecticidal activity, expand the insecticidal spectrum, and extend the duration of effectiveness. The parasporal crystals composed of Cry proteins produced by Bt can reach more than 20% of the cell dry weight, which is mainly due to the strong promoter of the cry gene ensuring the continuous and large-scale production of mRNA, and the stability of mRNA is crucial for the large-scale expression of cry genes (Shao Zongze, Yu J. Mechanism of overexpression of insecticidal crystal proteins of Bacillus thuringiensis [J]. Life Science 2000, 12 (4): 4.). Finding strong promoters to express Cry proteins has become a way to increase the production of crystal proteins. According to the different regulatory mechanisms of cry genes, they are divided into spore-dependent cry genes and non-spore-dependent cry genes. Therefore, some studies have screened strong promoters from cry gene promoters to guide cry gene expression, such as the promoter of cry8E gene (DU L, QIU L, PENG Q, et al. Identification of the promoter in the intergenic region between orf1 and cry8Ea1 controlled by sigma H factor [J]. Applied and environmental microbiology, 2012, 78 (12): 4164-8.). In addition, some studies have also attempted to screen strong promoters of non-cry genes to guide the expression of cry genes, such as P5014 (ZHANG X, GAO T, PENG Q, et al. A strong promoter of anon-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis [J]. Applied microbiology and biotechnology, 2018, 102 (8): 3687-99.) and PexsY (Zheng Qingyun, Wang Guannan, Zhang Zhe, et al. Expression of cry1Ac gene directed by promoter PexsY of gene encoding protein of bud outer wall matrix [J]. 2014, 54 (10): 8.).

[0005] In the early stage of the laboratory, a cdsR gene deletion mutant was constructed, and it was found that the mutant has the characteristics of no spores, no crystals and no lysis. Using the characteristics of this mutant as a chassis bacteria is very likely to solve the problems of inactivation of insecticidal crystal proteins and spore accumulation and environmental pollution. Therefore, the cdsR gene deletion mutant has the potential to be developed and utilized as an excellent chassis strain (ZHANG X, CHEN Y, YAN T, et al. Cell death dependent on holins LrgAB repressed by a novel ArsR family regulator CdsR [J]. Cell death discovery, 2024, 10 (1): 173.). In the ΔcdsR mutant, a strong promoter was screened to guide the expression of the cry gene, and an engineered strain was constructed to improve the performance of Bacillus thuringiensis in field applications, making it a more effective and sustainable product. When analyzing the total protein of the ΔcdsR strain, it was found that compared with the wild-type HD73 strain, the expression of a protein with a molecular weight of about 80KDa was significantly increased. According to the results of mass spectrometry, the protein is InhA1 (immune inhibitor A1), a metalloprotease of the M6 ​​family, which contains a highly conserved zinc binding sequence and is similar to many zinc-containing proteases from pathogens. Therefore, it has a high resistance to the humoral defense system of some insects. It is a virulence factor of Bacillus thuringiensis and plays an important role in enhancing the insecticidal activity of Cry proteins (GRANDVALET C, GOMINET M, LERECLUSD. Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation [J]. Microbiology (Reading, England), 2001, 147 (Pt 7): 1805-13). So far, the function of InhA1 has not been fully resolved. InhA expression is activated at the end of logarithmic growth and is continuously secreted throughout the cell life cycle. In HD73, there are two regulatory factors in the upstream region of the inhA gene: SinI and SinR. SinI inhibits SinR activity, and inhA transcription is directly or indirectly inhibited by sinR.In addition, inhA1 is directly negatively regulated by AbrB (GRANDVALET C, GOMINET M, LERECLUSD. Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation[J]. Microbiology (Reading, England), 2001, 147(Pt 7): 1805-13). Summary of the invention

[0006] The present invention has discovered a strong promoter PinhA1, which selects 556bp upstream of the inhA1 gene (the inhA1 gene encodes a metalloproteinase). The inhA1 gene promoter PinhA1 can efficiently guide the expression of Cry protein in a ΔcdsR mutant of Bacillus thuringiensis.

[0007] Since the expression level of InhA1 in the ΔcdsR strain was increased, the results of the β-galactosidase assay showed that the transcription level of PinhA1 was significantly increased after T4 compared with the wild-type strain. Using the previously reported high-activity promoter P5014 as a control, in ΔcdsR, the transcriptional activity of PinhA1 was higher than that of P5014, and it was able to guide the expression of cry1Ac, and the expression level was comparable to that of the wild-type strain. As an excellent chassis strain, ΔcdsR is an important resource for the development of cell factories. PinhA1 has high transcriptional activity in the ΔcdsR strain and can be used to guide the expression of cry genes, providing new ideas for the construction of broad-spectrum Bt engineering bacteria.

[0008] The specific details of the present invention are to construct a ΔcdsR (PinhA1-1Ac) strain, in which PinhA1 guides the expression of the cry1Ac gene and produces a large amount of insecticidal crystal protein. And the insecticidal activity of this strain against the larvae of the diamondback moth is equivalent to that of the HD73 wild-type strain. The above results show that the non-cry gene promoter PinhA1 has the ability to efficiently express Cry protein in the ΔcdsR mutant, and has great application potential in constructing Bacillus thuringiensis engineering strains. The present invention provides a new strategy for improving crystal proteins or other insecticides in future agricultural production, and brings new hope for the further development and utilization of biological pesticides.

[0009] The nucleotide sequence of promoter PinhA1 is shown in SEQ ID NO:1.

[0010] An expression vector contains the promoter PinhA1 and a functional gene.

[0011] The functional gene is the cry gene.

[0012] The cry gene is cry1Ac gene.

[0013] The backbone vector is pHT315 vector, and the expression vector is named pHT315-PinhA1-1Ac.

[0014] An engineered strain is obtained by transferring the aspore-free mutant strain ΔcdsR into the above expression vector.

[0015] The expression vector is pHT315-PinhA1-1Ac, which contains the inhA1 promoter and the cry1Ac gene. The artificial strain is named ΔcdsR (PinhA1-1Ac) strain.

[0016] The above promoter PinhA1 is used to guide the efficient expression of functional genes in Bacillus thuringiensis.

[0017] The functional gene is the cry gene.

[0018] The cry gene is cry1Ac gene.

[0019] The Bacillus thuringiensis is a spore-free and crystal-free ΔcdsR mutant.

[0020] The present invention identifies a new non-cry strong promoter PinhA1, which is derived from the HD73 wild strain and can effectively guide the expression of cry1Ac. Studies have shown that the effect of PinhA1 in guiding the expression of Cry1Ac protein is equivalent to the Cry1Ac protein produced by the HD73 wild strain itself. This conclusion is supported by the following evidence: First, SDS-PAGE analysis shows that the production of Cry1Ac expressed under the guidance of the inhA1 gene promoter (PinhA1) in the ΔcdsR mutant is equivalent to the production expressed by the HD73 wild strain itself ( Figure 4 ). Secondly, the strain ΔcdsR (PinhA1-1Ac) carrying PinhA1-directed cry1Ac can also accumulate regular bipyramidal crystal proteins in mother cells ( Figure 5). Finally, in terms of insecticidal activity against Plutella xylostella larvae, the insecticidal activity of the Cry1Ac protein directed by PinhA1 in the ΔcdsR mutant was also similar to that of the Cry1Ac protein in the HD73 wild strain (Table 3). The present invention found that the PinhA1 promoter can also direct the efficient expression of the Cry1Ac protein. The PinhA1 promoter is derived from Bacillus thuringiensis, and therefore can also direct the expression of other cry genes, and can also direct the efficient expression of other functional genes in Bacillus thuringiensis.

[0021] In conclusion, the present invention provides a good example for using non-cry gene promoters to efficiently guide the efficient expression of Cry1Ac. This inhA1 gene promoter PinhA1 can also be used for the expression of other cry genes, providing important clues for future Bt strain improvement and using non-cry gene promoters to increase Cry protein production and insecticidal activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 InhA1 protein was abundantly expressed in the ΔcdsR mutant.

[0023] The figure shows the quantitative analysis of T in the ΔcdsR mutant. 16 The arrows in the figure indicate the InhA1 protein bands.

[0024] Figure 2 Comparison of inhA1 gene promoter transcriptional activity in HD73 and ΔcdsR.

[0025] The circles indicate the transcription of the strong promoter PinhA1 in the ΔcdsR mutant, and the squares indicate the transcription of the strong promoter PinhA1 in the HD73 wild-type strain. The sample cells were cultured in SSM medium at 30°C and 220 rpm, and the β-galactosidase activity of three monoclones was measured. Each value represents the mean and standard error of at least three independent replicates.

[0026] Figure 3 The transcriptional activities of PinhA1 promoter and the strongest promoter P5014 in ΔcdsR were compared and analyzed.

[0027] The circles represent the transcription of the strong promoter PinhA1 in the ΔcdsR mutant, and the squares represent the transcription of the strongest promoter P5014 in the ΔcdsR mutant. The sample cells were cultured in SSM medium at 30°C and 220 rpm, and the β-galactosidase activity of three monoclones was measured. Each value represents the mean and standard error of at least three independent replicates.

[0028] Figure 4 The strong PinhA1 promoter directs the efficient expression and accumulation of Cry1Ac.

[0029] The figure shows SDS-PAGE analysis of the expression levels of Cry1Ac protein in the HD73 wild-type strain, ΔcdsR mutant strain, and ΔcdsR (PinhA1-1Ac) strain. The abbreviation PinhA1 is used in this figure to indicate the ΔcdsR (PinhA1-1Ac) strain. The values ​​marked below the figure were obtained by analysis using ImageJ software and represent the relative multiples of Cry1Ac protein production.

[0030] Figure 5 Optical microscopy was used to observe the ability of the strong promoter PinhA1 in the ΔcdsR mutant to direct the expression of Cry1Ac crystals. In the figure, the HD73 wild-type strain, the ΔcdsR mutant strain, and the ΔcdsR (PinhA1-1Ac) strain were cultured in SSM medium at 30°C until T 16 、T 24 and optical microscope photos (BX63) taken 3 days later, in which the arrows indicate the Cry1Ac protein crystals. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the embodiments.

[0032] Materials and methods:

[0033] 1. Bacterial strains, plasmids, and growth conditions

[0034] Bacillus thuringiensis Kurstaki HD73 (abbreviated as HD73 in this patent) (Lereclus D, Arantes O, Chaufaux J, Lecadet M (1989) Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS microbiology letters 51(1):211-217) was activated in 5 mL LB medium (1% NaCl, 0.5% yeast extract and 1% tryptone, solid LB medium needs to be supplemented with 1.5% agar). When detecting promoter activity, Schaeffer's sporulation medium (SCHAEFFER P, MILLET J, AUBERT J P. Catabolic repression of bacterial sporulation [J]. Proceedings of the National Academy of Sciences of the United States of America, 1965, 54 (3): 704-11.) was selected, wherein the components of SSM medium are 0.8% nutrient broth, 0.012% MgSO4, 0.1% KCl, 0.01μM MnCl2, 0.05μM CaCl2, 1μM FeSO4. The Escherichia coli JM109 strain used in the experiment was used for routine molecular cloning experiments, while the ET1 strain was used to obtain nonmethylated plasmid DNA for successful transformation into Bacillus thuringiensis (MACALUSO A, METTUS A M. Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA [J]. J Bacteriol, 1991, 173 (3): 1353-6., WANG G, ZHANG J, SONG F, et al. Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests [J]. Applied microbiology and biotechnology, 2006, 72 (5): 924-30.). Escherichia coli were cultured in LB medium at 37°C and 220 rpm.Antibiotics and concentrations used for the growth of Bacillus thuringiensis: erythromycin 5 μg / ml, kanamycin 50 μg / ml. Antibiotics and concentrations used for the growth of Escherichia coli: ampicillin 100 μg / ml. The bacterial strains and plasmids used in this study are summarized in Table 1. The strains and plasmids are available for public distribution.

[0035] Table 1 Strains and plasmids used in this study

[0036]

[0037]

[0038] a Antibiotic resistance is as follows: Erm R , erythromycin resistance; Amp R , ampicillin resistance.

[0039] [1]DU C,NICKERSON K W. Bacillus thuringiensis HD-73 spores havesurface-localized Cry1Ac toxin:Physiological and pathogenic consequences[J].Applied and environmental microbiology,1996,62(10):3722-6.

[0040] [2]ZHANG

[0041] [3]AGAISSE H, LERECLUS D. Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis[J]. Molecular Microbiology, 1994, 13(1):97-107.

[0042] [4]ARANTES O,LERECLUS D.Construction of cloning vectors for Bacillusthuringiensis[J].Gene,1991,108(1):115-9.

[0043] 2. DNA manipulation and transformation

[0044] Plasmid DNA was extracted from E. coli cells using a plasmid miniprep kit (Omega, Guangzhou, China), and restriction enzymes (Baoriyi Biotechnology Co., Ltd., Beijing, China) and seamless cloning enzymes (Novozyme Biotechnology Co., Ltd., Nanjing, China) were used for digestion and ligation according to the instructions. PCR amplification was performed using P520 high-fidelity DNA polymerase (Novozyme Biotechnology Co., Ltd., Nanjing, China) and ordinary Taq DNA polymerase (Novozyme Biotechnology Co., Ltd., Nanjing, China). DNA fragments were recovered from 1% agarose gel using a DNA gel recovery kit (Omega, Guangzhou, China). The transformation of Escherichia coli was carried out by heat shock method (Sambrook BJ, Russell DW (2015) Molecular Cloning A Laboratory Manual. 3rd ed edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), and the transformation of Bacillus thuringiensis was carried out by electroporation method (Lereclus D, Arantes O, Chaufaux J, Lecadet M (1989) Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS microbiology letters 51 (1): 211-217.)

[0045] 3. Construction of PinhA1 promoter and lacZ fusion vector

[0046] In order to analyze the transcriptional activity of the inhA1 promoter in the ΔcdsR mutant and the HD73 wild strain, the sequence of the inhA1 promoter was amplified from the HD73 strain. The inhA1 promoter (PinhA1) is a 556 bp sequence located upstream of the start codon of the inhA1 gene. The promoter is named PinhA1, and its nucleotide sequence is shown in SEQ ID NO: 1 from position 1 to 556, and is amplified using specific primers PinhA1-F / PinhA1-R (Table 2). It was then fused to the pHT304-18Z vector containing the lacZ gene through two restriction sites, PstI and BamHI (AGAISSE H, LERECLUSD. Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis [J]. Molecular Microbiology, 1994, 13 (1): 97-107.). The recombinant plasmid pHT304-PinhA1 was transformed into the wild type HD73 and the ΔcdsR mutant by electroporation (Gene Pulser II Apparatus, BIO-RAD). The positive strains of the transformed HD73 (PinhA1-lacZ) and ΔcdsR (PinhA1-lacZ) were screened for erythromycin resistance and then identified by PCR, and finally the correct strains were obtained for preservation.

[0047] Table 2 Specific primers used in this application

[0048]

[0049] a Restriction enzyme sites are underlined

[0050] 4. Construction of inhA1 promoter and cry1Ac gene fusion vector

[0051] The PinhA1 promoter was fused with the cry1Ac gene by overlapping PCR. The specific primers pHTinhA1-F / pHTinhA1-R and pHT1Ac-F / pHT1Ac-R (see Table 2) were used to amplify the PinhA1 promoter and cry1Ac gene fragments respectively (the gene accession number of the cry1Ac gene is AAB46989.1 (WONG HC, SCHNEPF HE, WHITELEY HR. Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene [J]. The Journal of biological chemistry, 1983, 258 (3): 1960-7.)). The specific PCR product obtained by amplification was recovered by gel recovery and used as a template for overlapping extension PCR using primers pHTPinhA1-F / pHT1Ac-R. The obtained fusion fragment PinhA1-cry1Ac was constructed into the pHT315 vector through the SalI and SphI sites to obtain the pHT315-PinhA1-1Ac plasmid. After being verified as correct by sequencing, it was transferred into the ΔcdsR mutant by electroporation. The constructed ΔcdsR (PinhA1-1Ac) strain was identified by erythromycin resistance screening and PCR method.

[0052] 5. β-galactosidase activity assay

[0053] To determine the β-galactosidase activity, Bacillus thuringiensis strains HD73 (PinhA1-lacZ) and ΔcdsR (PinhA1-lacZ) were inoculated into SSM medium and cultured at 30°C with shaking at 220 rpm. -2 Collect 2 ml of bacterial solution from each well until T9 (T0 indicates the end of the exponential growth phase, T nIndicates the nth hour after T0). The bacterial solution was centrifuged for 1 min, the supernatant was discarded and the precipitate was stored in a -40°C refrigerator for later use. The method for determining β-galactosidase activity is shown in (YANG H, WANGP, PENG Q, et al. Weak transcription of the cry1Ac gene in nonsporulating Bacillus thuringiensis cells [J]. Applied and environmental microbiology, 2012, 78 (18): 6466-74.), and the internationally unified Miller unit was used (MILLER J H. Experiments in Molecular Genetics [Z]. Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press. 1972).

[0054] 6. Analysis of Cry protein production

[0055] Bacillus thuringiensis HD73, ΔcdsR (PinhA1-1Ac), and ΔcdsR monoclonal were selected and inoculated into 5 mL LB medium to activate the strain. 1% of the activated strain was inoculated into 100 mL SSM liquid medium and grown at 30°C and 220 rpm until T 24 During the period, 1 mL of culture solution was centrifuged at 12000 rpm for 1 min to collect the cells. 300 μL of ddH2O was added to each sample for re-suspending, 50 μg of quartz sand (diameter 0.1 mm) was added to the cell re-suspended solution, and the cells were shaken for 3 min 30 s as total protein samples. 100 μL of 4×SDS-PAGE Loading Buffer was added, the cells were heated in a water bath for 10 min, and centrifuged at 12000 rpm for 1 min. The production of Cry protein was then detected by SDS-PAGE (4% polyacrylamide concentrated gel, 10% polyacrylamide separation gel, sample volume 8 μL) and Coomassie Brilliant Blue staining. The production was the yield of each strain at T 24 The amount of Cry protein contained in 26.7 μL bacterial solution at period.

[0056] 7. Determination of insecticidal activity

[0057] 1% of HD73, ΔcdsR (PinhA1-1Ac), and ΔcdsR strains were inoculated into 100 mL of SSM medium and cultured at 30°C and 220 rpm until T 24 At each stage, 20 mL of fermentation liquid was taken as the mother liquid and diluted to 1×10 6 PPM, 1×104 PPM, 5×10 3 PPM, 2.5×10 3 PPM, 2×10 3 PPM, 1.25×10 3 PPM, 1×10 3 PPM, 1×10 2 PPM, 10PPM, with sterile water as control, after adding 0.1% adhesive, evenly spread on cabbage leaves with a diameter of 6cm, put into a 9cm culture dish with a layer of absorbent paper and sprayed with water to keep it moist, and inoculate 30 second-instar Plutella xylostella larvae (XUE J, LIANG G, CRICKMORE N, et al. Cloning and characterization of a novel Cry1A toxin from Bacillus thuringiensis with high toxicity to the Asian cornborer and other lepidopteran insects [J]. FEMS microbiology letters, 2008, 280 (1): 95-101.) for each treatment, seal tightly and culture in a 25℃ incubator, count the number of dead insects after 48h, and calculate LC50 using Probit Analysis. Five replicates were performed for each concentration.

[0058] result

[0059] 1. InhA1 protein is highly expressed in the ΔcdsR mutant

[0060] Previous studies have found that the ΔcdsR mutant has many advantages, such as cell lethality and lack of spore formation, and has the potential to serve as an excellent chassis strain (ZHANG X, CHEN Y, YAN T, et al. Cell death dependent on holins LrgAB repressed by a novel ArsR family regulator CdsR[J]. Cell death discovery, 2024, 10(1): 173.). In order to compare the differences in total protein bands between the ΔcdsR mutant and the HD73 wild-type strain, SDS-PAGE detection revealed that the protein bands of the ΔcdsR mutant strain were quite different from those of the HD73 wild-type strain, especially a highly expressed protein band at about 80 kDa ( Figure 1). After protein general detection, it was found that the protein band with the largest coverage was InhA1 protein (coverage was 53%), which was annotated as M6 family metalloproteinase immunosuppressant InhA1.

[0061] 2. The transcriptional activity of inhA1 gene in ΔcdsR is much higher than that in HD73

[0062] To investigate the transcription of the inhA1 gene in the ΔcdsR mutant and HD73 wild type, firstly, the -556bp upstream of the inhA1 gene was selected as the promoter region of the inhA1 gene (base A in the start codon ATG was defined as +1), which was defined as the PinhA1 promoter. Subsequently, the inhA1 gene promoter and the lacZ reporter gene were constructed into a fusion expression vector (pHT304-PinhA1-lacZ) and transferred into the ΔcdsR mutant and HD73 wild type strains respectively ( Figure 2 A) to obtain ΔcdsR (PinhA1-lacZ) and HD73 (PinhA1-lacZ) strains. β-galactosidase reporter system detection found that the transcriptional activity of the inhA1 promoter in HD73 wild-type cells was -1 There was no transcriptional activity at T1, and the transcriptional activity gradually increased from T2 to T7, reaching a peak at T7 (about 81253U / mg protein). Then, the transcriptional activity of the PinhA1 promoter gradually decreased from T7 to T9. The transcriptional activity of the PinhA1 promoter in the ΔcdsR mutant was compared with that in the HD73 wild type. -1 There was no difference between the two until T4, but after T5, the transcriptional activity of PinhA1 promoter in ΔcdsR mutant was significantly higher than that in HD73 wild type, and the transcriptional activity in ΔcdsR mutant reached the peak at T8 (about 169726U / mg protein), and then began to decline ( Figure 2 (B). Interestingly, we also found that the peak transcriptional activity of PinhA1 was much higher in the ΔcdsR mutant than in HD73. In summary, the experimental results show that the transcription amount of the inhA1 gene in the ΔcdsR mutant is higher than that in the HD73 wild type.

[0063] 3. The PinhA1 promoter has extremely high transcriptional activity in the ΔcdsR mutant

[0064] To explore the reason why the inhA1 gene has extremely high expression in the ΔcdsR mutant, we selected the PinhA1 promoter and the P5014 strong promoter, which is one of the strongest promoters in Bacillus thuringiensis, for comparative analysis. The expression vectors of the PinhA1 promoter and the P5014 strong promoter fused with the lacZ reporter gene were respectively transferred into the ΔcdsR mutant. The results of the β-galactosidase reporter system showed that the transcriptional activity of the PinhA1 promoter in the ΔcdsR mutant was significantly higher than that of the P5014 strong promoter ( Figure 3 ). This experiment shows that the PinhA1 promoter has extremely high transcriptional activity in the ΔcdsR mutant. Therefore, we speculate that the PinhA1 promoter may have extremely high application potential in the ΔcdsR mutant.

[0065] 4. PinhA1 gene promoter directs cry1Ac expression and accumulation

[0066] In order to test whether the PinhA1 promoter can guide the efficient expression of cry1Ac in the Bacillus thuringiensis ΔcdsR mutant, we first constructed the pHT315-PinhA1-1Ac plasmid, which contains the PinhA1-cry1Ac fusion fragment ( Figure 4 A). The pHT315-PinhA1-1Ac fusion expression vector was introduced into the ΔcdsR mutant strain to successfully obtain the ΔcdsR (PinhA1-1Ac) strain. In order to confirm whether the ΔcdsR (PinhA1-1Ac) strain can express Cry1Ac protein in large quantities, we tested the Cry protein production of the ΔcdsR (PinhA1-1Ac) strain to determine whether the PinhA1 promoter can efficiently guide the expression of Cry1Ac protein and is suitable for production applications. The strain was first cultured in SSM medium until T 24 The cells were then collected and proteins were extracted for detection. SDS-PAGE experiments showed that both ΔcdsR (PinhA1-1Ac) and HD73 produced Cry1Ac proteins of ~130 kDa in size, and the protein yield of ΔcdsR (PinhA1-1Ac) was significantly greater than that of HD73 ( Figure 4 (B) This result indicates that the inhA1 gene promoter can correctly and efficiently guide the expression of the cry1Ac gene.

[0067] To further confirm the formation of Cry protein crystals, the obtained strains were observed by optical microscopy (BX63). The results showed that compared with ΔcdsR, both ΔcdsR(PinhA1-1Ac) and HD73 strains could produce obvious crystal inclusions. 16 (Wild strains do not lyse during this period) and T24 (The wild strain lyses during this period) can form typical bipyramidal crystals and ΔcdsR still does not lyse after 3 days. The ΔcdsR (PinhA1-1Ac) strain also does not lyse. Its cell wall can effectively wrap the crystals ( Figure 5 ). These results indicate that the inhA1 gene promoter can correctly and efficiently direct the expression of the cry1Ac gene without affecting the formation of the bipyramidal crystal structure, and that the ΔcdsR mutant, as a chassis strain, can protect the crystals by not breaking down its cell wall. Most importantly, the ability of the PinhA1 promoter to direct the expression of Cry protein crystals is comparable to the amount of Cry protein produced by the HD73 wild strain itself, and this promoter has great potential for application in industrial production applications.

[0068] 5. Cry1Ac expressed under the guidance of PinhA1 promoter has insecticidal activity

[0069] Compared with the ΔcdsR mutant, the ΔcdsR(PinhA1-1Ac) strain and the HD73 wild type had obvious accumulation of Cry1Ac protein, and the amount of Cry1Ac protein accumulated in the ΔcdsR(PinhA1-1Ac) strain was comparable to that in the HD73 wild type ( Figure 4 and Figure 5 ). So, is the insecticidal activity of the Cry1Ac protein expressed under the guidance of the PinhA1 promoter also comparable to that of the HD73 wild type? We fed second-instar diamondback moth larvae with cabbage treated with the ΔcdsR (PinhA1-1Ac) strain, the HD73 wild type, and the ΔcdsR mutant strain, and then calculated the half-lethal concentration (LC50). The results showed that the half-lethal concentration of the ΔcdsR (PinhA1-1Ac) strain was similar to that of the HD73 wild type, while the ΔcdsR mutant strain had no insecticidal activity (Table 3). This shows that the Cry1Ac expressed under the guidance of the PinhA1 promoter has good insecticidal activity, and the ΔcdsR (PinhA1-1Ac) strain can be used as a good candidate biological control agent.

[0070] Table 3 Insecticidal activity of Bacillus thuringiensis strains against Plutella xylostella

[0071] strain <![CDATA[Lethal concentration 50% LC 50 (PPM)]]> 95% Confidence Interval ΔcdsR(PinhA1-1Ac) 1020.23 645.74-1546.27 HD73 1042.33 681.69-1491.75 ΔcdsR NA NA

[0072] The Cry1Ac protein expressed under the guidance of the PinhA1 promoter of the present invention not only has an expression amount similar to that of the HD73 wild-type strain, but also has similar insecticidal activity. The PinhA1 promoter can be used to guide the efficient expression of Cry proteins, in order to obtain more efficient Bacillus thuringiensis engineered bacteria.

Claims

1. Promoter PinhA1, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. An expression vector comprising the above promoter PinhA1 and a functional gene.

3. The expression vector according to claim 2, wherein the functional gene is a cry gene. The expression vector according to claim 3 , wherein the cry gene is cry1Ac gene. 5 . The expression vector according to claim 4 , wherein the backbone vector is pHT315 vector, and the expression vector is named pHT315-PinhA1-1Ac.

6. An engineered bacterial strain obtained by transferring the aspore-free mutant strain ΔcdsR into the expression vector according to any one of claims 2 to 5. 7 . The engineered bacterial strain according to claim 6 , wherein the expression vector is pHT315-PinhA1-1Ac, which contains the inhA1 promoter and the cry1Ac gene, and the artificial strain is named ΔcdsR (PinhA1-1Ac) strain.

8. Use of the promoter PinhA1 according to claim 1 in directing the efficient expression of functional genes in Bacillus thuringiensis.

9. The use according to claim 8, wherein the functional gene is a cry gene. 10 . The use according to claim 9 , wherein the cry gene is cry1Ac gene, and the Bacillus thuringiensis is a ΔcdsR mutant.

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