1. The application of the aspartic protease gene in improving beauveria bassiana varieties is characterized in that: the yield and the toxicity of conidia of beauveria bassiana are improved by knocking out the aspartic protease gene BbASP, or the high-temperature tolerance of beauveria bassiana is improved by over-expressing the aspartic protease gene BbASP, the nucleotide sequence of the aspartic protease gene BbASP is shown as SEQ ID No.20, and the high temperature is higher than 30 ℃.

2. A method for improving yield and toxicity of conidia of beauveria bassiana is characterized in that: the mutant strain of the beauveria bassiana is obtained by knocking out the nucleotide sequence between 1468 th site and 2040 th site of the beauveria bassiana aspartic protease gene BbASP shown in SEQ ID NO.20, and the beauveria bassiana mutant strain has improved conidium yield and toxicity.

3. The method for improving conidium yield and virulence of beauveria bassiana according to claim 2, wherein the conidia are selected from the group consisting of: the knockout is a knockout by means of homologous recombination replacement.

4. The method for improving conidium yield and virulence of beauveria bassiana according to claim 3, wherein the conidia are selected from the group consisting of: the knockout by the homologous recombination replacement mode is the replacement of a herbicide resistance gene bar, and the nucleotide sequence of the bar gene is shown in SEQ ID No. 10.

5. A method for improving the high-temperature tolerance of beauveria bassiana is characterized in that: obtaining a mutant strain of beauveria bassiana by overexpressing a promoter pb3 and an aspartic protease gene BbASP open reading frame sequence in beauveria bassiana, the mutant strain of beauveria bassiana having an improved high temperature tolerance at a temperature above 30 ℃; the nucleotide sequence of the promoter pb3 is shown as SEQ ID NO.21, and the sequence of the BbASP open reading frame is shown as SEQ ID NO. 4.

6. The method for improving the high temperature tolerance of beauveria bassiana according to claim 5, wherein: the elevated temperature is above 30 ℃.

7. A beauveria bassiana mutant strain, which is characterized in that: the mutant strain knocks out nucleotide sequences between 1468 th site and 2040 th site of Beauveria bassiana aspartic protease gene BbASP, and the nucleotide sequence of the aspartic protease gene BbASP is shown in SEQ ID No. 20.

8. An improved strain of beauveria bassiana is characterized in that: the improved strain is obtained by over-expressing an aspartic protease gene BbASP in beauveria bassiana, wherein the nucleotide sequence of the aspartic protease gene BbASP is shown as SEQ ID No.20, the aspartic protease gene BbASP is regulated and expressed by a promoter pb3, and the nucleotide sequence of the promoter pb3 is shown as SEQ ID No. 21.

9. A fungal pesticide comprising the mutant strain of Beauveria bassiana of claim 7 or the improved strain of Beauveria bassiana of claim 8.

10. Use of the mutant strain of beauveria bassiana of claim 7 or the improved strain of beauveria bassiana of claim 8 in the preparation of a fungal pesticide.

Background

China belongs to the traditional agriculture kingdom, and the sustainable development of agriculture cannot be used without pesticide. Chemical pesticides are widely applied due to the advantages of good insecticidal effect, quick response, simple use and the like, but abuse of chemical pesticides can cause environmental pollution, pesticide residues, people and livestock mistaking poisoning and other hazards, so the demand of nuisanceless or low-toxicity pesticides in agricultural production is higher and higher, and among them, biological pesticides are one of the most effective alternative schemes. Beauveria bassiana (Beauveria bassiana) is a broad-spectrum entomopathogenic fungus, is easy to culture, has a wide pathogenic spectrum, and can infect more than 700 insects of 15 orders and 149 families. Because of its lethal effect and broad spectrum on insects, it has been considered to be studied as an agricultural-friendly biopesticide.

The beauveria bassiana as the biological pesticide has the following advantages: 1. the culture is simple and the propagation is rapid; 2. the host is not easy to generate resistance; 3. no harm to human, livestock, crops and ecological environment; 4. the sustained action time is long, and the beauveria bassiana can be propagated and diffused again by using a host, so the application development momentum of the beauveria bassiana in modern agriculture is good. The growth temperature range of the beauveria bassiana is 5-30 ℃, the optimal growth temperature is about 26 ℃, and when the environmental temperature is higher than 30 ℃, the spore germination and growth of the beauveria bassiana are inhibited to different degrees. Therefore, the application of the entomopathogenic fungi in the field is greatly limited due to the influence of the environmental factor of temperature in practical application. Therefore, the development of the beauveria bassiana high-temperature stress tolerance related gene, the analysis of the regulation mechanism and the optimization and improvement of the strain by taking the beauveria bassiana high-temperature stress tolerance related gene as a target point have important significance in improving the stress resistance of the strain and expanding the application of the strain.

Aspartic proteases are an important class of proteolytic enzymes, widely found in mammals, plants, bacteria, fungi, and viruses. The aspartic protease mainly plays the functions of degrading protein, promoting enzyme activation and the like. Among the human pathogenic fungi, Candida albicans, aspartic protease is one of the important virulence factors, playing an important role in the pathogenic process by degrading cell surface structures, which in turn adhere to and invade the host, and degrading proteins involved in immune defense. A beauveria bassiana T-DNA insertion mutant sensitive to high temperature is found in the early stage of a subject group, and the colony growth inhibition rate of the mutant is higher than that of a wild type at the high temperature of 32 ℃, which indicates that a gene damaged by the T-DNA insertion is possibly involved in the regulation and control of the beauveria bassiana tolerance to high temperature stress. The flanking sequence of the T-DNA insertion site of this mutant was amplified by the YADE (Y-shaped adaptor dependent extension) method, and the gene disrupted by the insertion of the T-DNA was named BbASP because it was a gene encoding Aspartic protease (Aspartic protease). At present, no report is found on the research of regulating the high temperature tolerance of the strain by the aspartic protease. How to improve the existing pathogenic insect fungi by means of genetic engineering and improve the toxicity and the stress resistance of the pathogenic insect fungi has important significance and good prospect.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an application of aspartic protease gene in improving beauveria bassiana strain; the second purpose of the invention is to provide a method for improving the yield and the toxicity of conidia of beauveria bassiana; the third purpose of the invention is to provide a method for improving the high-temperature tolerance of beauveria bassiana; the fourth purpose of the invention is to provide a beauveria bassiana mutant strain, and the fifth purpose of the invention is to provide an improved beauveria bassiana strain; the sixth purpose of the invention is to provide a fungal pesticide; the seventh purpose of the invention is to provide the application of the beauveria bassiana mutant strain or the beauveria bassiana improved strain in preparing fungal pesticides.

In order to achieve the purpose, the invention provides the following technical scheme:

1. the application of the aspartic protease gene in improving the beauveria bassiana variety improves the conidium yield and toxicity of the beauveria bassiana by knocking out the aspartic protease gene BbASP, or improves the high-temperature tolerance of the beauveria bassiana by over-expressing the aspartic protease gene BbASP, the nucleotide sequence of the aspartic protease gene BbASP is shown as SEQ ID NO.20, and the high temperature is higher than 30 ℃.

2. A method for improving yield and toxicity of conidia of beauveria bassiana is characterized in that a nucleotide sequence between 1468 th site and 2040 th site of a beauveria bassiana aspartic protease gene BbASP shown in SEQ ID No.20 is knocked out to obtain a beauveria bassiana mutant strain, and the beauveria bassiana mutant strain has the advantages of improving yield and toxicity of the conidia.

Preferably, the knockout is a knockout by means of homologous recombination replacement.

Preferably, the knockout by means of homologous recombination replacement is replacement of a herbicide resistance gene bar, and the nucleotide sequence of the bar gene is shown as SEQ ID No. 10.

3. A method for improving high temperature tolerance of beauveria bassiana, which comprises the steps of over-expressing a promoter pb3 and an aspartic protease gene BbASP open reading frame sequence in the beauveria bassiana to obtain a beauveria bassiana mutant strain, wherein the beauveria bassiana mutant strain has improved high temperature tolerance, and the high temperature tolerance is a condition that the temperature is higher than 30 ℃; the nucleotide sequence of the promoter pb3 is shown as SEQ ID NO.21, and the sequence of the BbASP open reading frame is shown as SEQ ID NO. 4.

Preferably, the elevated temperature is above 30 ℃.

4. A mutant strain of beauveria bassiana is provided, the mutant strain knocks out nucleotide sequences between 1468 th site and 2040 th site of a beauveria bassiana aspartic protease gene BbASP, and the nucleotide sequence of the aspartic protease gene BbASP is shown in SEQ ID No. 20.

5. An improved strain of beauveria bassiana, the improved strain is obtained by over-expressing an aspartic protease gene BbASP in beauveria bassiana, the nucleotide sequence of the aspartic protease gene BbASP is shown as SEQ ID NO.20, the aspartic protease gene BbASP is regulated and controlled to be expressed by a promoter pb3, and the nucleotide sequence of the promoter pb3 is shown as SEQ ID NO. 21.

6. A fungal pesticide contains the beauveria bassiana mutant strain or the beauveria bassiana improved strain.

7. The beauveria bassiana mutant strain or the beauveria bassiana improved strain is applied to preparation of a fungus insecticide.

The invention has the beneficial effects that: the invention discloses application of an aspartic protease gene in improving a beauveria bassiana variety, wherein the yield and the toxicity of conidia of the beauveria bassiana are improved by knocking out the aspartic protease gene BbASP, or the high-temperature tolerance of the beauveria bassiana is improved by over-expressing the aspartic protease gene BbASP, and the nucleotide sequence of the aspartic protease gene BbASP is shown as SEQ ID No. 20.

The beauveria bassiana mutant strain delta Bbasp obtained by knocking out aspartic protease gene BbASP has the advantages of increased conidium yield and enhanced toxicity. When the strain is cultured on a CZM medium at the temperature of 26 ℃, the culture time is 10d and 20d, the conidiophore yield of the knockout strain delta Bbasp is respectively higher than that of the wild type by 72.57 percent (P)<0.01) and 59.43% (P)<0.01). At the normal temperature of 26 ℃, no matter body wall immersion dyeing and body cavity injection, the host survival rate curve of the knockout strain delta Bbasp is lower than that of beauveria bassiana wild type, and the LT of the knockout strain delta Bbasp under the body wall immersion dyeing condition of 26 DEG C₅₀(103h) The length of the gene is shortened by 36.11h (P) compared with the wild type (139.11h)<0.001); LT for knocking out strain delta Bbasp under 26 ℃ coelomic injection condition₅₀(71.07h) is shortened by 4h (P) compared with wild type (75.04h)<0.01). Under the conditions of 32 ℃ high-temperature body wall infection and body cavity injection, the host survival rate curve of the knockout strain delta Bbasp is lower than that of the beauveria bassiana wild type, and the LT of the knockout strain delta Bbasp through body wall infection₅₀(192.90h) has a reduced 33.64h (P) compared with wild type (226.54h)<0.01); LT for knocking out strain delta Bbasp under coelom injection condition₅₀(142.33h) is shortened by 5.51h (P) compared with wild type (147.84h)<0.05). And the test insects which die at the same time under the conditions of body wall infection and body cavity injection at the temperature of 26 ℃ knock out the strain delta Bbasp thalli earlier than the wild type.

The beauveria bassiana improved strain OEBbasp obtained by overexpression of aspartic protease gene BbASP has enhanced high-temperature resistance. Under the high-temperature stress of 32 ℃, the colony diameter of the excess strain OEBbasp is slightly larger than that of the wild type, the inhibition rate (47%) is about 2% lower than that of the wild type, and the growth rate of the OEBbasp is faster than that of the wild type. After conidia of the OEBbasp are stressed at the high temperature of 43 ℃ for 2h and 4h, the germination rates of the excess strain OEBbasp are respectively improved by 7 percent (P <0.01) and 11.34 percent (P <0.01) compared with the wild type.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the expression analysis of conidium (A) and hypha (B) BbASP genes under high temperature stress;

FIG. 2 shows pK₂-bar support schematic;

FIG. 3 is a schematic diagram (A) of BbASP gene homologous knockout vector construction and a restriction enzyme digestion verification diagram (B);

FIG. 4 shows pK₂-schematic diagram of PtrpC-sur-TtrpC vector;

FIG. 5 shows the construction of BbASP gene reversion complementary vector (A) and the result of enzyme digestion verification (B);

FIG. 6 shows a BbASP gene overexpression vector construction diagram (A) and a restriction enzyme digestion verification result (B);

FIG. 7 shows a screening and verification diagram of BbASP gene homologous knockout and reversion complementation strains ((A) PCR verification results of homologous knockout and reversion complementation strains, wherein lanes 1-3 are wild type, knockout strain and reversion strain respectively, M is Marker 2000, (B) southern blot verification results, and lanes 1-3 are wild type WT, knockout strain delta Bbasp and reversion strain delta Bbasp respectively, (C) Real-time fluorescence quantitative PCR verification results, (D) Real-time PCR verification results, and lanes 1-3 are wild type, knockout strain and reversion strain respectively;

FIG. 8 shows the screening of the over-expressed improved strain (A: PCR verification of over-transformant, lanes 1-10: over-expressed transformant, WT: beauveria bassiana wild type, M: Marker 2000; B: analysis of BbASP gene expression in over-expressed transformant; C: southern blot verification, lane 1: No.3 over-transformant.);

FIG. 9 is a graph of colony growth (A) and relative growth inhibition (B) for each strain under high temperature stress (asterisks indicate significant differences calculated by one-way ANOVA, ". x." indicates that the differences are extremely significant, P < 0.001.);

FIG. 10 shows the spore germination rate (A) at room temperature, the germination time (B) at room temperature and the spore germination rate (C) after high temperature stress for each strain;

FIG. 11 shows the spore yields of 10d and 20d under normal temperature and high temperature stress of each strain;

FIG. 12 shows the survival rate and the half-lethal time of test insects under different bioassay conditions;

FIG. 13 shows the hypha emergence times of test insects dead at the same time under different biological test conditions.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1 BbASP Gene cloning

Upstream and downstream primers were designed based on aspartic protease sequences in NCBI:

ASP-F：5'-ATGTCGCTCAGAAACATCGTC-3'(SEQ ID NO.1)；

ASP-R：5'-TTACTTGAGATTAGCGAAGCCC-3'(SEQ ID NO.2)；

using the genome DNA of wild type Beauveria Bassiana (Beauveria Bassiana) Bb0062 as a templateMax DNA Polymerase is subjected to PCR amplification, gel recovery is carried out after the size of a product is verified to be correct through gel electrophoresis, then the fragment is connected to a cloning vector pEASY-Blunt, and the BbASP gene is obtained after the enzyme digestion and sequencing verification are correct.

The BbASP genome DNA has the total length of 1208bp (SEQ ID NO.3), contains two introns, has the ORF sequence with the total length of 1068bp (SEQ ID NO.4), codes 355 amino acids (SEQ ID NO.5), and has the predicted protein molecular weight of 37.5 kDa.

Example 2 expression analysis of BbASP Gene under high temperature stress

A, B two culture modes are adopted to detect the difference of the expression quantity of BbASP genes in conidia and hyphae of beauveria bassiana under the stress of normal temperature (26 ℃) and high temperature (32 ℃).

(A) Taking 50 μ L of 1 × 10⁷culturing spore suspension of beauveria bassiana wild strain in a CZM solid culture medium at 26 ℃ and 32 ℃ for 14d respectively, extracting conidium RNA, performing reverse transcription to obtain cDNA, detecting the expression quantity of the BbASP gene by taking a gamma-Actin gene as an internal standard, and taking no stress as a control;

(B) taking 50 μ L of 1 × 10⁷Adding conidia/mL of beauveria bassiana wild type strain spore suspension into 1/4SDY liquid medium, shake-culturing at 26 ℃ and 200rpm for 3d, stressing at 32 ℃ for 4h, extracting hypha RNA, performing reverse transcription to obtain cDNA, detecting BbASP gene expression quantity by using a gamma-Actin gene as an internal standard, and using no stress as a control.

Real-time fluorescent quantitative PCR reaction system (10 μ Ι _): iQ SYBR Green Supermix (BIO-RAD) 5. mu.L, primers 0.5. mu.L each, reverse transcribed cDNA as template 1. mu.L, and sterile water 3. mu.L. The real-time fluorescent quantitative PCR program was as follows: at 95 ℃ for 3 min; 95 ℃, 10s, 56 ℃, 30s, 72 ℃, 20s, 39 cycles; the dissolution profile was from 65 ℃ to 95 ℃ with 0.5 ℃ increase per cycle, 5s reaction. The internal reference gene is a beauveria bassiana gamma-Actin gene. The qRT-PCR primers used were:

BbASP-F：5'-AATCTGGCCACTGATTCGGG-3'(SEQ ID NO.6)；

BbASP-R：5'-AATCGTAGGGGAGTTGCTGC-3'(SEQ ID NO.7)；

γ-Actin-F：5'-TTGGTGCGAAACTTCAGCGTCTAGTC-3'(SEQ ID NO.8)；

γ-Actin-R：5'-TCCAGCAAATGTGGATCTCCAAGCAG-3'(SEQ ID NO.9)；

as shown in FIG. 1, the BbASP gene expression level in conidia under high temperature stress was up-regulated by 4.54 times compared with the control without stress (FIG. 1, A); the BbASP gene expression level in the hyphae under high temperature stress is up-regulated by 0.7 times compared with the non-stress control (FIG. 1, B). The results show that the expression of the BbASP gene is induced by high-temperature stress.

Example 3 construction of BbASP Gene homology knockout vector

pK is selected as homologous knock-out skeleton vector₂-PtrpC-bar-TtrpC (pK for short)₂Bar) was the backbone vector pK from this laboratory₂Is obtained by modification on the basis of₂The schematic diagram of the-PtrpC-bar-TtrpC vector is shown in figure 2, bar is a glufosinate resistance gene, the nucleotide sequence of the bar gene is shown in SEQ ID NO.10, a section of sequence is selected as a homologous arm at the 5 'end and the 3' end of the ORF region of the BbASP gene respectively, a primer is designed, and underlining represents an enzyme cutting site:

LB-F：5'-CGGAATTCGCATGCTCACTTTGCAACTTC-3'(EcoRⅠ，SEQ ID NO.11)；

LB-R：5'-CGGAATTCCGATGACTGACGCTGAATTG-3'(EcoRⅠ，SEQ ID NO.12)；

RB-F：5'-GCTCTAGAGGTCAAGTTTATCGCGACGTC-3'(XbaⅠ，SEQ ID NO.13)；

RB-R：5'-CCCAAGCTTCTTTGCAGCAATCCATGAGTC-3'(HindⅢ，SEQ ID NO.14)；

respectively amplifying left and right arm fragments of the BbASP gene, wherein the lengths of the left and right homologous arms are BbASPLB: 731bp, BbASPRB: 812 bp. The left arm BbASPLB was single digested with EcoRI and ligated to the vector pK₂On bar, construction of pK₂-BbASPLB-bar. The right-arm BbASPRB was double digested with Xba I and Hind III and ligated to pK₂On a BbASPLB-bar vector, enzyme digestion and sequencing verification are carried out, and the verification primers are as follows:

P1-F：5'-GACAATGTCTCACTCCAAGC-3'(SEQ ID NO.15)；

P1-R：5'-GTCGTCTATGGCACCAATGG-3'(SEQ ID NO.16)；

confirmation of homologous knock-out vector pK₂The construction of the BbASPLB-bar-BbASPRB is successful.

The schematic diagram of the BbASP gene homologous knockout vector construction is shown in FIG. 3 and A, the enzyme digestion verification result of the left arm and the right arm is shown in FIG. 3 and B, 2 fragments of 731bp BbASP LB (lane 1) and 812bp BbASPRB (lane 2) are cut off, and the successful construction of the BbASP gene homologous knockout vector is shown.

Example 4 construction of BbASP Gene Reversal complementation vector

Restoring and complementing boneThe carrier is pK₂-Ptrpc-sur-Ttrpc (pK for short)₂Sur) was the backbone vector pK from the laboratory₂Is obtained by modification on the basis of₂The schematic diagram of the-Ptrpc-sur-Ttrpc vector is shown in FIG. 4, sur is a chlorimuron-ethyl resistance gene, and the nucleotide sequence of the sur is shown in SEQ ID NO. 17. Selecting a BbASP gene (1208bp, SEQ ID NO.4) and 2993bp of a promoter and a terminator thereof, and performing amplification reaction by using a primer:

PH-F：5'-CCCAAGCTTCTACCAGTACGTCCTTGTTCC-3'(Hind III，SEQ ID NO.18)；

PH-R：5'-CCCAAGCTTGTGCAACAACATTGGAGTGCC-3'(Hind III，SEQ ID NO.19)；

amplifying to obtain target fragment (PBbASPT, SEQ ID NO.20), recovering target band after gel electrophoresis verification, and respectively digesting the fragment PBbASPT and the vector pK by restriction enzyme Hind III₂After sur, the ligation product is genetically transformed into Escherichia coli by T4 DNA Ligase ligation (see Takara T4 DNA Ligase instruction manual), and after Hind III digestion and sequencing verification, the BbASP gene reverts to the complementary vector pK₂The construction of sur-PBbASPT is successful, the construction schematic diagram of the BbASP gene reversion complementary vector and the enzyme digestion verification are shown in figure 5.

Example 5 construction of BbASP Gene overexpression vector

The overexpression framework vector is selected from pK₂-sur-pb₃(11926bp), the nucleotide sequence of pb3 is shown in SEQ ID NO. 21. The pK2-sur-pb3 vector was obtained by inserting pb3 gene between the BamH I sites of pK2-sur vector. Designing a primer:

PO-F：5'-CGGGATCCATGTCGCTCAGAAACATCGTC-3'(BamHⅠ，SEQ ID NO.22)；PO-R：5'-GATATCTTACTTGAGATTAGCGAAGCCC-3'(EcoRⅤ，SEQ ID NO.23)；

the wild cDNA of beauveria bassiana is used as a template to amplify BbASP gene ORF (1068bp, SEQ ID NO.3), and restriction enzymes BamH I and EcoR V are used for respectively digesting a target fragment and pK₂-sur-pb₃After the vector was verified and recovered, the ligation product was inherited through E.coli by T4 DNA Ligase ligation (see Takara T4 DNA Ligase manual for ligation systems)After transformation, enzyme digestion and sequencing verification, the BbASP gene overexpression vector pK₂-sur-pb₃The BbASP is successfully constructed, the construction diagram of the BbASP gene overexpression vector and the enzyme digestion verification are shown in the figure 6.

Example 6 genetic transformation of Beauveria bassiana and selection of transgenic strains

The BbASP gene homologous knockout vector, the reversion complementation vector and the overexpression vector prepared in the embodiment 3-5 are respectively transformed into the agrobacterium tumefaciens AGL-1 competent cell by an electric shock method.

After the transformed agrobacterium is subjected to YEB liquid culture medium amplification culture, plasmids are extracted, and after PCR detection shows positive, genetic transformation is carried out on beauveria bassiana under the mediation of the agrobacterium.

Screening BbASP gene homologous knockout strains (delta Bbasp) and revertants (delta Bbasp: Bbasp):

the BbASP gene homologous knockout vector is transferred into a beauveria bassiana wild type by adopting an agrobacterium-mediated transformation method, and if homologous recombination occurs, 573bp interchange occurs between the bar gene and the left arm and the right arm of the BbASP gene, so that the BbASP gene is damaged. Since the homologous knockout strain contains the bar gene, it can be grown in selection medium containing PPT (glufosinate) resistance. The homologous knockout strain amplified only a band of 1.9kb in length, whereas the wild type amplified only a band of about 945bp, as verified by the selection primer P1-F/P1-R (FIG. 7, A).

Then, a successful pK is constructed in an agrobacterium-mediated manner₂And transferring the SUR-PBbASPT reversion complementation vector into a BbASP gene homologous knockout strain, wherein the homologous knockout strain contains a bar resistance gene, and the reversion complementation vector contains a SUR resistance gene, so that the culture medium added with PPT and SUR is used for screening. The correct revertant complementation mutant should amplify two bands of 1.9kb and 945bp, verified by specific primers P1-F/P1-R (FIG. 7, A). Meanwhile, the knocked-out 563bp sequence is used as a probe to carry out southern blot verification, bands can appear in the wild strain and the revertant strain, and the homologous knocked-out strain can not be hybridized into the bands at the same position (figure 7, B). The homologous knockout strain and the revertant strain are subjected to Real-time PCR detection to find the revertantThe BbASP gene expression quantity of the strain is similar to that of the wild strain, and the BbASP gene of the homologous knockout strain is not expressed (figure 7, C, D).

BbASP gene overexpression mutant strain (OEBbasp) screening:

overexpression vector pK is transformed by agrobacterium-mediated transformation method₂-sur-pb₃BbASP is transferred into a beauveria bassiana wild strain, if the transformation is successful, the strain contains SUR resistance genes, so the strain is used for screening on a culture medium containing SUR resistance. To be provided with

pb₃-F：5'-AGCAAGATGGCCAGACATGG-3'(SEQ ID NO.24)；

PO-R (SEQ ID NO.23) was used as a screening primer, and a simple template made of hypha was used to screen and verify the transformed strain, and a band of about 1.5kb could be amplified from a successful transformed strain while a band could not be amplified from the wild type (FIG. 8, A). Selecting 10 transformants capable of being amplified, extracting RNA and carrying out reverse transcription to obtain cDNA, detecting the BbASP gene expression quantity in the transformants, wherein the BbASP gene expression quantity of the No.3 transformant is 147 times of that of a wild type (figure 8, B), and selecting the strain as an overexpression mutant strain for subsequent study. Meanwhile, taking the excess fragment as a probe, selecting a No.3 transformant for southern blot verification, and obtaining a result shown in FIGS. 8 and C.

Example 7 phenotypic analysis of BbASP mutant strains under high temperature stress

BbASP gene affecting growth of colony under high temperature stress

Culturing beauveria bassiana wild type WT, knockout strain delta Bbasp and recovery strain delta Bbasp, Bbasp and excess strain OEBbasp in CZM culture medium for 14d, and preparing spore suspension (1 × 10) of each strain⁷conidia/mL), inoculating 2 μ L of spore suspension into the center of the CZM medium, culturing for 8d under constant temperature culture conditions of 26 ℃ and 32 ℃, respectively, observing colony diameter and counting Relative growth inhibition (Relative growth inhibition) RGI, and calculating: relative inhibition ratio RGI ═ (no stress colony diameter-colony diameter under stress)/no stress colony diameter, with three replicates per group of data.

The result shows that under the condition of 26 ℃, the colony growth conditions of the knockout strain delta Bbasp and the replying strain delta Bbasp are not obviously different from the colony growth conditions of the wild type Bbasp and the excess strain OEBbasp (figure 9, A), and therefore, the gene is supposed not to influence the growth of the beauveria bassiana colony under the normal temperature condition.

Under the high-temperature stress of 32 ℃, the colony diameter of the knockout strain delta Bbasp is obviously smaller than that of a wild type, and the relative growth inhibition rate (61%) of the knockout strain delta Bbasp is increased by about 12% compared with that (49%) of the wild type after 8d culture (P < 0.01); the recovery strain delta Bbasp is consistent with the growth condition of the wild type; after the gene is over-expressed, the growth rate of the strain is fastest, the colony diameter of the over-expression strain OEBbasp is slightly larger than that of a wild type, and the inhibition rate (47%) is about 2% lower than that of the wild type (figure 9, B). The result shows that the BbASP gene influences the growth of beauveria bassiana colonies under high-temperature stress and influences the high-temperature tolerance of the beauveria bassiana.

BbASP gene influences conidium germination rate under high-temperature stress

In order to verify whether the beauveria bassiana deleted BbASP gene can influence the germination of conidia of the beauveria bassiana, wild type WT of the beauveria bassiana, a knockout strain delta Bbasp, a recovery strain delta Bbasp, a Bbasp and an excess strain OEBbasp suspension (1 multiplied by 10)⁸conidia/ml), taking 100 mu l of the mixture, coating the mixture on a CZM plate, culturing the mixture in a constant-temperature incubator at 26 ℃ for 8 hours, observing spore germination every 2 hours until each mutant completely germinates in 20 hours, counting 100 spores for one repetition every time, taking three repetitions of each strain, and drawing a strain germination rate curve by using Graphpad prism 5 software.

As a result, under the normal culture condition of 26 ℃, the conidium germination rates of the four strains after 18h are basically consistent and approach to 100% (FIG. 10, A), and the SPSS software analyzes GT50 when each strain conidium germinates under the normal temperature condition of 26 ℃, and finds that the conidia germinates of the four strains are basically the same and approach to 11h (FIG. 10, B).

In addition, conidium suspension (1 × 108conidia/mL, 100 μ L) of each strain is taken, subjected to high-temperature stress at 43 ℃ for 2h and 4h, and then coated on a PDA culture medium, the conidium suspension is cultured on a constant-temperature culture medium at 26 ℃ for 20h, then the germination rate is counted, 100 spores are counted each time to be one repetition, three repetitions are taken for each strain, and the normal culture condition is processed at 26 ℃ for a control group. The results are shown in FIG. 10 and C, the germination rates of conidia of the four strains are obviously reduced after 43 ℃ stress, wherein the Δ Bbasp of the knockout strain is reduced most obviously under 2h and 4h stress, and is respectively reduced by 7.33% (P <0.01) and 48.66% (P <0.001) compared with the wild type, and the germination rates of the conidia of the reverting strain Δ Bbasp are not obviously different from the germination rates of the wild type after the high temperature stress, and the germination rates of the excess strains OEBbasp are respectively improved by 7% (P <0.01) and 11.34% (P < 0.01).

The result shows that the germination rate of conidia of the knockout strain delta Bbasp is not greatly different from that of a wild type at the normal temperature of 26 ℃, and the BbASP gene does not influence the conidia germination of beauveria bassiana at the normal temperature; under the stress of 43 ℃, the high temperature resistance of conidia of the knockout strain delta Bbasp is obviously lower than that of a wild type, the high temperature resistance of conidia of the excess strain OEBbasp is higher than that of the wild type, and BbASP genes influence the conidia germination of beauveria bassiana under the high-temperature stress.

BbASP gene influencing output of beauveria bassiana conidia

In order to further explore the influence of BbASP on the growth and development of beauveria bassiana at high temperature, the yield of conidia of wild type and three mutant strains at 10d and 20d times, temperature and high temperature on a CZM culture medium is counted, and the specific method comprises the following steps: culturing 14d beauveria bassiana wild type, delta BbASP and delta BbASP in a CZM culture medium to prepare spore suspension (1 × 10)⁷conidia/mL), adding 65 μ L of spore suspension into 25mL of unset CZM solid culture medium, mixing well, pouring into a sterile culture dish, repeating three times per bacterium, and culturing at 26 deg.C and 32 deg.C for 10d and 20d respectively. After 10d and 20d, three samples were randomly taken from each medium using a 10mm punch, added to 3mL Tween-80 (0.05% v/v), vortexed thoroughly and mixed, and the number of spores was counted using a blood count plate.

As a result, as shown in fig. 11, it was found that, in CZM medium at room temperature condition of 26 ℃, conidia yields of the knockout strain Δ Bbasp were both higher than those of the wild type at 72.57% (P <0.01) and 59.43% (P <0.01) regardless of 10d and 20d, respectively, whereas the revertant-complemented mutant did not significantly differ from the wild type in 10d and 20d conidia yields in CZM medium, the over-expressed mutant did not significantly differ from the wild type in 10d conidia yield in CZM medium, and the over-expressed mutant decreased in 20d conidia yield from that of the wild type in CZM medium (fig. 11, A, B). Under the high temperature condition of 32 ℃, the knockout strain delta Bbasp has no difference, no matter 10d or 20d, the conidium yield of the knockout strain delta Bbasp is not obviously different from that of a wild type, while the recovery strain delta Bbasp has the advantages that the conidium yield of 10d and 20d of Bbasp and the over strain OEBbasp in a CZM culture medium under the condition of 32 ℃ is obviously reduced compared with that of the wild type (figure 11, C, D), and is suspected to be caused by the damaged gene of the T-DNA insertion site. The results show that BbASP affects the yield of conidia of beauveria bassiana and is affected by high temperature.

Example 8 Effect of BbASP Gene on virulence of Beauveria bassiana

BbASP gene affecting virulence of beauveria bassiana

The pathogenicity of the fungus is an important index for detecting the effect of the insecticidal fungus, so that the virulence of each strain treated by two inoculation modes of lower body wall infection and body cavity injection under the conditions of 26 ℃ and 32 ℃ is respectively detected, and the used test insect is larva of three-instar greater wax moth (Galleria mellonella).

Body wall infestation was as follows: taking beauveria bassiana wild type, delta BbASP and delta BbASP which are cultured on a CZM culture medium for 14d, preparing 15mL spore suspension (1 × 10 spore suspension)⁷conidia/mL) with Tween-80 (0.05% v/v) as negative control. 35 galleria mellonella hubner is soaked in conidium suspension, the conidium suspension is slightly shaken for 15s, redundant bacteria liquid is filtered by an iron wire filter screen, and each strain is repeated three times. The method comprises the following steps of immersing and dyeing the greater wax moth, placing the greater wax moth in a 150mm sterile culture dish, respectively placing the greater wax moth in culture boxes at 26 ℃ and 32 ℃ for culture, humidifying cotton balls, placing the cotton balls in the culture dish to keep humidity, counting the death number of the greater wax moth from 72 hours, and then counting every 24 hours. The survival rate of the strains and the hosts is plotted by Graphpad prism 5 software, and the half lethal time LT of each strain is calculated by SPSS software₅₀。

The body cavity injection method comprises the following steps: preparing conidium of each strain into spore suspension (1 × 10)⁶conidia/mL), also with Tween-80 (0.05% v/v) as a negative control. Injecting greater wax moth with 1mL injector at the position between the second and third ventral poda, and injecting 5 μ each with microsyringeAnd L. Three replicates per strain, 35 greater wax moth per replicate. After the injection, the cells were also placed in a sterile 150mm petri dish and incubated at 26 ℃ and 32 ℃ in an incubator, respectively, and after 48 hours, the number of deaths was counted every 12 hours. The survival rate of the strains and the host is plotted by Graphpad prism 5 software, and the half-lethal time of each strain is calculated by SPSS software.

The results show that the host survival rate curve of the knockout strain delta Bbasp is lower than that of beauveria bassiana wild type WT (figure 12, A, C) at the normal temperature of 26 ℃, no matter body wall staining and body cavity injection, and the semi-Lethal Time (LT) of the greater wax moth infected by four strains is analyzed₅₀) The LT of the strain delta Bbasp is knocked out under the condition of 26 ℃ body wall staining₅₀(103h) The length of the gene is shortened by 36.11h (P) compared with the wild type (139.11h)<0.001) (fig. 12, B); LT for knocking out strain delta Bbasp under 26 ℃ coelomic injection condition₅₀(71.07h) is shortened by 4h (P) compared with wild type (75.04h)<0.01) (fig. 12, D).

Under the conditions of high-temperature body wall infection at 32 ℃ and body cavity injection, the host survival rate curve of the knockout strain delta Bbasp is lower than that of beauveria bassiana wild type WT (figure 12, E, G), and LT of the knockout strain delta Bbasp is infected through body wall₅₀(192.90h) has a reduced 33.64h (P) compared with wild type (226.54h)<0.01) (fig. 12, F); LT for knocking out strain delta Bbasp under coelom injection condition₅₀(142.33h) is shortened by 5.51h (P) compared with wild type (147.84h)<0.05) (fig. 12, H).

The results show that the BbASP gene is damaged to cause the obvious increase of the virulence of the strain, wherein the virulence of the mutant delta Bbasp strain knocked out under the body wall staining mode of normal temperature 26 ℃ or high temperature 32 ℃ is greatly increased compared with the wild type, the virulence of the mutant delta Bbasp strain knocked out under the body cavity injection condition of normal temperature 26 ℃ or high temperature 32 ℃ is slightly increased compared with the wild type, and the BbASP gene participates in the regulation and control of the virulence of beauveria bassiana.

BbASP gene influences hypha penetration after death of host

According to bioassay experiments, the toxicity of the knockout strain delta Bbasp is higher than that of wild WT, test insects which die at the same time in two bioassay modes of body wall infection and body cavity injection are selected, the passing-out time of beauveria bassiana is observed, the test insects are observed once every 24 hours, and photographing records are carried out. As a result, it was found that the cells of the test insects which died at the same time in the conditions of body cavity injection and body wall infection at 26 ℃ were earlier in cell-out time than the wild type cells of the test insects (FIG. 13, A, B). Since the growth of beauveria bassiana is inhibited under the high-temperature condition of 32 ℃, the worm body can not penetrate out no matter the body wall is impregnated or the body cavity is injected by a biological test method (figure 13, C, D), which shows that the BbASP gene influences the penetration of the worm body after the death of the host.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> university of southwest

Application of <120> aspartic protease gene in improving beauveria bassiana variety

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtcgctca gaaacatcgt c 21

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ttacttgaga ttagcgaagc cc 22

<210> 3

<211> 1208

<212> DNA

<213> Beauveria bassiana (Beauveria bassiana)

<400> 3

atgtcgctca gaaacatcgt cgcctgccta gccgcggctc tagccgtcgc cgcagactcg 60

tcgcacccga tgcattataa aaaggtagat gcagaaactg ccaaaagcgt tgctgccgtt 120

agaaaccatg cgtcgggacg cgatggagcg cttgtgaatg agcaggtaag ttgagttgct 180

tgggctcatt tcaggcttcg accaattgaa cggttgctaa gtctatacaa cttcagggat 240

tctggttctc ccacttcacc gttggcgcca gcgaaaacct cgaactcctg attgacactg 300

gctcgtccga tgccatgctg aaccctggtg tctacaagcc cagctctacc tctgaggacc 360

taaaacgtcg attcagcatt tcatatgcga cgacgaaccc cgatggcacg ggaaccctat 420

ccgtgagtgc tccaaattga atgctacaac gaaacgtgcg tatcttgctt ctctaacagt 480

cctcttccta ggccttcggt caagtttatc gcgacgtcat cacccagctc ggtgccaacc 540

ttgtcgtccc gcagcaagcc attggtgcca tagacgaccc caaaacgcca gccacgtttc 600

cccgtgatgg cctcattggc tacgccggca aaggcggcgc tgccctgcgc gagagctcct 660

tcttcttctc cctctgcgcg tccaacgccc tcaccgagtg ccgattcggc ctcgccctcc 720

gcaccgacgg cactggccag ctgcactacg gcactgtcgt aaaggaggag tttgacggcg 780

agctcaccac cgtcgacctg accggctact ggtccatcaa cggcggcgtc accgtcaacg 840

gcaaggtcat tgccgacaat ctcaatctgg ccactgattc gggtacgact gtcatctttg 900

gcccaacgag cgtggtcaga gaagtctaca agtccgccgg catcaccgag gtctcgacag 960

caaacggcat cgaaggacac tacagctgca gcaactcccc tacgattggc ttcaacctcg 1020

gcggcaagaa cttcaatatt gaccccaagg cgcttgcctt taagaaggag ggcgacaatt 1080

gcaccgctag cctcatgggc acccaagact ttggcagcat gtggctcgtt ggtcaggcct 1140

ttttccaagg tcgctacatc gaccacaatg gttctgggaa aaccatgggc ttcgctaatc 1200

tcaagtaa 1208

<210> 20

<211> 1068

<212> DNA

<213> Beauveria bassiana (Beauveria bassiana)

<400> 20

atgtcgctca gaaacatcgt cgcctgccta gccgcggctc tagccgtcgc cgcagactcg 60

tcgcacccga tgcattataa aaaggtagat gcagaaactg ccaaaagcgt tgctgccgtt 120

agaaaccatg cgtcgggacg cgatggagcg cttgtgaatg agcagggatt ctggttctcc 180

cacttcaccg ttggcgccag cgaaaacctc gaactcctga ttgacactgg ctcgtccgat 240

gccatgctga accctggtgt ctacaagccc agctctacct ctgaggacct aaaacgtcga 300

ttcagcattt catatgcgac gacgaacccc gatggcacgg gaaccctatc cgccttcggt 360

caagtttatc gcgacgtcat cacccagctc ggtgccaacc ttgtcgtccc gcagcaagcc 420

attggtgcca tagacgaccc caaaacgcca gccacgtttc cccgtgatgg cctcattggc 480

tacgccggca aaggcggcgc tgccctgcgc gagagctcct tcttcttctc cctctgcgcg 540

tccaacgccc tcaccgagtg ccgattcggc ctcgccctcc gcaccgacgg cactggccag 600

ctgcactacg gcactgtcgt aaaggaggag tttgacggcg agctcaccac cgtcgacctg 660

accggctact ggtccatcaa cggcggcgtc accgtcaacg gcaaggtcat tgccgacaat 720

ctcaatctgg ccactgattc gggtacgact gtcatctttg gcccaacgag cgtggtcaga 780

gaagtctaca agtccgccgg catcaccgag gtctcgacag caaacggcat cgaaggacac 840

tacagctgca gcaactcccc tacgattggc ttcaacctcg gcggcaagaa cttcaatatt 900

gaccccaagg cgcttgcctt taagaaggag ggcgacaatt gcaccgctag cctcatgggc 960

acccaagact ttggcagcat gtggctcgtt ggtcaggcct ttttccaagg tcgctacatc 1020

gaccacaatg gttctgggaa aaccatgggc ttcgctaatc tcaagtaa 1068

<210> 5

<211> 355

<212> PRT

<213> Beauveria bassiana (Beauveria bassiana)

<400> 5

Met Ser Leu Arg Asn Ile Val Ala Cys Leu Ala Ala Ala Leu Ala Val

1 5 10 15

Ala Ala Asp Ser Ser His Pro Met His Tyr Lys Lys Val Asp Ala Glu

20 25 30

Thr Ala Lys Ser Val Ala Ala Val Arg Asn His Ala Ser Gly Arg Asp

35 40 45

Gly Ala Leu Val Asn Glu Gln Gly Phe Trp Phe Ser His Phe Thr Val

50 55 60

Gly Ala Ser Glu Asn Leu Glu Leu Leu Ile Asp Thr Gly Ser Ser Asp

65 70 75 80

Ala Met Leu Asn Pro Gly Val Tyr Lys Pro Ser Ser Thr Ser Glu Asp

85 90 95

Leu Lys Arg Arg Phe Ser Ile Ser Tyr Ala Thr Thr Asn Pro Asp Gly

100 105 110

Thr Gly Thr Leu Ser Ala Phe Gly Gln Val Tyr Arg Asp Val Ile Thr

115 120 125

Gln Leu Gly Ala Asn Leu Val Val Pro Gln Gln Ala Ile Gly Ala Ile

130 135 140

Asp Asp Pro Lys Thr Pro Ala Thr Phe Pro Arg Asp Gly Leu Ile Gly

145 150 155 160

Tyr Ala Gly Lys Gly Gly Ala Ala Leu Arg Glu Ser Ser Phe Phe Phe

165 170 175

Ser Leu Cys Ala Ser Asn Ala Leu Thr Glu Cys Arg Phe Gly Leu Ala

180 185 190

Leu Arg Thr Asp Gly Thr Gly Gln Leu His Tyr Gly Thr Val Val Lys

195 200 205

Glu Glu Phe Asp Gly Glu Leu Thr Thr Val Asp Leu Thr Gly Tyr Trp

210 215 220

Ser Ile Asn Gly Gly Val Thr Val Asn Gly Lys Val Ile Ala Asp Asn

225 230 235 240

Leu Asn Leu Ala Thr Asp Ser Gly Thr Thr Val Ile Phe Gly Pro Thr

245 250 255

Ser Val Val Arg Glu Val Tyr Lys Ser Ala Gly Ile Thr Glu Val Ser

260 265 270

Thr Ala Asn Gly Ile Glu Gly His Tyr Ser Cys Ser Asn Ser Pro Thr

275 280 285

Ile Gly Phe Asn Leu Gly Gly Lys Asn Phe Asn Ile Asp Pro Lys Ala

290 295 300

Leu Ala Phe Lys Lys Glu Gly Asp Asn Cys Thr Ala Ser Leu Met Gly

305 310 315 320

Thr Gln Asp Phe Gly Ser Met Trp Leu Val Gly Gln Ala Phe Phe Gln

325 330 335

Gly Arg Tyr Ile Asp His Asn Gly Ser Gly Lys Thr Met Gly Phe Ala

340 345 350

Asn Leu Lys

355

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aatctggcca ctgattcggg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aatcgtaggg gagttgctgc 20

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttggtgcgaa acttcagcgt ctagtc 26

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tccagcaaat gtggatctcc aagcag 26

<210> 9

<211> 558

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tctagtatga gcccagaacg acgcccggcc gacatccgcc gtgccaccga ggcggacatg 60

ccggcggtct gcaccatcgt caaccactac atcgagacaa gcacggtcaa cttccgtacc 120

gagccgcagg aaccgcagga gtggacggac gacctcgtcc gtctgcggga gcgctatccc 180

tggctcgtcg ccgaggtgga cggcgaggtc gccggcatcg cctacgcggg cccctggaag 240

gcacgcaacg cctacgactg gacggccgag tcgaccgtgt acgtctcccc ccgccaccag 300

cggacgggac tgggctccac gctctacacc cacctgctga agtccctgga ggcacagggc 360

ttcaagagcg tggtcgctgt catcgggctg cccaacgacc cgagcgtgcg catgcacgag 420

gcgctcggat atgccccccg cggcatgctg cgggcggccg gcttcaagca cgggaactgg 480

catgacgtgg gtttctggca gctggacttc agcctgccgg taccgccccg tccggtcctg 540

cccgtcaccg agatctaa 558

<210> 10

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cggaattcgc atgctcactt tgcaacttc 29

<210> 11

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cggaattccg atgactgacg ctgaattg 28

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gctctagagg tcaagtttat cgcgacgtc 29

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cccaagcttc tttgcagcaa tccatgagtc 30

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gacaatgtct cactccaagc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gtcgtctatg gcaccaatgg 20

<210> 16

<211> 2048

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

acgactgaga acagattcga aatgcttcgt actgttggcc gcaaagccct gaggggctca 60

tccaagggat gttctcgaac catctcgact ctcaagcccg ccacggcaac tattgccaag 120

cccggcagca ggaccctttc gacgccagcg acggcaacag caacgtaagt cagaatcacg 180

agcgagcgaa caaacaggca gagttacaaa cgccatcact acgccttgtc ttttcgtctc 240

tgttacgtgt cgccaccccc atgcgagcta cacgtctgcg cctgaagtca caaggcttgc 300

aatttcatga cgatgaacaa cactgacact tctgttacag agcacctcga actaagccca 360

gcgccagctt caatgctcgc cgcgatcccc agcctctggt caaccctcgc tcaggtgagg 420

cagacgaatc gtaagttgcg ccaacaccgc tcctacaccg ccaagaccca taccgccgtc 480

gcatttggac aagcacaaga ctgatttgac cgtggcatag attcattggc aagacgggag 540

gagagatttt ccacgagatg atgctgaggc aaaacgtcaa gcatatctgt aagcgtctaa 600

ttctacaaat tcctaccctc gatttcaacc acatattcaa ccacatattc tgacacatga 660

gtcacagtcg gttaccctgg cggtgctatc cttcccgtgt tcgacgcgat ctacaactcg 720

aagcacatcg actttgttct gcccaagcat gagcaaggcg ccggccacat ggcagagggc 780

tatgctcgcg cttcaggcaa acccggcgtt gttctcgtca cctccggccc cggtgccaca 840

aatgtcatca ctcccatggc cgacgctctt gccgacggta cacctctggt tgtattctca 900

ggacaggttg ttacctctga tattggaagc gacgccttcc aggaggccga cgtcataggc 960

atctcccggt cttgcaccaa gtggaacgtc atggttaaga gcgctgacga gctcccgagg 1020

agaattaacg aggcctttga gattgccacc agtgggcgac ctgggcctgt cttggtcgat 1080

ctgcccaagg acgtcacggc tagtgtgctg aggagggcta tccccaccga gacctcgatt 1140

ccctctatta gcgcagcagc acgggctgtc caagaggcag gccgaaagca gcttgagcac 1200

tccatcaaac gcgtagccga tctcgtcaac attgccaaga agcccgtcat atatgccggc 1260

caaggtgtca ttttgtcgga aggcggcgtt gaacttctca aggcgcttgc cgacaaggcc 1320

tcgattcctg tcaccaccac tctgcatggt ctgggagcct ttgacgagct cgacgagaag 1380

gcactgcaca tgcttggtat gcacggttcg gcttatgcca acatgtccat gcaagaggcc 1440

gatttgatca ttgcccttgg tggccgcttc gatgaccgtg tcactggcag catccccaaa 1500

tttgctcctg ccgccaagct agctgctgct gaaggacgcg gaggtattgt ccacttcgag 1560

attatgccca agaacatcaa caaggtcgtc caagcaacag aggccattga gggcgacgtt 1620

gcttcgaact tgaagctgtt gctccccaag attgaacaac gatccatgac cgatcgcaag 1680

gagtggttcg accagatcaa ggagtggaag gagaagtggc ctctgtcaca ttatgagagg 1740

gccgagcgta gtggtctcat caagcctcag actctgatcg aggagctgag caacctgact 1800

gctgaccgca aggacatgac ctacatcaca accggtgttg gccagcacca aatgtggaca 1860

gcacaacatt tcaggtggag gcacccacgg tccatgatca cctctggcgg tttgggaacc 1920

atgggatatg gtctgccggc agcgattggc gccaaggttg ctaggccaga tgctttggtc 1980

attgacgtcg acggcgacgc atcgttcaac atgactctga cagagctttc gacggcggca 2040

cagttcaa 2048

<210> 17

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cccaagcttc taccagtacg tccttgttcc 30

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cccaagcttg tgcaacaaca ttggagtgcc 30

<210> 19

<211> 2993

<212> DNA

<213> Beauveria bassiana (Beauveria bassiana)

<400> 19

ctaccagtac gtccttgttc ctctctggag cccgtctctg agcagagctg agcagagcag 60

agcagagcag agcgggggac gtatcttaag gggggggggt gggggggggg cgtcaactct 120

aacagtaata caagattctc tgtcctcgac accaacatga ttggcttcgc cgagatcccc 180

aagggcgctc aaggcaaaat gcacgagtac ctttttcctt tactggaact ggcggaaggc 240

acgtcgaata tacaatgacc atgctcggtc gtctaccgcg gttactttga tatcatgagc 300

catcccagct ctgacagttg ggtctcgttg cggcggcagc accgccatcc tcaacatgaa 360

cgctgcgtgc accatttgcc tgccctcatt gctgtgagtt gacgactgcg aagacggatc 420

taatcccaaa actcaccccc aagtatcggt gcaataacca tgacaaccca cccccttagc 480

aatagaaacc aatttgctaa tgagcggctg ttttcatcta gtcgatcacg ttagcggcaa 540

gtttcgggtc aagtttggca tgcagcggag agttctgccc taaagagtag gaacgtgttg 600

tggcatgtat catggcttct tctttttctt tctttttttt tttttttctc tttttttctt 660

catggttctt tgcagagccg tctggttggt gtcctcaatc gtttgcgcag cttcagtgtc 720

ccccgatgga ggaggggcat gctcactttg caacttcatc ctaaagaaca tcaacttact 780

tgttcacctc caggggctag ctttctcctt tttgtttgtt ctggttattt ctttgaaaaa 840

ccaaatgtcg aaaaaaatct tggatcaacg caaggtgtga aacacattgg gactaataag 900

tcggtcaaag actctcccag ccttcccttc gggtcttgcc tcccgcatca ttttttagcg 960

cagcacacgg taacgccact cctcggacgt cggcaacagt caacctgcca gatgtggaag 1020

gtcgggcaga cttacaaaac tagttcaact agtgcatgga attttcctgc ctccctcttg 1080

gttacgcgaa ccggctctcc tcccccggta cagcgcggct tacagaagag cccgtcatct 1140

ggtctagagc ttgggctctc cgccagtata catttgtgtg atccgccatc aggctataac 1200

ttgtgtgtct ctctcgttgg catgcagcac tgctcacatt gccgtcttgc gcgcgggccg 1260

cttttaggtt tgccggccga cacgttgtca aactttatag agggtcaact cgatggacaa 1320

tgtctcactc caagcacaaa tcctcgccac aaaagcaatc tctcaccctc cctggttgta 1380

gcatatctcc tgaaaaactt ttttctgatg tcgcgggttg ccatctgaaa aaaaaaaaaa 1440

aaaaaaacaa ttcagcgtca gtcatcgatt gtcatctaca gtgcgagtcg atcttgtgct 1500

gcatcgagaa ggtcaacacg gcatcgtcca caatcaatgc aacgccggcg acgctacaca 1560

aggaagcggt ttcatgcatg tgttcatgta tagtcaacta tatatcccca actctctccc 1620

ctccaaactc tatcagacag cgattcctcg tttcccgcat tgtcaccaca aggtctcgtc 1680

atcatgtcgc tcagaaacat cgtcgcctgc ctagccgcgg ctctagccgt cgccgcagac 1740

tcgtcgcacc cgatgcatta taaaaaggta gatgcagaaa ctgccaaaag cgttgctgcc 1800

gttagaaacc atgcgtcggg acgcgatgga gcgcttgtga atgagcaggg attctggttc 1860

tcccacttca ccgttggcgc cagcgaaaac ctcgaactcc tgattgacac tggctcgtcc 1920

gatgccatgc tgaaccctgg tgtctacaag cccagctcta cctctgagga cctaaaacgt 1980

cgattcagca tttcatatgc gacgacgaac cccgatggca cgggaaccct atccgccttc 2040

ggtcaagttt atcgcgacgt catcacccag ctcggtgcca accttgtcgt cccgcagcaa 2100

gccattggtg ccatagacga ccccaaaacg ccagccacgt ttccccgtga tggcctcatt 2160

ggctacgccg gcaaaggcgg cgctgccctg cgcgagagct ccttcttctt ctccctctgc 2220

gcgtccaacg ccctcaccga gtgccgattc ggcctcgccc tccgcaccga cggcactggc 2280

cagctgcact acggcactgt cgtaaaggag gagtttgacg gcgagctcac caccgtcgac 2340

ctgaccggct actggtccat caacggcggc gtcaccgtca acggcaaggt cattgccgac 2400

aatctcaatc tggccactga ttcgggtacg actgtcatct ttggcccaac gagcgtggtc 2460

agagaagtct acaagtccgc cggcatcacc gaggtctcga cagcaaacgg catcgaagga 2520

cactacagct gcagcaactc ccctacgatt ggcttcaacc tcggcggcaa gaacttcaat 2580

attgacccca aggcgcttgc ctttaagaag gagggcgaca attgcaccgc tagcctcatg 2640

ggcacccaag actttggcag catgtggctc gttggtcagg cctttttcca aggtcgctac 2700

atcgaccaca atggttctgg gaaaaccatg ggcttcgcta atctcaagta agcgggtcat 2760

taccaaaaaa aaaaaaaagg cgtatataac tatgcatgct ctaatgttta tggaaatgcc 2820

aagagtacat agactcatgg attgctgcaa agaaaacagg tgcccaacgg tcctcgggct 2880

tttacaaagt tccttccctc gaactttccg actgcgtata tatatataca aaagactaaa 2940

gagcgtgtcg cttcgcttga accgatgtag gaggcactcc aatgttgttg cac 2993

<210> 21

<211> 1153

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gttgggtatg ctccggcgcg accgagcaag atggccagac atgggatcaa aattagttga 60

ttcgcctttt tgcttggaca aagattacga gagcagtgag tgccatccca gtggtcaacc 120

atggcaattc cgtcttggcc cacgaggcac tcaacgaact tttgctagtc aggaggaggt 180

tggaacaagg ggaagctcgt ccatttgcat cattgatgac caagagatta gtcgacgagc 240

aggatcttgg ccgttcacag taataagcaa agacttattt ttttgcgtag cgggcaagcg 300

agatggcggc caaagtttgc gcagacacac acacgggttg acaggttgcc cgcacgcagg 360

caggcaacac gaagcttggg agctcgacgt cagtaaagtg ggggagggga agcaaccaga 420

acaagagcaa gtaagtggga tgaagcttgc tgcaacggaa gctcgagcgt cgatggagct 480

gtattgacag gtgaatacag atggattcat tgtgttgtga tagaagccaa agaacctgtg 540

tggctaattg accaggatag ctatcactaa atatgtactc aagcatcgag ggtgatatgt 600

actgttgtga cgacgagcgt tgcccctctg cccctccatg gggacctgca gcaacttgag 660

ccagcccact ggtcgagtaa ataggtatga gcaaggcggt gctggaggag ggggcgacaa 720

tcagaccagc gcctgcaact ctgcccctaa aaacaacctc agtggctgtt gacgaccttc 780

tagcgcatgt ttttcagtga caatggcctt ttttaccctt gctctggcag agccccagaa 840

tccgtgccca cgactacaaa accatttgcc cgcccctctt ccgccgccgc gacacccttt 900

ttcttctctc catatacctc atcctcgaat cgaaagacca aaagtgagtt ttcccattcg 960

ttccattcca tctcgagctt cattgctgct tcttctccct cgtcatctac ccctccatag 1020

ctccctcacc agagaagctg caccgttttc ccctcaacga ccccgccatc cgccaccaaa 1080

cagctacccc tcgctaacca ccaaacgcct caacaggttt atctcttcca cctcaccctt 1140

ttaatcaata aca 1153

<210> 22

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cgggatccat gtcgctcaga aacatcgtc 29

<210> 23

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gatatcttac ttgagattag cgaagccc 28

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

agcaagatgg ccagacatgg 20