CN115029369B - Preparation method and application of bacillus thuringiensis engineering bacteria for preventing and treating pine wood nematode disease - Google Patents

Abstract

The invention relates to a bacillus thuringiensis efficient expression vector, engineering bacteria, and a preparation method and application thereof. The constructed bacillus thuringiensis expression vector can efficiently express exogenous proteins in host cells. The constructed engineering strain has nematicidal capacity and masson pine in-vivo colonization capacity.

Description

Preparation method and application of bacillus thuringiensis engineering bacteria for preventing and treating pine wood nematode disease

Technical Field

The invention relates to the technical field of biology, in particular to a bacillus thuringiensis efficient expression vector, engineering bacteria, and a preparation method and application thereof.

Background

Pine wood nematode disease (Pine wilt disease) is an international major quarantine disease, is the first disease in forestry in China, has pathogenic effect of pine wood nematode (Bursaphelenchus xylophilus) (Limophelia and Zhang Xingyao, 2018) and has spread to 17 provinces (district, city) since invasion into China in 1982 (national forestry and grassland office bulletin (No. 5 of 2021)), causing large-area pine tree death, bringing about huge economic and ecological losses and seriously threatening the safety of the ecological system in China. At present, the microbial pesticide is used for biological control of pine wood nematodes, is not only suitable for aviation spraying and application, but also is environment-friendly, and can well meet the large-scale prevention and control requirements, so that the pine wood nematodes are paid more attention to.

Bacillus thuringiensis (Bacillus thuringiensis, bt) products are currently the most widely used microbial pesticides in the world. At the same time as the cells form spores, the cells produce companion crystals (Insecticidal Crystal Proteins, ICPs) at one or both ends of the cells, which are released outside the cells upon release of the spores. The companion crystal is formed mainly by Cry toxins, the largest class of insecticidal proteins produced by Bacillus. Nematicidal companion crystal proteins mainly include three subclasses: the Cry5 subclass (Cry 5B, cry5C, cry5E, cryl2A, cryl3A, cryl4A, cry A and Cry 21B), the Cry6 subclass (Cry 6A and Cry 6B) and the Cry55 subclass (Cry 55A) (Pinos et al, 2021, yu et al, 2015, xiang-Qian et al, 2008). Cry, when ingested by nematodes, breaks their intestinal tracts, forming vacuolated structures in the intestinal cells, causing intestinal contractions to separate from the body wall, and also can form perforations in the cell membrane leading to nematode death (Pinos et al 2021). Based on the specific insecticidal activity of the companion crystal nematodes, the Bt microbial inoculum and Cry transgenic plants show good control effects in the control of root-knot nematodes, cyst nematodes and pine wood nematodes, and have wide application prospects (Yu et al, 2015, li et al, 2007). Several Bt strains have been reported to have a significant lethal effect on pine wood nematodes (rowland et al, 2007, xu Huachao et al, 2010), and demonstrate a significant synergistic effect when used in combination with the use of the strain esceya vermicola as the line-killing fungus strain of i (Li Enjie et al, 2019). Three Cry crystal proteins Cry1Ea, cry5Ba and Cry6A have higher toxicity to pine wood nematodes (Huang et al, 2018, lei Mengying, 2015, wang Diandong, etc. 2013), and the botrytis cinerea transformed into Cry6A gene has obvious inhibition effect on pine wood nematodes by genetic engineering means.

Parasitic harm exists in pine stems of pine nematodes, so Bt is applied to control of pine nematodes, and not only has nematicidal activity, but also can adapt to a woodland ecological system and colonize the pine bodies. The research and development of Bt engineering bacteria provides a way for solving the problem, and Bacillus thuringiensis G033A is the first engineering bacteria for obtaining pesticide registration evidence in China, is also the first Bt product for preventing and controlling coleopteran pests, and has high toxicity to lepidopteran pests. The cry3Aa7 gene promoter and the coding region containing target coleopteran pests are constructed to recombinant plasmid Pstk-3A, and the plasmid is introduced into a strain G03 with high toxicity to the lepidopteran pests to obtain Bt engineering bacteria G033A, so that engineering bacteria G033A capable of simultaneously controlling the lepidopteran pests and the coleopteran pests is obtained, and the insecticidal spectrum is enlarged (Wang Yuhang and the like, 2020). The research and development and industrialization of bacillus thuringiensis G033A provide an important reference for the research of preventing and treating pine wood nematode diseases by using Bt. By using genetic engineering means and taking widely existing Bt in pine tree as an initial strain, the Bt expresses the nematicidal Cry protein, thereby obtaining the Bacillus thuringiensis engineering strain with the capability of killing pine wood nematodes and the capability of colonizing pine tree bodies, and having wide application prospect in preventing and treating pine wood nematode diseases.

Disclosure of Invention

In response to the problems identified above, one aspect of the present application provides one or more bacillus thuringiensis expression vectors.

In another aspect, the present application provides Bacillus thuringiensis engineering strains comprising the expression vectors.

In another aspect, the present application provides methods of preparing the expression vectors or engineered strains.

In another aspect, the present application provides the use of the expression vector or engineered strain for one or more of the following:

(1) Use of sfGFP to express green fluorescent protein;

(2) Use in expressing a nematicidal Cry protein;

(3) Application in killing nematodes;

(4) The application in preventing and/or treating pine wood nematode disease.

Effects achieved by the present application include, but are not limited to:

the bacillus thuringiensis expression vector constructed by the application can efficiently express exogenous proteins in host cells, such as green fluorescent protein sfGFP or nematicidal protein Cry6A;

the application utilizes the high-efficiency expression vector to construct engineering strain Bxy P3C6 in wild Bxy strain, expresses nematicidal protein Cry6A, has obvious nematicidal capacity, and can colonize and spread in a masson pine body at a certain distance by spraying and injecting two inoculation means.

The engineering strain has nematicidal capacity and masson pine in-vivo colonization capacity.

Drawings

Fig. 1: PCR detection of Bxy sfGFP, bxy19P3C6

Fig. 2: fluorescent microscopic observation of pP3A-sfgfp expression protein

Fig. 3: SDS-PAGE detection of protein expression

Fig. 4: morphology observation of Bxy P3C6 trophic crystallins

Fig. 5: determination of nematicidal activity of engineering bacterium Bxy P3C6

Fig. 6: morphology observation of pine wood nematodes

Fig. 7: determination of the colonization Capacity of the Strain according to the present application

Detailed Description

In order to make the technical scheme and the beneficial effects of the invention more obvious and understandable, the following detailed description is given by way of example. Unless otherwise defined, technical and scientific terms used herein have the same meaning as technical and scientific terms in the technical field to which this application belongs.

In one aspect, the present application provides one or more expression vectors.

In some embodiments, the expression vector is a vector pP3A-sfGFP, the sequence of which is shown in SEQ ID NO. 1, that directs the expression of green fluorescent protein sfGFP.

In some embodiments, the expression vector pP3A-sfgfp is prepared by:

the recombinant plasmid pP3A-sfGFP for guiding the high-efficiency expression of the green fluorescent protein sfGFP in bacillus thuringiensis is constructed by utilizing a promoter (GenBank: AJ 237900.1) of Cry3Aa gene for regulating and controlling the expression of Cry proteins in the nutrition period of the bacillus thuringiensis.

In some embodiments, the expression vector is a vector pP3A-Cry6A directing expression of nematicidal protein Cry6A, the sequence of which is shown in SEQ ID NO. 2.

In some embodiments, the expression vector pP3A-cry6a is prepared by:

(1) Constructing a recombinant plasmid pP3A-sfGFP for guiding the efficient expression of green fluorescent protein sfGFP in bacillus thuringiensis by utilizing a promoter (GenBank: AJ 237900.1) of Cry3Aa gene for regulating and controlling the expression of Cry proteins in the nutrition period of the bacillus thuringiensis;

(2) The sfgfp gene is replaced by the nematicide Cry6A gene by homologous recombination means to obtain the expression vector shown in SEQ ID NO. 2.

In another aspect the present application provides an engineered strain comprising the one or more expression vectors.

In some embodiments, the engineered strain is obtained by introducing an expression vector described herein into a host cell.

In some embodiments, the host cell is a bacillus thuringiensis, e.g., wild-type Bxy, 19 bacillus thuringiensis, such as bacillus thuringiensis Bxy, deposited with the China general microbiological culture Collection center under the accession number CGMCC No. 24656.

In some embodiments, the engineered strain is an engineered strain Bxy sfGFP comprising the expression vector pP3A-sfGFP.

In some embodiments, the engineered strain is an engineered strain Bxy P3C6 comprising the expression vector pP3A-cry6a.

In another aspect, the present application provides the use of the expression vector or engineered strain for one or more of the following:

(1) Use of sfGFP to express green fluorescent protein;

(2) Use in expressing a nematicidal Cry protein;

(3) Application in killing nematodes;

(4) The application in preventing and/or treating pine wood nematode disease.

In some embodiments, the expression vector pP3A-sfGFP or engineered strain Bxy19sfGFP is capable of expressing the green fluorescent protein sfGFP with high efficiency.

In some embodiments, the expression vector pP3A-Cry6A or engineered strain Bxy19P3C6 is capable of highly expressing a nematicidal Cry protein, such as a nematicidal Cry6A protein.

In some embodiments, the expression vector pP3A-cry6a or engineered strain Bxy19P3C6 is used to kill nematodes.

In some embodiments, the expression vector pP3A-cry6a or engineered strain Bxy19P3C6 is used for preventing and/or treating pine wood nematode disease.

On the other hand, the application provides bacillus thuringiensis Bxy which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 24656 and the preservation time of 2022, 04 and 08.

The sequence information referred to in this application is as follows:

SEQ ID NO:1：

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattccttaaggaacgtacagacggcttaaaagcctttaaaaacgtttttaaggggtttgtagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaacctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataaaaaaagcacctgaaaaggtgtctttttttgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttattttagttgctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgttaaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaatcaagggttttagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaacttctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaaagaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtcacaaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaagaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattaataaaaatcttgttggttttatgcgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaagaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagttcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacggattttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtggtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccgatgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgttgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctccaaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaagaaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttggtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggacaaccggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgcttacacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcccaaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaaggaccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgacgagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaactttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtggatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagtaattatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatgagcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggcattgccagtcggggatattaaaaagagtataggtttttattgggataaagtaggtttcactttggttcaccatgaagatggattcgcagttctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgcctcgtagtaatgattcaccggtttgtacaggtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggcattttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagcttttttcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctcttgtatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaattttattcttgctgagtctggctttcggtaagctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgcgcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttatgcagtttgtagaatgcaaaaagtgaaatcagctggactaaaaggcatgcaatttcataatcaaagagagcgaaaaagtagaacgaatgatgatattgaccatgagcgaacacgtgaaaattatgatttgaaaaatgataaaaatattgattacaacgaacgtgtcaaagaaattattgaatcacaaaaaacaggtacaagaaaaacgaggaaagatgctgttcttgtaaatgagttgctagtaacatctgaccgagatttttttgagcaactggatccagcttaattaaagataatatctttgaattgtaacgcccctcaaaagtaagaactacaaaaaaagaatacgttatatagaaatatgtttgaaccttcttcagattacaaatatattcggacggactctacctcaaatgcttatctaactatagaatgacatacaagcacaaccttgaaaatttgaaaatataactaccaatgaacttgttcatgtgaattatcgctgtatttaattttctcaattcaatatataatatgccaatacattgttacaagtagaaattaagacacccttgatagccttactatacctaacatgatgtagtattaaatgaatatgtaaatatatttatgataagaagcgacttatttataatcattacatatttttctattggaatgattaagattccaatagaatagtgtataaattatttatcttgaaaggagggatgcctaaaaacgaagaacattaaaaacatatatttgcaccgtctaatggatttatgaaaaatcattttatcagtttgaaaattatgtattatgataagaaagggaggaagaaaaatgagcaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtccgtggagagggtgaaggtgatgctacaaacggaaaactcacccttaaatttatttgcactactggaaaactacctgttccgtggccaacacttgtcactactctgacctatggtgttcaatgcttttcccgttatccggatcacatgaaacggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaacgcactatatctttcaaagatgacgggacctacaagacgcgtgctgaagtcaagtttgaaggtgatacccttgttaatcgtatcgagttaaagggtattgattttaaagaagatggaaacattcttggacacaaactcgagtacaactttaactcacacaatgtatacatcacggcagacaaacaaaagaatggaatcaaagctaacttcaaaattcgccacaacgttgaagatggttccgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtcgacacaatctgtcctttcgaaagatcccaacgaaaagcgtgaccacatggtccttcttgagtttgtaactgctgctgggattacacatggatgagctctacaaatagaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

SEQ ID NO:2：

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattccttaaggaacgtacagacggcttaaaagcctttaaaaacgtttttaaggggtttgtagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaacctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataaaaaaagcacctgaaaaggtgtctttttttgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttattttagttgctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgttaaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaatcaagggttttagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaacttctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaaagaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtcacaaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaagaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattaataaaaatcttgttggttttatgcgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaagaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagttcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacggattttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtggtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccgatgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgttgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctccaaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaagaaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttggtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggacaaccggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgcttacacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcccaaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaaggaccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgacgagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaactttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtggatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagtaattatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatgagcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggcattgccagtcggggatattaaaaagagtataggtttttattgggataaagtaggtttcactttggttcaccatgaagatggattcgcagttctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgcctcgtagtaatgattcaccggtttgtacaggtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggcattttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagcttttttcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctcttgtatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaattttattcttgctgagtctggctttcggtaagctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgcgcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttatgcagtttgtagaatgcaaaaagtgaaatcagctggactaaaaggcatgcaatttcataatcaaagagagcgaaaaagtagaacgaatgatgatattgaccatgagcgaacacgtgaaaattatgatttgaaaaatgataaaaatattgattacaacgaacgtgtcaaagaaattattgaatcacaaaaaacaggtacaagaaaaacgaggaaagatgctgttcttgtaaatgagttgctagtaacatctgaccgagatttttttgagcaactggatccagcttaattaaagataatatctttgaattgtaacgcccctcaaaagtaagaactacaaaaaaagaatacgttatatagaaatatgtttgaaccttcttcagattacaaatatattcggacggactctacctcaaatgcttatctaactatagaatgacatacaagcacaaccttgaaaatttgaaaatataactaccaatgaacttgttcatgtgaattatcgctgtatttaattttctcaattcaatatataatatgccaatacattgttacaagtagaaattaagacacccttgatagccttactatacctaacatgatgtagtattaaatgaatatgtaaatatatttatgataagaagcgacttatttataatcattacatatttttctattggaatgattaagattccaatagaatagtgtataaattatttatcttgaaaggagggatgcctaaaaacgaagaacattaaaaacatatatttgcaccgtctaatggatttatgaaaaatcattttatcagtttgaaaattatgtattatgataagaaagggaggaagaaaaggtaccattataggtaagagaggaatgtacacatgattattgatagtaaaacgactttacctagacattcacttattcatacaattaaattaaattctaataagaaatatggtcctggtgatatgaccaatggaaatcaatttattatttcaaaacaagaatgggctactattggagcatatattcagactggattaggtttaccagtaaatgaacaacaattaagaacacatgttaatttaagtcaggatatatcaatacctagtgatttttctcaattatatgatgtttattgttctgataaaacttcagcagaatggtggaataaaaatttatatcctttaattattaaatctgctaatgatattgcttcatatggttttaaagttgctggtgatccttctattaagaaagatggatattttaaaaaattgcaagatgaattagataatattgttgataataattccgatgatgatgcaatagctaaagctattaaagattttaaagcgcgatgtggtattttaattaaagaagctaaacaatatgaagaagctgcaaaaaatattgtaacatctttagatcaatttttacatggtgatcagaaaaaattagaaggtgttatcaatattcaaaaacgtttaaaagaagttcaaacagctcttaatcaagcccatggggaaagtagtccagctcataaagagttattagaaaaagtaaaaaatttaaaaacaacattagaaaggactattaaagctgaacaagatttagagaaaaaagtagaatatagttttctattaggaccattgttaggatttgttgtttatgaaattcttgaaaatactgctgttcagcatataaaaaatcaaattgatgagataaagaaacaattagattctgctcagcatgatttggatagagatgttaaaattataggaatgttaaatagtattaatacagatattgataatttatatagtcaaggacaagaagcaattaaagttttccaaaagttacaaggtatttgggctactattggagctcaaatagaaaatcttagaacaacgtcgttacaagaagttcaagattctgatgatgctgatgagatacaaattgaacttgaggacgcttctgatgcttggttagttgtggctcaagaagctcgtgattttacactaaatgcttattcaactaatagtagacaaaatttaccgattaatgttatatcagattcatgtaattgttcaacaacaaatatgacatcaaatcaatacagtaatccaacaacaaatatgacatcaaatcaatatatgatttcacatgaatatacaagtttaccaaataattttatgttatcaagaaatagtaatttagaatataaatgtcctgaaagtaattttatgatatattggtataataattcggattggtataataattcggattggtataataattaaaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

example 1: construction of bacillus thuringiensis efficient expression vector

The recombinant plasmid pP3A-sfGFP for guiding the efficient expression of the green fluorescent protein sfGFP in bacillus thuringiensis is constructed by utilizing a promoter (GenBank: AJ 237900.1) of Cry3Aa gene for regulating and controlling the expression of Cry proteins in the nutrition period of the bacillus thuringiensis, and the plasmid is confirmed to be constructed correctly by sequencing, wherein the sequence is shown as SEQ ID NO. 1.

The plasmid pP3A-sfgfp is taken as a starting plasmid, and the sfgfp gene is replaced by a nematicide protein Cry6A gene (GenBank: DQ 835612.1) by homologous recombination means to obtain a recombinant plasmid pP3A-Cry6A for guiding the expression of nematicide protein Cry6A. The plasmid is sequenced to confirm that the plasmid is constructed correctly, and the sequence is shown as SEQ ID NO. 2.

Example 2: obtaining of fluorescent marker Strain Bxy, 19sfGFP and line-killing engineering Strain Bxy, 19P3C6

The correctly identified plasmid pP3A-sfGFP was introduced into the obtained pine endophyte bacillus thuringiensis Bxy (deposited with China general microbiological culture Collection center, with the preservation number of CGMCC No.24656, and the preservation time of 2022, 04 month and 08) and subjected to PCR detection to obtain a fluorescence marker strain Bxy19sfGFP (FIG. 1). Fluorescence microscopic observation shows that the promoter Pcry3A has higher transcriptional activity in bacillus thuringiensis Bxy (figure 2), and the recombinant plasmid pP3A-sfgfp can successfully express target proteins in bacillus thuringiensis Bxy.

The identified correct plasmid pP3A-cry6a was introduced into the endophyte Bacillus thuringiensis Bxy of pine, and the engineering strain Bxy P3C6 was obtained by PCR detection (FIG. 1).

Example 3: identification of expression of nematicide and observation of crystal morphology

Wild bacteria Bxy (deposited in China general microbiological culture Collection center with the preservation number of CGMCC No.24656 and the preservation time of 2022, 04 and 08) and engineering bacteria Bxy P3C6 are inoculated in a beef extract peptone liquid culture medium (NA) (peptone 10g/L, beef powder 3g/L, sodium chloride 5g/L and agar 20 g/L), and after culturing for 3 days in a shaking table at 28 ℃ at 220rpm, the thalli are collected by centrifugation, and are washed 3 times with sterile water and resuspended. The cell concentration was measured by OD600 and adjusted to be uniform by dilution with sterile water. The expression of crystallins in both samples was examined by SDS-PAGE. As can be seen from FIG. 3, the engineering bacterium Bxy P3C6 produced Cry6A protein with a molecular weight of about 54 kDa. Meanwhile, a small amount of collected engineering bacteria Bxy P3C6 is coated on a glass slide, naturally dried at room temperature, the glass slide is lightly baked for 6-8 times by an alcohol lamp, the carbolic acid reddish dye is washed for 1-2min, the dyeing liquid is washed clean by sterile water, and the glass slide is dried and observed by an oil mirror, so that rice grain crystals are observed at two ends of the thallus (figure 4).

Example 4: insecticidal Activity assay

Wild bacteria Bxy (preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24656 and the preservation time of 2022, 04 and 08) and engineering bacteria Bxy P3C6 are inoculated in NB culture medium, cultured in a shaking table at 28 ℃ for 24 hours at 220rpm, and then the cells are collected by centrifugation and washed 3 times with sterile water for resuspension. And (3) measuring the concentration of the thalli through OD600, diluting with sterile water to adjust the concentration of the thalli to be consistent, and centrifuging to remove the thalli to obtain a fermentation supernatant for detecting nematicidal activity. mu.L of supernatant, 10. Mu.L of nematode solution (about 1000 nematodes) and 4. Mu.L of chloramphenicol solution (1 mg/mL) were added to each well of a 96-well plate, and the mixture was subjected to dark culture at 25℃for 24 hours to count the mortality of nematodes. And the nematodes before and after the experiment were placed under a microscope to observe the morphology of the nematodes. The two strains were each subjected to 10 biological replicates. The results show that the supernatant of engineering bacteria Bxy19P3C6 has significant nematicidal activity, which is significantly higher than that of the wild type strain (fig. 5). Microscopic observation revealed that pine wood nematodes were intact in epidermis, but that intestinal cells were lysed, similar to the Cry6A perforation mechanism reported (fig. 6).

Example 5: determination of colonisation Capacity

Fluorescent marker strain Bxy sfGFP was inoculated into NB medium, incubated at 220rpm in a shaker at 28℃for 24 hours, and after centrifugation, the cells were collected and resuspended after 3 washes with sterile water. The cell concentration was determined by OD600 and adjusted to OD600 = 1.0 by dilution with sterile water for the colonization ability detection experiment. Bxy19sfGFP was inoculated into 3-year old pinus massoniana seedlings in the greenhouse by both spray inoculation and injection inoculation. In the spraying experiment, diluted thalli are uniformly sprayed on the surface of pinus massoniana seedlings by using a spray pot, 100mL of each pinus massoniana seedling is sprayed, 5 days, 10 days, 20 days and 30 days after spraying, the leaf part is sampled, meanwhile, the stem part is drilled to obtain the epidermis of the stem and the wood dust in the stem, and the colonization condition of the strain on the leaf part, the stem surface and the stem is detected by a PCR technology. In the injection experiment, electric rotation is utilized to drill holes in pinus massoniana seedling stems, the aperture is about 2mm, absorbent cotton is plugged in, a pipetting gun is used for injecting 40ul of diluted thalli, a sealing film is used for sealing the hole openings, drilling and sampling are carried out on seed points and positions 5cm and 10cm away from the seed points after 5 days, 10 days, 20 days and 30 days of injection inoculation, and wood chips are taken for carrying out PCR experiment to detect bacterial strain colonization. The results showed (FIG. 7) that the fluorescence-labeled Bxy sfGFP was allowed to survive on the leaf surface for 20 days, stem surface for 30 days or more, and inside the stem by spraying, for about 10 days, by spraying. By means of injection inoculation, the plant can survive for more than 30 days at an inoculation point, and can be transported and spread in a short distance, and can spread to a position 5cm away from the inoculation point after 5 days of injection inoculation, and can spread to a position 10cm away from the inoculation point after 10 days of injection inoculation. Strains spread over short distances can survive for more than 20 days.

It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the disclosure. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.

Sequence listing

<110> institute for ecological protection and repair of China forestry science institute

<120> a high-efficiency expression vector, engineering bacteria, and preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7678

<212> DNA

<213> Artificial Sequence

<400> 1

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccttaaggaa cgtacagacg 420

gcttaaaagc ctttaaaaac gtttttaagg ggtttgtaga caaggtaaag gataaaacag 480

cacaattcca agaaaaacac gatttagaac ctaaaaagaa cgaatttgaa ctaactcata 540

accgagaggt aaaaaaagaa cgaagtcgag atcagggaat gagtttataa aataaaaaaa 600

gcacctgaaa aggtgtcttt ttttgatggt tttgaacttg ttctttctta tcttgataca 660

tatagaaata acgtcatttt tattttagtt gctgaaaggt gcgttgaagt gttggtatgt 720

atgtgtttta aagtattgaa aacccttaaa attggttgca cagaaaaacc ccatctgtta 780

aagttataag tgactaaaca aataactaaa tagatggggg tttcttttaa tattatgtgt 840

cctaatagta gcatttattc agatgaaaaa tcaagggttt tagtggacaa gacaaaaagt 900

ggaaaagtga gaccatggag agaaaagaaa atcgctaatg ttgattactt tgaacttctg 960

catattcttg aatttaaaaa ggctgaaaga gtaaaagatt gtgctgaaat attagagtat 1020

aaacaaaatc gtgaaacagg cgaaagaaag ttgtatcgag tgtggttttg taaatccagg 1080

ctttgtccaa tgtgcaactg gaggagagca atgaaacatg gcattcagtc acaaaaggtt 1140

gttgctgaag ttattaaaca aaagccaaca gttcgttggt tgtttctcac attaacagtt 1200

aaaaatgttt atgatggcga agaattaaat aagagtttgt cagatatggc tcaaggattt 1260

cgccgaatga tgcaatataa aaaaattaat aaaaatcttg ttggttttat gcgtgcaacg 1320

gaagtgacaa taaataataa agataattct tataatcagc acatgcatgt attggtatgt 1380

gtggaaccaa cttattttaa gaatacagaa aactacgtga atcaaaaaca atggattcaa 1440

ttttggaaaa aggcaatgaa attagactat gatccaaatg taaaagttca aatgattcga 1500

ccgaaaaata aatataaatc ggatatacaa tcggcaattg acgaaactgc aaaatatcct 1560

gtaaaggata cggattttat gaccgatgat gaagaaaaga atttgaaacg tttgtctgat 1620

ttggaggaag gtttacaccg taaaaggtta atctcctatg gtggtttgtt aaaagaaata 1680

cataaaaaat taaaccttga tgacacagaa gaaggcgatt tgattcatac agatgatgac 1740

gaaaaagccg atgaagatgg attttctatt attgcaatgt ggaattggga acggaaaaat 1800

tattttatta aagagtagtt caacaaacgg gccagtttgt tgaagattag atgctataat 1860

tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct gaataagaac 1920

ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga aaagggaatg 1980

agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat tgttcatgaa 2040

attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg tgtttatggc 2100

tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg tgtcatgtca 2160

acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt ggaagtgaat 2220

tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga ttggccgctt 2280

acacatggtc aatttttctc tattttgccg atttatgatt caggtggata cttagagaaa 2340

gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat ttgtgccctt 2400

atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt gcaaggaccg 2460

acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat gttgattggt 2520

ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc agttaagcaa 2580

tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg tcaactttcc 2640

gactctgaga aacttctgga atcgctagag aatttctgga atgggattca ggagtggaca 2700

gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac gatgacctct 2760

aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt agtaattatc 2820

atggctgtca tggcgcatta acggaataaa gggtgtgctt aaatcgggcc attttgcgta 2880

ataagaaaaa ggattaatta tgagcgaatt gaattaataa taaggtaata gatttacatt 2940

agaaaatgaa aggggatttt atgcgtgaga atgttacagt ctatcccggc attgccagtc 3000

ggggatatta aaaagagtat aggtttttat tgggataaag taggtttcac tttggttcac 3060

catgaagatg gattcgcagt tctaatgtgt aatgaggttc ggattcatct atgggaggca 3120

agtgatgaag gctggcgcct cgtagtaatg attcaccggt ttgtacaggt gcggagtcgt 3180

ttattgctgg tactgctagt tgccgcattg aagtagaggg aattgatgaa ttatatcaac 3240

atattaagcc tttgggcatt ttgcacccca atacatcatt aaaagatcag tggtgggatg 3300

aacgagactt tgcagtaatt gatcccgaca acaatttgat tagctttttt caacaaataa 3360

aaagctaaaa tctattatta atctgttcag caatcgggcg cgattgctga ataaaagata 3420

cgagagacct ctcttgtatc ttttttattt tgagtggttt tgtccgttac actagaaaac 3480

cgaaagacaa taaaaatttt attcttgctg agtctggctt tcggtaagct agacaaaacg 3540

gacaaaataa aaattggcaa gggtttaaag gtggagattt tttgagtgat cttctcaaaa 3600

aatactacct gtcccttgct gatttttaaa cgagcacgag agcaaaaccc ccctttgctg 3660

aggtggcaga gggcaggttt ttttgtttct tttttctcgt aaaaaaaaga aaggtcttaa 3720

aggttttatg gttttggtcg gcactgccgc gcctcgcaga gcacacactt tatgaatata 3780

aagtatagtg tgttatactt tacttggaag tggttgccgg aaagagcgaa aatgcctcac 3840

atttgtgcca cctaaaaagg agcgatttac atatgagtta tgcagtttgt agaatgcaaa 3900

aagtgaaatc agctggacta aaaggcatgc aatttcataa tcaaagagag cgaaaaagta 3960

gaacgaatga tgatattgac catgagcgaa cacgtgaaaa ttatgatttg aaaaatgata 4020

aaaatattga ttacaacgaa cgtgtcaaag aaattattga atcacaaaaa acaggtacaa 4080

gaaaaacgag gaaagatgct gttcttgtaa atgagttgct agtaacatct gaccgagatt 4140

tttttgagca actggatcca gcttaattaa agataatatc tttgaattgt aacgcccctc 4200

aaaagtaaga actacaaaaa aagaatacgt tatatagaaa tatgtttgaa ccttcttcag 4260

attacaaata tattcggacg gactctacct caaatgctta tctaactata gaatgacata 4320

caagcacaac cttgaaaatt tgaaaatata actaccaatg aacttgttca tgtgaattat 4380

cgctgtattt aattttctca attcaatata taatatgcca atacattgtt acaagtagaa 4440

attaagacac ccttgatagc cttactatac ctaacatgat gtagtattaa atgaatatgt 4500

aaatatattt atgataagaa gcgacttatt tataatcatt acatattttt ctattggaat 4560

gattaagatt ccaatagaat agtgtataaa ttatttatct tgaaaggagg gatgcctaaa 4620

aacgaagaac attaaaaaca tatatttgca ccgtctaatg gatttatgaa aaatcatttt 4680

atcagtttga aaattatgta ttatgataag aaagggagga agaaaaatga gcaaaggaga 4740

agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca 4800

caaattttct gtccgtggag agggtgaagg tgatgctaca aacggaaaac tcacccttaa 4860

atttatttgc actactggaa aactacctgt tccgtggcca acacttgtca ctactctgac 4920

ctatggtgtt caatgctttt cccgttatcc ggatcacatg aaacggcatg actttttcaa 4980

gagtgccatg cccgaaggtt atgtacagga acgcactata tctttcaaag atgacgggac 5040

ctacaagacg cgtgctgaag tcaagtttga aggtgatacc cttgttaatc gtatcgagtt 5100

aaagggtatt gattttaaag aagatggaaa cattcttgga cacaaactcg agtacaactt 5160

taactcacac aatgtataca tcacggcaga caaacaaaag aatggaatca aagctaactt 5220

caaaattcgc cacaacgttg aagatggttc cgttcaacta gcagaccatt atcaacaaaa 5280

tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt cgacacaatc 5340

tgtcctttcg aaagatccca acgaaaagcg tgaccacatg gtccttcttg agtttgtaac 5400

tgctgctggg attacacatg gatgagctct acaaatagaa gcttggcgta atcatggtca 5460

tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 5520

agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 5580

cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5640

caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5700

tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5760

cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5820

aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5880

gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5940

agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 6000

cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 6060

cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 6120

ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 6180

gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 6240

tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 6300

acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 6360

tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 6420

attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 6480

gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 6540

ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6600

taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6660

ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6720

ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6780

gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6840

ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6900

gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 6960

tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 7020

atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 7080

gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 7140

tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 7200

atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 7260

agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 7320

ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 7380

tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 7440

aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 7500

tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 7560

aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 7620

accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 7678

<210> 2

<211> 8426

<212> DNA

<213> Artificial Sequence

<400> 2

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccttaaggaa cgtacagacg 420

gcttaaaagc ctttaaaaac gtttttaagg ggtttgtaga caaggtaaag gataaaacag 480

cacaattcca agaaaaacac gatttagaac ctaaaaagaa cgaatttgaa ctaactcata 540

accgagaggt aaaaaaagaa cgaagtcgag atcagggaat gagtttataa aataaaaaaa 600

gcacctgaaa aggtgtcttt ttttgatggt tttgaacttg ttctttctta tcttgataca 660

tatagaaata acgtcatttt tattttagtt gctgaaaggt gcgttgaagt gttggtatgt 720

atgtgtttta aagtattgaa aacccttaaa attggttgca cagaaaaacc ccatctgtta 780

aagttataag tgactaaaca aataactaaa tagatggggg tttcttttaa tattatgtgt 840

cctaatagta gcatttattc agatgaaaaa tcaagggttt tagtggacaa gacaaaaagt 900

ggaaaagtga gaccatggag agaaaagaaa atcgctaatg ttgattactt tgaacttctg 960

catattcttg aatttaaaaa ggctgaaaga gtaaaagatt gtgctgaaat attagagtat 1020

aaacaaaatc gtgaaacagg cgaaagaaag ttgtatcgag tgtggttttg taaatccagg 1080

ctttgtccaa tgtgcaactg gaggagagca atgaaacatg gcattcagtc acaaaaggtt 1140

gttgctgaag ttattaaaca aaagccaaca gttcgttggt tgtttctcac attaacagtt 1200

aaaaatgttt atgatggcga agaattaaat aagagtttgt cagatatggc tcaaggattt 1260

cgccgaatga tgcaatataa aaaaattaat aaaaatcttg ttggttttat gcgtgcaacg 1320

gaagtgacaa taaataataa agataattct tataatcagc acatgcatgt attggtatgt 1380

gtggaaccaa cttattttaa gaatacagaa aactacgtga atcaaaaaca atggattcaa 1440

ttttggaaaa aggcaatgaa attagactat gatccaaatg taaaagttca aatgattcga 1500

ccgaaaaata aatataaatc ggatatacaa tcggcaattg acgaaactgc aaaatatcct 1560

gtaaaggata cggattttat gaccgatgat gaagaaaaga atttgaaacg tttgtctgat 1620

ttggaggaag gtttacaccg taaaaggtta atctcctatg gtggtttgtt aaaagaaata 1680

cataaaaaat taaaccttga tgacacagaa gaaggcgatt tgattcatac agatgatgac 1740

gaaaaagccg atgaagatgg attttctatt attgcaatgt ggaattggga acggaaaaat 1800

tattttatta aagagtagtt caacaaacgg gccagtttgt tgaagattag atgctataat 1860

tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct gaataagaac 1920

ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga aaagggaatg 1980

agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat tgttcatgaa 2040

attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg tgtttatggc 2100

tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg tgtcatgtca 2160

acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt ggaagtgaat 2220

tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga ttggccgctt 2280

acacatggtc aatttttctc tattttgccg atttatgatt caggtggata cttagagaaa 2340

gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat ttgtgccctt 2400

atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt gcaaggaccg 2460

acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat gttgattggt 2520

ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc agttaagcaa 2580

tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg tcaactttcc 2640

gactctgaga aacttctgga atcgctagag aatttctgga atgggattca ggagtggaca 2700

gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac gatgacctct 2760

aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt agtaattatc 2820

atggctgtca tggcgcatta acggaataaa gggtgtgctt aaatcgggcc attttgcgta 2880

ataagaaaaa ggattaatta tgagcgaatt gaattaataa taaggtaata gatttacatt 2940

agaaaatgaa aggggatttt atgcgtgaga atgttacagt ctatcccggc attgccagtc 3000

ggggatatta aaaagagtat aggtttttat tgggataaag taggtttcac tttggttcac 3060

catgaagatg gattcgcagt tctaatgtgt aatgaggttc ggattcatct atgggaggca 3120

agtgatgaag gctggcgcct cgtagtaatg attcaccggt ttgtacaggt gcggagtcgt 3180

ttattgctgg tactgctagt tgccgcattg aagtagaggg aattgatgaa ttatatcaac 3240

atattaagcc tttgggcatt ttgcacccca atacatcatt aaaagatcag tggtgggatg 3300

aacgagactt tgcagtaatt gatcccgaca acaatttgat tagctttttt caacaaataa 3360

aaagctaaaa tctattatta atctgttcag caatcgggcg cgattgctga ataaaagata 3420

cgagagacct ctcttgtatc ttttttattt tgagtggttt tgtccgttac actagaaaac 3480

cgaaagacaa taaaaatttt attcttgctg agtctggctt tcggtaagct agacaaaacg 3540

gacaaaataa aaattggcaa gggtttaaag gtggagattt tttgagtgat cttctcaaaa 3600

aatactacct gtcccttgct gatttttaaa cgagcacgag agcaaaaccc ccctttgctg 3660

aggtggcaga gggcaggttt ttttgtttct tttttctcgt aaaaaaaaga aaggtcttaa 3720

aggttttatg gttttggtcg gcactgccgc gcctcgcaga gcacacactt tatgaatata 3780

aagtatagtg tgttatactt tacttggaag tggttgccgg aaagagcgaa aatgcctcac 3840

atttgtgcca cctaaaaagg agcgatttac atatgagtta tgcagtttgt agaatgcaaa 3900

aagtgaaatc agctggacta aaaggcatgc aatttcataa tcaaagagag cgaaaaagta 3960

gaacgaatga tgatattgac catgagcgaa cacgtgaaaa ttatgatttg aaaaatgata 4020

aaaatattga ttacaacgaa cgtgtcaaag aaattattga atcacaaaaa acaggtacaa 4080

gaaaaacgag gaaagatgct gttcttgtaa atgagttgct agtaacatct gaccgagatt 4140

tttttgagca actggatcca gcttaattaa agataatatc tttgaattgt aacgcccctc 4200

aaaagtaaga actacaaaaa aagaatacgt tatatagaaa tatgtttgaa ccttcttcag 4260

attacaaata tattcggacg gactctacct caaatgctta tctaactata gaatgacata 4320

caagcacaac cttgaaaatt tgaaaatata actaccaatg aacttgttca tgtgaattat 4380

cgctgtattt aattttctca attcaatata taatatgcca atacattgtt acaagtagaa 4440

attaagacac ccttgatagc cttactatac ctaacatgat gtagtattaa atgaatatgt 4500

aaatatattt atgataagaa gcgacttatt tataatcatt acatattttt ctattggaat 4560

gattaagatt ccaatagaat agtgtataaa ttatttatct tgaaaggagg gatgcctaaa 4620

aacgaagaac attaaaaaca tatatttgca ccgtctaatg gatttatgaa aaatcatttt 4680

atcagtttga aaattatgta ttatgataag aaagggagga agaaaaggta ccattatagg 4740

taagagagga atgtacacat gattattgat agtaaaacga ctttacctag acattcactt 4800

attcatacaa ttaaattaaa ttctaataag aaatatggtc ctggtgatat gaccaatgga 4860

aatcaattta ttatttcaaa acaagaatgg gctactattg gagcatatat tcagactgga 4920

ttaggtttac cagtaaatga acaacaatta agaacacatg ttaatttaag tcaggatata 4980

tcaataccta gtgatttttc tcaattatat gatgtttatt gttctgataa aacttcagca 5040

gaatggtgga ataaaaattt atatccttta attattaaat ctgctaatga tattgcttca 5100

tatggtttta aagttgctgg tgatccttct attaagaaag atggatattt taaaaaattg 5160

caagatgaat tagataatat tgttgataat aattccgatg atgatgcaat agctaaagct 5220

attaaagatt ttaaagcgcg atgtggtatt ttaattaaag aagctaaaca atatgaagaa 5280

gctgcaaaaa atattgtaac atctttagat caatttttac atggtgatca gaaaaaatta 5340

gaaggtgtta tcaatattca aaaacgttta aaagaagttc aaacagctct taatcaagcc 5400

catggggaaa gtagtccagc tcataaagag ttattagaaa aagtaaaaaa tttaaaaaca 5460

acattagaaa ggactattaa agctgaacaa gatttagaga aaaaagtaga atatagtttt 5520

ctattaggac cattgttagg atttgttgtt tatgaaattc ttgaaaatac tgctgttcag 5580

catataaaaa atcaaattga tgagataaag aaacaattag attctgctca gcatgatttg 5640

gatagagatg ttaaaattat aggaatgtta aatagtatta atacagatat tgataattta 5700

tatagtcaag gacaagaagc aattaaagtt ttccaaaagt tacaaggtat ttgggctact 5760

attggagctc aaatagaaaa tcttagaaca acgtcgttac aagaagttca agattctgat 5820

gatgctgatg agatacaaat tgaacttgag gacgcttctg atgcttggtt agttgtggct 5880

caagaagctc gtgattttac actaaatgct tattcaacta atagtagaca aaatttaccg 5940

attaatgtta tatcagattc atgtaattgt tcaacaacaa atatgacatc aaatcaatac 6000

agtaatccaa caacaaatat gacatcaaat caatatatga tttcacatga atatacaagt 6060

ttaccaaata attttatgtt atcaagaaat agtaatttag aatataaatg tcctgaaagt 6120

aattttatga tatattggta taataattcg gattggtata ataattcgga ttggtataat 6180

aattaaaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 6240

cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 6300

agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 6360

gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 6420

gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 6480

ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 6540

aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 6600

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6660

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6720

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6780

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 6840

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 6900

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 6960

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 7020

gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 7080

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 7140

cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 7200

tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 7260

tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 7320

ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 7380

cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 7440

cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 7500

accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 7560

ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 7620

ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 7680

tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 7740

acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 7800

tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 7860

actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 7920

ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 7980

aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 8040

ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 8100

cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 8160

aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 8220

actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 8280

cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 8340

ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 8400

taggcgtatc acgaggccct ttcgtc 8426

Claims (7)

1. An expression vector has a sequence shown in SEQ ID NO. 2.

2. The method for preparing the expression vector according to claim 1, which comprises the following preparation steps:

(1) Constructing a recombinant plasmid pP3A-sfGFP for guiding the efficient expression of the green fluorescent protein sfGFP in bacillus thuringiensis by utilizing a promoter of Cry3Aa gene for regulating and controlling the expression of Cry proteins in the vegetative phase of the bacillus thuringiensis;

(2) The sfgfp gene is replaced by the nematicide Cry6A gene by homologous recombination means to obtain the expression vector shown in SEQ ID NO. 2.

3. An engineered strain of bacillus thuringiensis comprising the expression vector of claim 1.

4. The engineered strain of claim 3, which is obtained by introducing the expression vector of claim 1 into a host cell, wherein the host cell is wild type Bxy bacillus thuringiensis, and is deposited with the China general microbiological culture Collection center with the accession number CGMCC No.24656 for 2022, 04, and 08.

5. Use of the expression vector of claim 1, or the engineered strain of any one of claims 3-4, for expression of a nematicidal Cry protein.

6. The use according to claim 5 wherein said nematicidal Cry protein is a nematicidal Cry6A protein.

7. Use of the expression vector of claim 1, or the engineered strain of any one of claims 3-4, in one or more of the following:

(1) Application in killing nematodes;

(2) The application in preventing and/or treating pine wood nematode disease.

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