CN110747220B - Antibacterial peptide gene promoter P BbAFP1 Application in improving entomogenous fungi spores - Google Patents
Antibacterial peptide gene promoter P BbAFP1 Application in improving entomogenous fungi spores Download PDFInfo
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Abstract
The invention discloses an application of an antibacterial peptide gene promoter in improving entomogenous fungi spores, belongs to the field of molecular biology, provides a new choice for improving and utilizing entomogenous fungi by a molecular biology means, and can improve the tolerance of the spores to adverse environments such as ultraviolet resistance and stress resistance by utilizing the expression of a regulatory gene in the later period of sporulation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a beauveria bassiana antibacterial peptide gene promoter P BbAFP1 Use in the modification of entomogenous fungi spores.
Background
Entomopathogenic fungi are one of the main factors for controlling natural pests (Charnley, 1997), have the characteristics of various varieties, rapid growth, capability of infecting different development stages of hosts and the like, and become an important component in biological pesticides (Li Zeng Zhi, 1997). Entomopathogenic fungi also have the advantages of environmental friendliness, difficulty in host resistance generation and the like, have been developed into biological insecticides, and are widely applied to prevention and control of agricultural and forestry pests and urban sanitary insects. Conidiospores are effective components of fungal pesticides, begin an infection process by adhering to the body wall of a host, penetrating through the body wall of the host after germination, and then largely generate on the body surface of the stiff insects to begin the next infection process. The fungus, when lethal to an insect, is generally capable of producing a large number of spores in its body, causing spread, forming an epidemic when appropriate, and being compatible with a wide variety of other natural enemies and many insecticides. Fungal insecticides have been widely used in China and European and American countries (Von Ming., 1998), and to date, over 50 registered products have been produced, including over ten fungi including Beauveria bassiana (Beauveria bassiana), metarhizium anisopliae (Metarhizium anisopliae), paecilomyces fumosoroseus (Paecilomyces fumosoroseus), and Verticillium lecanii (Verticillium lecanii). Recently, entomopathogenic fungi, in particular strains improved by genetic engineering, have shown great potential in the control of urban sanitary pests such as mosquitoes, flies, cockroaches and invading pests such as fire ants, showing that entomopathogenic fungi have broad application prospects in the field of biocontrol (Wang et al, 2007 fan et al, 2012a,2012b fan et al, 2011. Therefore, the regulation and control of the spore development of the entomogenous fungi by utilizing the genetic engineering have important significance on the popularization and the application of the entomogenous fungi, the environmental safety and the like. However, in general, the fungal pesticide has a low share in the pesticide market and the biopesticide market due to the problems of unstable control effect of entomopathogenic fungi in the application process, time process for knocking down pests and the like.
Among the entomopathogenic microorganisms, the fungal infestation mechanism is particularly pronounced, where it does not have to be ingested by the host but instead invades the host directly through the exoskeleton or epidermis. The infection process comprises the following steps: conidia attach to the body wall of the insect and germinate to form an infection structure (such as an attachment cell); hyphae penetrate through the body wall of the insect and grow in the insect body to cause disease to the insect; finally, the fungus takes dead polypides as a nutrient source, conidia are generated in a large amount, the conidia infect other insects after spreading, and another round of infestation process begins (Chanley, 2003). In the process, spores are the beginning of infection and the end point of completing one round of infection, and the yield, the activity and the stress resistance of the spores directly determine the control effect and the market position of the fungal biocontrol preparation. The genetic engineering is utilized to improve the spores of the entomopathogenic fungi, so that the tolerance of the entomopathogenic fungi to adverse conditions such as ultraviolet and high temperature is improved when the entomopathogenic fungi are applied in fields, or the spore yield is improved, and the method has important value for the application of the entomopathogenic fungi. Existing studies have shown that the choice of promoter before and after spore production is critical in determining success or failure. For a long time, constitutive promoters have been the main promoters in the transgenic research of entomopathogenic fungi. However, constitutive promoters, when expressed at non-target positions and for a long time, tend to consume energy of the organism and even adversely affect the growth of the organism. Therefore, in order to study the characteristics of entomopathogenic fungi before and after spore production by genetic engineering, promoters specifically expressed at different stages of the entomopathogenic fungi, such as before, during, and after spore production, are urgently needed. However, to date, there has been no report on expression of a promoter in the late sporulation stage (spore maturation stage) of an entomopathogenic fungus.
Disclosure of Invention
In order to solve the technical problems, the invention discloses a beauveria bassiana antibacterial peptide gene promoter P BbAFP1 The promoter provides a new choice for improving and utilizing entomogenous fungi by a molecular biological means, and can improve the characteristics of the spores on adverse environments such as ultraviolet resistance, stress resistance and the like by utilizing the expression of a regulatory gene in the later period of sporulation.
The invention is realized by the following technical scheme:
antibacterial peptide gene promoter P BbAFP1 Use in the modification of entomogenous fungi spores.
Specifically, the application is the application in regulating and controlling gene expression after sporulation of the entomogenous fungi or improving entomogenous fungi spores.
Specifically, the antibacterial peptide gene promoter P BbAFP1 Is derived from Beauveria bassiana.
Further, the entomogenous fungus is beauveria bassiana.
Specifically, the antibacterial peptide gene promoter P BbAFP1 Has a nucleotide sequence shown as SEQ ID No. 1:
SEQIDNO.1 P BbAFP1 promoter sequence (983 bp)
ACGGAATAGATCAATCGTGGTAGGAAAGACTTTTTCCAGAGGCAGTGCTGTGTCGAGTAAATGTCGTG TAAAAATTTCTTCTCCCTTCTACAGTCTGCCGGAATCTTTTTTCCGCACGATGCACGGCATTGCAATGC ATCAGATAACTAAACGAACAATGAAGCAAAGGCAGCGTCTAAAGGCTACAAAGTTCAAAGACATGCA TGGTCCTTGTCCGCGAGCGGCAGTATAGATCACCTTAATGGGCCAACGGCCATCGCACGATTCTTACTT GATGTTTCCGGAATCGCTCTCAAAGCTGCTTGATCTGGCCCtTTTTTTCGCACCCCCACACGATTTGCA ACTTCACACGAAATCACAGTGCGCGGCAACTGACGATCGACAACCGGCGCATGCCAAATGTGGTATC AATAATATCAGGCTGGCTCGGTGCACGGTCGGGGTGACAGTCGACGCTATAAATTCATGTACGTCTGA CCGAGGCCCTCCGGCATCGAGCAATTAGTAGCAATGAGCAGCGCACGTATGACTAAAGTGTTGTCAC AATCTTGTCTGGTGGCGCGATGGGCCGGAAGATCTCTCAGGCATTCGCTCCGTCGGGGGCCAATCGCA AACCATCAGTGCATCGCCTGCGTGCATCATAACACGGGGCGGGAATGGCCTAGACTAGAGATGATCGG TGATGTTACATAGGGTTGCATATATACGTGACGGTCGATGAGGCCCCCAGCCTGATTTCAGATTCCTTA ATCAGTGAACCAAGCCCAAATAGGTACGGAGAGATGTATCCTCTGGCAAGCTCGTCGGTAGAAGCCC AATTCGAAACAGCCCGACTATCCCATCATGTACGATATAAAAAGGCTTCACGACCGCTCCAAAACATC ACCTTACAAGTTACACCTCACCAAGAAGCATTCTGATATATCGTTCCCAATCATCACTTAGAAGCTTTC CCAATAAAATCACCTcAAAATTTTGCAAA
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention relates to an antibacterial peptide gene promoter P BbAFP1 Use in modification of entomogenous fungi spores, promoter P BbAFP1 The promoter provides a new choice for improving and utilizing entomogenous fungi by molecular biology means, and can improve the tolerance of the spores to adverse environments such as ultraviolet resistance and stress resistance by utilizing the expression of the regulatory gene in the late sporulation stage, but does not influence the characteristics of vegetative growth or sporulation and the like of the entomogenous fungi.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 shows the present invention P BbAFP1 1, constructing an eGFP eukaryotic expression vector: ptrpc is a promoter of an aspergillus nidulans tryptophan synthetic gene C; sur is a resistance gene for screening the drug chlorimuron-ethyl; ttrpc is the C terminator of the tryptophan synthesis gene of Aspergillus nidulans; the eGFP is a gene for enhancing green fluorescent protein.
FIG. 2 is a schematic representation of a solidIn the medium CZA (2, 4, 8d), P BbAFP1 Representative picture of eGFP expression in B.
FIG. 3 shows the present invention in liquid medium CZB (2, 3, 5d), P BbAFP1 Representative picture of eGFP expression in B.
FIG. 4 shows RT-PCR quantitative analysis of P BbAFP1 Expression in solid CZA medium (8 d) and liquid medium CZB (4 d), error bar = SE.
FIG. 5 is P BbAFP1 FIG. is a fluorescent observation of eGFP strains in insect cadaver hyphae and conidia: wild type (no signal) and strains constitutively expressing eGFP (using the b. Basiana constitutive promoter PgpdA) were used as negative and positive controls, respectively.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example 1
P BbAFP1 The acquisition of eGFP strains
1.P BbAFP1 Cloning of promoter and vector construction
Artificially synthesized primer pair P3/P4 (P3: 5-; GACCTGCAGGCATGCAAGCTTGACGAATCAATCAATCGGTG-; P4: 5-; TTGCTCACCATGGTGGCGGCTTTGCAATTTTTGAGGGTGATTTTT-; P3') and amplified by using the genome DNA of the wild type beauveria bassiana BbAFP1 A promoter fragment. The amplification system is as follows: 10 XPHUSION Taq PCR Buffer (containing Mg) 2+ ) mu.L of 2.5. Mu.L, 10mmol/L dNTP 2. Mu.L, 1. Mu.L of each of 10. Mu. Mol/L primers (P3/P4), 1. Mu.L of genomic DNA template (10 ng/. Mu.L), 0.2. Mu.L of 5U/. Mu.L Phusion polymerase, and water was added to the 25. Mu.L system. PCR reaction parameters: 98 deg.C (2 min); 30 cycles of 98 deg.C (10 sec), 56 deg.C (15 sec), 72 deg.C (30 sec); 72 deg.C (3 min). The P was recovered by 1.2% agarose electrophoresis BbAFP1 Promoter fragment (P) BbAFP1 About 1.0 kb).
The primer P5/P6 (P5: 5ACAGCTCGTCCAT-3') PCR-amplified eGFP fragment with pK2-BarGFP (Zhang et al, 2010) as template. The amplification system is as follows: 10 XPPhusion Taq PCRbuffer (containing Mg) 2+ ) mu.L of 2.5. Mu.L, 2. Mu.L of 10mmol/L dNTP, 1. Mu.L of each of 10. Mu. Mol/L primer (P5/P6), 1. Mu.L of genomic DNA template (10 ng/. Mu.L), 0.2. Mu.L of 5U/. Mu.L Phusion polymerase, and water was added to the 25. Mu.L system. PCR reaction parameters: 98 deg.C (2 min); 30 cycles of 98 deg.C (10 sec), 56 deg.C (15 sec), 72 deg.C (50 sec); 72 deg.C (3 min). Recovering the eGFP-TtrpC fragment obtained.
The plasmid pK2-Bar was digested with HindIII and the pK2-Bar fragment was recovered. Will P BbAFP1 The promoter, eGFP and pK2-Bar fragments were cloned seamlessly, using the kit Clon express MultiS One cloning kit from Vazyme. The obtained pk2-Bar-P BbAFP1 The eGFP (FIG. 1) plasmid was transferred to Agrobacterium AGL-1 by electroporation.
2. Genetic transformation and selection of fungi
Wild type beauveria bassiana is used as a transformation receptor, and the transformation is carried out by utilizing an agrobacterium tumefaciens mediated genetic transformation method according to the method of Fang et al (Fang et al, 2004), wherein the used culture medium and the specific transformation method are as follows:
media used during transformation:
IM solid medium: (400mL of 2.5 XMM salt solution/L, 5mL of glycerin/L, 8.5g/L MES, 15g/L agar powder, 200. Mu. Mol/L acetosyringone and 5mmol/L glucose added before use)
IM liquid medium: (400mL 2.5 XMM salt solution/L, 5mL glycerin/L, MES 8.5g/L, before use 200. Mu. Mol/L acetosyringone and 10mmol/L filter-sterilized glucose are added, and stored in the dark)
CZM medium (1L): 30g/L; naNO 3 ，2g/L；MgSO4·7H2O，0.5g/L；K 2 HPO 4 ，1g/L； FeSO 4 7H2O,0.001g/L; KCl,0.5g/L; the pH was adjusted to 7.0. 15g/L of agar powder; sterilizing at 121 deg.C for 15min.
A single colony of Agrobacterium tumefaciens containing the plasmid vector was inoculated into YEB liquid medium (containing 50. Mu.g/mL carbenicillin and 50. Mu.g/mL kanamycin), and cultured overnight (16 to 20 hours) with shaking at 200rpm at 28 ℃. Taking 3mL of bacterial liquid at 6000rpm and 10min centrifugation, supernatant was discarded, and the cells were resuspended in an equal amount of IM broth (containing 200. Mu.M acetosyringone and 10mM Glucose) and adjusted to OD 660 0.15, and then shake-cultured at 28 ℃ and 180rpm for 6 hours. In this period, the microfiltration membrane (peninsula, shanghai) was first plated on the IM solid medium and prepared with Tween-80 (0.5% v/v) to a concentration of 2X 10 4 Spore suspension per ml. And (3) mixing the spore suspension and the pre-cultured agrobacterium liquid equally, coating 100 mu l of the liquid on a microporous filter membrane on an IM solid culture medium flat plate, and co-culturing for 48h at 22 ℃. After co-cultivation, the microporous filter membrane is transferred to a one-screen culture medium CZM +60 mu g/ml chlorimuron for further screening, and is cultured at 26 ℃ for about 5 days until resistant colonies appear. The positive colonies were verified by PCR. The transformant amplified an approximately 1.9kb fragment. PCR amplification System: 10 XEx Taq PCR Buffer (containing Mg) 2+ ) 2 μ L of 5mmol/L dNTP 2 μ L, primer P5 (P) BbAFP1 Upstream primer, 5 'GACGGAATAGATCAATCGGTG-3') 1. Mu.L, primer Pgfp-2 (eGFP downstream: 5-. Procedure for amplification: pre-denaturation at 94 ℃ for 5min, pre-denaturation at 94 ℃ for 30s, denaturation at 56 ℃ for 30s, and pre-denaturation at 72 ℃ for 3min, wherein the pre-denaturation is repeated for 32 times and the pre-denaturation is extended for 10min at 72 ℃.
3. P in beauveria bassiana BbAFP1 Determination of expression Pattern of promoters
P BbAFP1 Is an antibacterial peptide gene from beauveria bassiana, and in order to analyze the activity characteristics of a promoter thereof, wild beauveria bassiana is inoculated into a culture medium, and the culture medium adopts a liquid culture medium CZB and a solid culture medium CZA. Wherein, the CZA is that agar is added into the CZB to turn the CZB into solid, conidia are generated by the growth of beauveria bassiana in a solid culture medium CZA, and the conidia are provided with antibacterial peptide; and the liquid generates blastospores which do not generate the antibacterial peptide. P BbAFP1 The promoter is conidiophore-specific and is produced late in spore development.
1) P in CZA Medium BbAFP1 The eGFP gene expression detection of the eGFP strain
Inoculation of P BbAFP1 eGFP strains in CZA medium at multiple growth timesSpots were fluorescence observed to study P BbAFP1 The expression pattern of the gene. The detection mode is as follows: constructing a gene containing P by taking an enhanced green fluorescent protein (eGFP) gene as a reporter gene BbAFP1 The transformant strain of the eGFP fusion gene is the strain P BbAFP1 When the promoter is expressed, the eGFP reporter gene is expressed, green fluorescent protein is generated in spores of the recombinant beauveria bassiana, and the recombinant strain can emit bright green fluorescence under the irradiation of long-wave ultraviolet rays, namely a GFP green fluorescent signal.
The observation result showed that P BbAFP1 That is, when eGFP strain was cultured on CZA for 4 days, no GFP signal was detected in both conidia and hyphae at the beginning of conidia production, but the expression level was significantly increased in conidia appearing on day 8, and the specificity was expressed in the spores (as shown in FIG. 2), and no GFP signal was observed in the hyphae. Furthermore, P BbAFP1 That hyphae and blastospores of the eGFP strain in CZB medium had no GFP signal (FIG. 3).
2) RT-PCR detection
Extracting RNA at different time points, reverse transcribing to cDNA (TaKaRa, primeScript RT Reagent Kit with gDNA Eraser), and measuring P by Real-time PCR BbAFP1 The expression pattern of the gene. The RT primer pair is P1: 5 'GAGCAGTTTGAGGCCCAGAGA-3', P2:5 'AGTTGGGGCACTTTGACAAAAAG-3'; RT-PCR reaction system: 5.0. Mu.L Universal SYBR Green Supermix (Bio-RAD), 4.0. Mu.L cDNA, 0.5. Mu.L P1, 0.5. Mu.L P2, total volume 10. Mu.L; RT-PCR reaction parameters: 95 deg.C (3 min); and 40 cycles of 95 deg.C (10 sec), 56 deg.C (20 sec), and 72 deg.C (20 sec). The actin of beauveria bassiana is an internal standard gene, the primer sequences are Pact-1; on CZB medium, the wild type Beauveria bassiana typically starts sporulating at 3 d.
RT-PCR results showed that BbAFP1 was expressed in a high amount in CZA medium and P was expressed when cultured in CZB BbAFP1 The gene was hardly expressed (FIG. 4). The results in connection with 1) indicate P BbAFP1 The promoter is used for promoting expression in late sporulationThe heterologous promoter is not expressed until sporulation in the CZA medium, but is expressed in a higher amount after sporulation.
3) In the process of infecting a host by beauveria bassiana PBbAFP1, eGFP gene expression detection of eGFP strain
Beauveria bassiana infection starts infection by adhering to the wall of a host body, and then proliferates in the form of worm bodies in the blood cavity of the host. When the host dies, the fungus grows on the dead worm and produces spores. To analyze the expression of BbAFP1 in a pathogenic host, P was introduced BbAFP1 The eGFP strain is inoculated to the greater wax moth. The PgpdA is a fusion gene of a constitutive promoter PgpdA and eGFP. Compared with the PgpdA, eGFP BbAFP1 That is, eGFP is also expressed in the insect cells, but is present only in the conidia (see FIG. 5).
P in synthetic beauveria bassiana BbAFP1 The results of the expression pattern determination of the promoter indicate that P BbAFP1 The promoter has good specificity and is specifically expressed only in the late sporulation stage. The promoter has important application value, can adjust the expression of a specific gene in the post spore production period of the beauveria bassiana, thereby effectively controlling the spore characteristics of the beauveria bassiana and not influencing the growth, the spore production and the like of fungi.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<110> university of southwest
Application of <120> antibacterial peptide gene promoter PBbAFP1 in improvement of entomogenous fungi spore
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 982
<212> DNA
<213> Artificial sequence (1)
<400> 1
acggaataga tcaatcgtgg taggaaagac tttttccaga ggcagtgctg tgtcgagtaa 60
atgtcgtgta aaaatttctt ctcccttcta cagtctgccg gaatcttttt tccgcacgat 120
gcacggcatt gcaatgcatc agataactaa acgaacaatg aagcaaaggc agcgtctaaa 180
ggctacaaag ttcaaagaca tgcatggtcc ttgtccgcga gcggcagtat agatcacctt 240
aatgggccaa cggccatcgc acgattctta cttgatgttt ccggaatcgc tctcaaagct 300
gcttgatctg gccctttttt tcgcaccccc acacgatttg caacttcaca cgaaatcaca 360
gtgcgcggca actgacgatc gacaaccggc gcatgccaaa tgtggtatca ataatatcag 420
gctggctcgg tgcacggtcg gggtgacagt cgacgctata aattcatgta cgtctgaccg 480
aggccctccg gcatcgagca attagtagca atgagcagcg cacgtatgac taaagtgttg 540
tcacaatctt gtctggtggc gcgatgggcc ggaagatctc tcaggcattc gctccgtcgg 600
gggccaatcg caaaccatca gtgcatcgcc tgcgtgcatc ataacacggg gcgggaatgg 660
cctagactag agatgatcgg tgatgttaca tagggttgca tatatacgtg acggtcgatg 720
aggcccccag cctgatttca gattccttaa tcagtgaacc aagcccaaat aggtacggag 780
agatgtatcc tctggcaagc tcgtcggtag aagcccaatt cgaaacagcc cgactatccc 840
atcatgtacg atataaaaag gcttcacgac cgctccaaaa catcacctta caagttacac 900
ctcaccaaga agcattctga tatatcgttc ccaatcatca cttagaagct ttcccaataa 960
aatcacctca aaattttgca aa 982
<210> 2
<211> 41
<212> DNA
<213> Artificial sequence (2)
<400> 2
gacctgcagg catgcaagct tgacggaata gatcaatcgt g 41
<210> 3
<211> 44
<212> DNA
<213> Artificial sequence (3)
<400> 3
ttgctcacca tggtggcggc tttgcaaaat tttgaggtga tttt 44
Claims (5)
1. Antibacterial peptide gene promoter P BbAFP1 Use in modification of entomogenous fungi spores; the entomogenous fungi is beauveria bassiana; antibacterial peptide gene promoter P BbAFP1 Has the nucleotide sequence shown as SEQ ID No. 1.
2. The use according to claim 1, wherein the use is in regulating gene expression after sporulation of the entomogenous fungus or in modifying entomogenous fungus spores.
3. Use according to claim 1, characterized in that the antibacterial peptide gene promoter P BbAFP1 Is derived from Beauveria bassiana.
4. Use according to claim 1, characterized in that it comprises the following steps:
(1) Cloning of P Using wild type Beauveria bassiana genomic DNA BbAFP1 A promoter;
(2) P cloned in the step (1) BbAFP1 The promoter, eGFP and pK2-Bar fragment were cloned seamlessly to obtain pK2-Bar-
P BbAFP1 An eGFP plasmid;
(3) The pk2-Bar-PBbAFP1 is characterized in that an eGFP plasmid is transferred into agrobacterium AGL-1 by an electric shock transformation method, wild entomogenous fungi is taken as a transformation receptor, and the agrobacterium tumefaciens-mediated genetic transformation method is utilized for transformation;
in the step (3), the entomogenous fungi is beauveria bassiana.
5. The method of claim 4Characterized in that, in step (1), P is cloned BbAFP1 The primer pair P3/P4 used by the promoter has nucleotide sequences shown as SEQ ID No. 2 and SEQ ID No. 3.
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