CN112480220A - Random recombinant mutant protein of Sip1Aa protein of bacillus thuringiensis - Google Patents

Abstract

The invention relates to a Bacillus thuringiensis Sip1Aa protein random recombinant mutant protein, belonging to the technical field of biology. The invention obtains the Sip1Aa high-efficiency soluble insecticidal protein mutant M6 and M8 proteins, respectively changes an amino acid, improves the insecticidal activity of the obtained mutant protein M8 by 4.02 times and the insecticidal activity of M6 by 2.19 times, effectively overcomes the problem of searching a gene with high toxicity of the bacillus thuringiensis expression protein to the bullion, and delays the generation of drug resistance of pests to engineering bacteria and transgenic plants.

Description

Random recombinant mutant protein of Sip1Aa protein of bacillus thuringiensis

Technical Field

The invention belongs to the technical field of biological control, and particularly relates to random recombinant mutant protein of Bt insecticidal gene with high toxicity to coleoptera agricultural pests.

Background

Bacillus thuringiensis (Bt) is a gram-positive bacterium, widely exists in nature, and generally has a short rod shape, and is single-grown or short-chain. In 1911, the German biologist Bellen (Berlinier) isolated the same bacterium from diseased larvae of Mediterranean pink borer, Scotch, Germany and named Bacillus thuringiensis. The bacillus thuringiensis has been studied for over 100 years, and reports on the bacillus thuringiensis relate to aspects of biomolecular science, cytology, classification nomenclature, effective components of Bt and insecticidal mechanism thereof, and the like, and the research reports are many tens of thousands.

At present, fewer reports on Bt Sip proteins are made at home and abroad. In the course of Donovan et al investigating the insecticidal activity of Bt strains against coleopteran insects, the culture supernatants of some strains were found to be Lethal (LC)₅₀) The gene has insecticidal activity on Colorado Potato Beetle (CPB) larvae at 0.12 (0.09-0.15) mu.g/mL, comprises 1104bp, encodes 367 amino acids, is named as Sip1A, and has 46% of similarity with Mtx3 mosquito killing protein (Donovan W P, Engleman J T, Donovan J C, et al]Appl Microbiol Biotechnol,2006,72(4): 713-719). In 2012, the laboratory cloned and identified sip gene comprising 1038bp and encoding 345 amino acid sequences from Bt strain QZL26, but its insecticidal activity has not been reported (Liu Yangjie, Lihao, Liu Rong Mei, etc.. Bt novel gene sip cloning, expression and bioinformatics analysis [ J]Biotechnological notification 2012(12) 101-. In 2013, Murawska et al applied genome sequencing technology and found that the strain IS5056 contained the sip gene, but they were not studied in depth. In 2015, 1188bp Sip protein which encodes 395 amino acids and is cloned and expressed from Bt strain DQ89 by Zhang jin wave shows that the protein has higher poisoning effect on bulla and LC thereof₅₀The value was 1.542. mu.g/mL (Zhang jin Bo, Liaotao, Liurongmei, etc.. Bt Strain DQ89 sip gene cloning, expression and insecticidal activity analysis [ J]The Chinese journal of biological control 2015,31(4): 598-. In addition, sandmonanthus chevalis clones sip1Aa gene from Bt strain QZL38, constructs its truncated mutant, removes the first 90bp signal peptide, and can produce 37.6kDa soluble eggWhite and tested for LC against bulleyana₅₀The value was 1.051. mu.g/mL (Sha J, Zhang J, Chi B, et al. Sip1Abgene from a native Bacillus thuringiensis strain QZL38 and its induced activity acquisition of collagen bowring Baly [ J].Biocontrol Science&Technology,2018,28(5):459-467.)。

Theoretically, PCR amplification is an exponential growth process, but in practice, the amplification curve of PCR is not a standard "J" shaped exponential curve, but rather a curve that approximates a "S" shape. This occurs because the number of amplification cycles increases with PCR. The amplification scale is exponentially and rapidly increased, and the elements of various PCR reactions such as Taq enzyme, dNTPs, primers, even DNA templates and the like are gradually short of demand, so that the PCR efficiency is lower and lower, and the increase rate of products is also gradually slowed down. When all the raw materials required for PCR are consumed, the PCR is in a plateau phase, and the amount of PCR products is saturated. Due to the complex interaction among various factors, the opportunity and height of different PCR reaction systems entering the platform phase are greatly different.

And obtaining an amplification curve of the plasmid through fluorescent quantitative PCR, detecting the amplification process of the whole PCR, observing a base line phase, a logarithmic phase and a platform phase of PCR reaction, determining the cycle number of PCR entering exponential amplification under the condition of different template amounts, and calculating to obtain the exponential amplification cycle number required for reaching the expected mutation rate. Selecting a plasmid with a proper concentration capable of generating as much product quantity as possible as a template of error-prone PCR, carrying out PCR amplification by using Taq DNA polymerase, carrying out double enzyme digestion reaction with a vector plasmid after PCR product gel is recovered and purified, carrying out T4 ligase connection, then transforming to an escherichia coli LM109 competent cell, screening positive clone, and storing a transformant, thus obtaining the sip1Aa random recombinant library.

In the application, the Sip1Aa mutant protein with high activity is expected to be obtained by a random recombination method, and for the research on key amino acid residues of bacillus thuringiensis playing a poisoning effect on pests, the higher the mutation rate is, the better the mutation rate is, so that the selection of the proper mutation rate is important. In addition, the relationship between the structure and function of insecticidal proteins is studiedIt is generally advisable to study mutations of individual amino acid residues, i.e.about 1.5 base mutations per segment of the gene of interest. If only proteins with significant changes in properties are desired, it is possible to increase the amount of Mg in the PCR system²⁺The concentration can increase the mutation rate and expand the capacity of the mutation library. Even so, when the number of mutated bases in each segment of the target gene exceeds 6, the protein often loses its original function.

In addition, in previous studies on Cry proteins, a series of mutants were constructed by random mutagenesis, in which the mutants with increased insecticidal activity were all at low mutation frequencies, and when the mutation frequency was too high, most of the mutations were harmful and beneficial mutations could hardly be screened (Vartanian JP, Henry M, Wain-Hobson S. hypermutagenic PCR incorporation of all mutations and a sizeable breeding of transitions. nucleic Acids Res,1996,24(14): 2627) 2631.). Meanwhile, if the mutation frequency is too low, the wild-type protein will occupy the advantages of the mutation library, and the ideal mutants cannot be screened. Therefore, this study selects a low mutation frequency that would expect 1.5 base mutations per segment of the gene of interest, and constructs a random recombinant library of sip1 Aa.

Disclosure of Invention

In order to delay insect resistance and obtain mutant proteins with higher insecticidal activity, the research utilizes an error-prone PCR technology to construct a random recombination library of the Sip1Aa protein, randomly selects 100 positive transformants for sequence determination, and generates 25 mutants in total, which are respectively named as M1-M25. A total of 29 base mutations occurred, with an average of 1.2 base mutations per mutant. Compared with the Sip1Aa protein, the insecticidal activity of the mutant M1(A31, Y118 and D227), M5(K168) and M21(I307) is obviously reduced, the activity is reduced by 4-6 times, meanwhile, the mutant M8(R174S) with improved insecticidal activity on simian beetle is obtained, the activity is improved by 4.02 times, and the activity of the mutant M6(F346V) is improved by 2.19 times, and the research result provides reference for molecular modification of the Sip1Aa protein and research on key sites of the insecticidal activity.

Mutant M8(R174S), the amino acid sequence of which is as shown in SEQ ID No. 3.

A gene encoding the mutant M8 (R174S).

Mutant M6(F346V), the amino acid sequence of which is shown in SEQ ID No. 5.

A gene encoding the mutant M6(F346V) described above.

The gene sequence is shown in SEQ ID No.4 and SEQ ID No. 6.

The mutant SEQ ID No.3 and SEQ ID No.5 show application in killing great ape beetle pests.

The mutant SEQ ID No.3 and SEQ ID No.5 are prepared into an insecticide to kill the great ape beetles.

The invention constructs the random recombination library of the Sip1Aa protein by using the error-prone PCR technology, randomly selects 100 positive transformants for sequence determination, generates 25 mutants in total and is respectively named as M1-M25. A total of 29 base mutations occurred, with an average of 1.2 base mutations per mutant. Compared with wild type Sip1Aa protein, the insecticidal activity of the mutant M1(A31, Y118 and D227), M5(K168) and M21(I307) is obviously reduced, the LC50 value is increased by 4-6 times, and meanwhile, the mutant M8(R174S) with improved insecticidal activity to the simian beetle is obtained, and the LC50 value is reduced by 4 times; and the activity of the M6(F346V) mutant was increased 2.19-fold. The research result provides reference for molecular modification of the Sip1Aa protein and research of key sites of insecticidal activity.

Drawings

FIG. 1 fluorescent quantitative PCR amplification/cycling plot,

FIG. 2 shows the result of PCR amplification,

m: DL2000 DNA Marker, 1-2: the concentration of the pAc19sip1Aa plasmid template is 3.5 ng/. mu.L, 35 and 28 cycles of PCR amplification are carried out respectively; 3-4: plasmid template concentration of pAc19sip1Aa is 35 ng/. mu.L, PCR is respectively amplified for 35 and 13 cycles,

FIG. 3 shows the error-prone PCR product and the enzyme digestion of the vector,

m: DL5000 and DL2000, 1: xho I and EcoR I enzyme digestion plasmid pAc19sip1Aa, then recycling 3kb carrier; 2: after the Xho I and EcoR I enzyme digestion error-prone PCR glue recovery product, a 1kb target band is recovered,

figure 4 shows the distribution of the abrupt points,

FIG. 5 random mutation library protein expression analysis,

M:Premixed Protein Marker(Low),CK-:pUC19,P:pAc19Sip1Aa,1:M 1,2:M 2,3:M 5,4:M 6,5:M 7,6:M 8,7:M 10,8:M 11,9:M 13,10:M 17,11:M 18,12:M 19,13:M 21and 14:M 25，

FIG. 6 analysis of mutation site structure.

Detailed Description

The present invention will be described in further detail with reference to examples.

1. Materials and reagents

1.1 strains and plasmids

The strains and plasmids used in the experiments are detailed in Table 1 and can be distributed to the public.

TABLE 1 strains and plasmids

1.2 primers

Random mutation primer

Designing random mutation primers according to the sequence of the sip1Aa gene, introducing Xho I enzyme cutting sites at the 5 'end of an upstream primer, and introducing EcoR I enzyme cutting sites at the 5' end of a downstream primer. The primer sequences are shown in Table 2.

TABLE 2 primer names and sequences

1.3 Medium and antibiotics

Liquid LB: tryptone 1%, NaCl 1%, yeast extract 0.5%; ampicillin (Ampicillin) 100mg of Ampicillin (Ampicillin) was dissolved in 1ml of sterile water and diluted 1000-fold for use in 0.22 μm filter sterilization.

1.5 enzymes and Biochemical reagents

Protein Marker was purchased from TaKaRa corporation; 2 × Taq Mix DNA polymerase was purchased from CWBIO; the DNA gel recovery kit was purchased from Axygen; other reagents are domestic analytical pure reagents.

1.6 test insects

The standardized great ape beetle test insect used in the experiment is a gift from the plant protection research of the Chinese academy of agricultural sciences.

2 method of experiment

2.1 plasmid concentration determination

mu.L of the plasmid was collected and the concentration was determined using a NanoDrop apparatus. Plasmid concentration gradients were diluted to 35 ng/. mu.L and 3.5 ng/. mu.L.

2.2 fluorescent quantitative PCR reaction

The plasmid pAc19sip1Aa transformed with E.coli JM109 was used as a template, sip1Aa-Xho I and sip1Aa-EcoR I were used as a primer pair, and qpCR amplification of a sip1Aa gene fragment was performed, and the reaction system was as follows:

reaction procedure: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, 40 cycles.

2.3 error-prone PCR reactions

A3.5 ng/. mu.L pAc19sip1Aa plasmid is used as a template, sip1Aa-Xho I and sip1Aa-EcoR I are used as primer pairs, and a sip1Aa gene fragment is amplified, wherein the reaction system is as follows:

PCR reaction procedure: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 59.8 ℃ for 30s, and extension at 72 ℃ for 1 min. 28 cycles of amplification and final extension at 72 ℃ for 10 min.

After the reaction is finished, the target fragment is recovered by glue.

2.4 enzyme digestion

Xho I and EcoRI are used for carrying out double enzyme digestion on pAc19sip1Aa plasmid and error-prone PCR gel recovery products simultaneously, and the reaction system is as follows:

reaction conditions are as follows: the reaction was carried out at 37 ℃ for 30 min.

After the reaction is finished, the enzyme digestion product is recovered by agarose gel.

2.5 ligation reaction

Connecting the double-restriction enzyme digestion vector fragment pAc19 after gel recovery with the error-prone PCR gel recovery product through T4 ligase, wherein the system is as follows:

reaction conditions are as follows: 16 ℃ overnight.

After the reaction, the Escherichia coli JM109 was transformed to be competent, and a random recombinant library was established.

2.6 sequencing and analysis

Screening positive clones in the random recombinant library, and randomly picking 100 positive clones for sequence determination.

2.7 random recombination library protein expression and extraction

(1) Selecting a recombinant single colony to be cultured in 5mL of liquid LB culture medium containing ampicillin at 37 ℃ and 220rpm/min for 12 h;

(2) inoculating into 100mL 2 XLB liquid culture medium according to 1% inoculum size, culturing at 37 deg.C and 220rpm/min for 16 h;

(3) centrifuging at 4 deg.C and 8000rpm/min for 10min, and collecting thallus;

(4) the precipitate was suspended in 5mL of 1 XTE solution;

(5) performing ultrasonic disruption in ice-water bath (80%, 3s, 3s, 10 min);

(6) centrifuging at 4 deg.C and 12000rpm/min for 10min, and collecting supernatant; SDS-PAGE detects protein expression.

(7) Suspending the precipitate with 2% Triton-100, centrifuging at 4 deg.C and 12000rpm for 10min, discarding the supernatant, and repeating for 3 times;

(8) suspending with sterile water, precipitating, centrifuging at 4 deg.C and 12000rpm/min for 10min, and removing supernatant;

(9) the pellet was suspended in 5mL PBS and stored until use.

2.8 SDS-PAGE electrophoresis

The polyacrylamide gel was formulated as shown in Table 3.

TABLE 3 formulation of SDS Polyacrylamide gels

Protein sample treatment: mixing 100 mu L of protein sample with 40 mu L of 5 Xloading buffer solution, then carrying out boiling water bath for 10min, and centrifuging at 12000rpm/min for 10 min;

loading and electrophoresis: and (3) taking 10 mu L of supernatant for sample loading, performing 80V pre-electrophoresis for 20min, and changing the condition of 120V electrophoresis until the protein sample is concentrated to a straight line.

Dyeing: after electrophoresis, the gel was removed, placed in a glass dish to which 100mL of SI had been added, heated with a microwave oven over a high fire for 30s, taken out and placed on a shaker, and decolorized at 70rpm/min for 5 min. The SI was decanted off and the gel was transferred to a glass dish to which 100mL SII and 2mL SIII had been added, heated on a microwave oven with a high fire for 30S, removed and placed on a shaker and stained at 70rpm/min for about 20min until a clear band appeared. The gel was removed and transferred to a glass dish containing distilled water and decolorized overnight at 70 rpm/min.

Imaging: the gel is stored by photographing through a gel imaging system.

2.9 insecticidal Activity assay

pAc19-Sip1Aa protein and random recombinant protein were diluted to different concentrations, and the virulence analysis was performed on the simian diabrotica using fresh pakchoi by leaf dip bioassay with the protein expressed from pUC19 in E.coli as negative control. The specific method comprises the following steps:

(1) selecting fresh and uniform-sized pakchoi leaves, cleaning with distilled water, and placing on filter paper for airing;

(2) putting a protein sample to be detected into a sterile culture dish, adding 0.1% household detergent into the sample, soaking the leaves in the protein sample for about 30s, and turning over the leaves once;

(3) placing the leaves on a preservative film for airing;

(4) putting the dried leaves into culture dishes paved with filter paper wetted by sterilized water respectively;

(5) gently inoculating the larva of the great ape concha into the brush pen, inoculating 16 heads of the larva to each leaf, and repeating the treatment for three times;

(6) placing in a biochemical incubator at 27 deg.C, spraying appropriate amount of sterilized water in the incubator in the morning and evening to maintain the humidity of the incubator air, and controlling the light cycle to 14/10 (light/dark);

counting the number of dead insects and the number of live insects in 48h, and calculating the mortality, correcting the mortality and LC by adopting SPSS software₅₀(95% confidence interval).

3 results of the test

3.1 determination of initial template concentration for PCR amplification

The concentration of pAc19sip1Aa plasmid was 268.3 ng/. mu.L as measured by a microspectrophotometer, Narodrop. The plasmid was diluted to 35 ng/. mu.L and 3.5 ng/. mu.L, and 1. mu.L of plasmid was taken as a template, and fluorescence quantitative PCR was performed using sip1Aa-Xho I and sip1Aa-EcoR I as primer pairs, while negative control was performed using water as a template, and each condition was repeated 3 times. The qPCR amplification results are shown in figure 1.

Since different PCR exponential amplification cycles lead to different mutation frequencies, the amplification of the pAc19sip1Aa plasmid was investigated by quantitative fluorescence PCR. According to the results of FIG. 1, when the concentration of pAc19sip1Aa plasmid template was 35 ng/. mu.L, PCR was in the exponential amplification phase at 10-22 cycles; when the concentration of pAc19sip1Aa plasmid template was 3.5 ng/. mu.L, PCR was in the exponential amplification phase at 25-35 cycles.

According to the calculation result, when the initial concentration of the pAc19sip1Aa plasmid template is 35 ng/. mu.L, 13 PCR cycles are needed to obtain the mutation rate of 1.5 bases; when the initial concentration of pAc19sip1Aa plasmid template was 3.5 ng/. mu.L, 28 PCR cycles were required. The results of PCR amplification under these conditions are shown in FIG. 2.

As can be seen from FIG. 2, the amount of PCR product increased with the increase of the number of PCR cycles with the same amount of template before the PCR amplification reached saturation. After the PCR products are subjected to gel recovery, concentration measurement shows that the initial concentration of the pAc19sip1Aa plasmid template is 35 ng/. mu.L, and the concentration of the products obtained by 13 PCR cycles of amplification is obviously lower than that of the products obtained by 28 PCR cycles of amplification, wherein the initial concentration of the pAc19sip1Aa plasmid template is 3.5 ng/. mu.L. For the convenience of subsequent gel recovery, enzyme digestion and other operations, pAc19sip1Aa plasmid template with initial concentration of 3.5 ng/. mu.L and 28 PCR cycles were selected as conditions for establishing random recombination library.

3.2 Generation of random recombinant libraries

The pAc19sip1Aa plasmid at an initial concentration of 3.5 ng/. mu.L was used as a template for error-prone PCR reactions, and after 28 cycles of PCR amplification, the product was recovered in gel. Then carrying out double enzyme digestion on the gel recovery product by using restriction enzymes Xho I and EcoR I, and recovering a target band with the size of 1 kb; at the same time, the pAc19sip1Aa plasmid was double digested with restriction enzymes Xho I and EcoR I, and the vector fragment of 3kb in size was recovered from the gel, as shown in FIG. 3. And connecting the gel recovery products of the two products according to a reaction system of T4 ligase, carrying out overnight reaction at 16 ℃, transforming E.coli JM109 competent cells, screening positive clones, and storing 1000 positive transformants in total to establish the sip1Aa random recombinant library.

3.3 random recombination library sequence analysis

In order to verify the base change of the random mutation target fragment, 100 positive transformants are randomly selected from the random mutation library for sequence determination, and the sequencing result is analyzed. Among them, 25 mutants were subjected to base change and named M1-M25, respectively. The statistics of the nucleotide and amino acid changes in the mutants are shown in Table 4.

TABLE 4 mutant base and amino acid sequences in random recombination libraries

Of the 100 transformants sequenced, 11 mutants had nucleotide changes but did not cause amino acid changes. The total number of mutants with nucleotide changes and amino acid changes is 14, accounting for 14%. Mutants with missense mutations were M1, M2, M5, M6, M7, M8, M10, M11, M13, M17, M18, M19, M21 and M25, respectively. Among the 25 mutants obtained, no mutation occurred and no mutation of base insertion and deletion was observed, even though a stop codon was generated after the mutation.

Sequencing analysis of 100 positive transformants showed that 25 mutants out of 100 transformants had base mutations, resulting in 29 nucleotide site mutations (Table 4), and that each mutant had 1.2 base mutations on average, which was substantially identical to the expected 1.5 base mutations per mutant. Of the 29 mutated nucleotide sites, most of the base substitutions occurred AT → GC, accounting for 82.8% of the total mutated sites, with less mutations of guanine and cytosine, only 3.4%, consistent with the previously reported propensity of Taq DNA polymerase to have AT → GC mismatches when performing PCR reactions.

3.4 distribution of abrupt change sites

Statistical analysis of 29 base mutation sites in the random mutation library using Origin 8.0 is shown in FIG. 4. Between 91 bp and 1005bp of the nucleotide sequence of sip1Aa, the base sites with mutation are distributed more uniformly, and no mutation hot spots are found. Except for the 1036 th base, 1 mutation is carried out on each base site. In the mutant M6, the 1036 th thymine is mutated into guanine, so that 346 th phenylalanine is replaced by valine; in the mutant M13, the 1036 th thymine was mutated to cytosine, resulting in the 346 th phenylalanine being mutated to leucine.

3.5 random mutant library protein expression

The proteins expressed by the recombinant strains in the random recombinant library in Escherichia coli JM109 were extracted, and SDS-PAGE analysis of the expression of the mutant proteins with missense mutation was performed, as shown in FIG. 5. Sip1Aa protein expressed under the guidance of cry1Ac promoter is used as a positive control, and pUC19 expressed in escherichia coli is used as a negative control. The results showed that all mutants in the random mutant library were successfully expressed in E.coli, and the expressed product contained 37.6kDa protein of interest, which was identical in size to Sip1Aa protein (FIG. 5).

3.6 measurement of biological Activity

Preliminary activity tests were first carried out on 14 mutants and the results showed that the lethality of the muteins M6(F346V) and M8(R174S) to bullosa was higher than that of the pAcSip1Aa protein and that the insecticidal activity of the muteins M1(A31G, Y118C, D227E), M2(N305I, T324A), M5(K168R) and M21(I307T) to bullosa was lower than that of pAcSip1 Aa. The muteins M7(F89L), M10(K168N), M11(I45V), M13(F364L), M17(D141G), M18(N317D), M19(K193K, E235G), M25(N75S) and pAcSip1Aa have no significant difference in the lethality to bullous apes.

TABLE 5 insecticidal results of Sip1Aa random muteins against Bullenia apetala

Mutants M1, M2, M5, M6, M8 and M21 were purified and used for counterscreening of the apes beetles, with PBS buffer as negative control. The protein concentration of the rescreened solution is set to be six concentration gradients of 0.1 mu g/mL, 1 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL and 50 mu g/mL, three times of repetition is set for each concentration, 16 head worms are inoculated in each repetition, after the solution is cultured in a climatic chamber at the temperature of 27 ℃ for 48 hours, the death condition of the insects is counted, and LC is calculated₅₀The value is obtained. The results of the biological activity assay rescreening are shown in Table 5.

TABLE 5 quantitative determination of the biological Activity of the Sip1Aa random mutein against the great Simian Blastoma

As can be seen from Table 6, the activity of the mutant M8 is greatly improved, the insecticidal activity against the great ape beetle is obviously better than that of pAc19Sip1Aa, the activity is improved by 4.02 times, and the insecticidal activity against the great ape beetle is improved by 2.19 times by the mutant M6. The insecticidal activity of the mutants M2 on the great ape beetle is slightly lower than that of pAc19Sip1Aa, M1, M5 and M21 on the great ape beetle, and the insecticidal activity is obviously lower than that of pAc19Sip1Aa protein, and is reduced by 4 times, 5 times and 6 times.

3.7 analysis of mutant site Structure

The structural analysis of the M1(A31, Y118, D227), M5(K168), M8(R174) and M21(I307) sites of mutants in a random recombination library of Sip1Aa protein was performed using PDB Viewer software.

Y118 and D260 are capable of forming two intramolecular hydrogen bonds, each having a bond length of

And

(a in FIG. 6). When threonine 118 is replaced by cysteine, C118 and D260 can only form a hydrogen bond with the length of

(b in FIG. 6). D227 and Y176 form two hydrogen bonds with the bond lengths of

And

(c in FIG. 6). When the 227 th aspartic acid is mutated into glutamic acid, E227 can form two hydrogen bonds with Y176, but the bond length is changed, namely

And

as shown at d in fig. 6. The mutation of A31G had no significant effect on the structure of the Sip1Aa protein. These mutations result in a reduction in the stability of mutant M1 compared to the wild-type Sip1Aa protein, resulting in a reduction in insecticidal activity against the macaca apelta.

The two bonds of the K168 and V236 forms in mutant M5 are of length

And

hydrogen bonds (e in fig. 6). When lysine 168 was replaced by arginine, the hydrogen bond disappeared (f in FIG. 6). The hydrogen bond formed by I307 and T306 in mutant M21 is as long as

(i in FIG. 6). After I307 was substituted with threonine, the hydrogen bond disappeared. Hydrogen bonding can maintain steric stabilization. The mutants M5 and M21 lose stable intramolecular hydrogen bonds, possibly resulting in the change of the spatial structure of the protein and reducing the insecticidal activity of the protein on the great ape beetle.

The arginine at position 174 in mutant M8 is located on the β 11 fold, as indicated by g in FIG. 6. When the basic arginine at position 174 was replaced with a neutral serine (h in FIG. 6), the mutant protein was increased 4-fold compared to the wild-type Sip1Aa protein. It is possible that at this position, neutral amino acids are more favorable for the Sip1Aa protein to exert a poisoning effect in the great ape beetle.

4 conclusion

The invention adopts the random recombination technology to transform the Sip1Aa protein, the insecticidal activity of the mutants M8 and M6 is obviously improved, the activity of the mutant M8 is improved by 4.02 times, and the insecticidal activity of the mutant M6 on the great ape beetle is improved by 2.19 times. In addition, because the promoter of the vector is changed, the expression quantity of the protein M8 and M6 of the Sip1Aa mutant is improved, the solubility is high, and the stability is good.

Sequence listing

<110> northeast university of agriculture

<120> Sip1Aa protein random recombinant mutant protein

<141> 2020-12-02

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 364

<212> PRT

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<400> 1

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Met Ile Thr Thr Phe Val Thr Pro Ser Met Ala Val Phe Ala Ala Glu

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Thr Lys Ser Pro Asn Leu Asn Ala Ser Gln Gln Ala Ile Thr Pro Tyr

35 40 45

Ala Glu Ser Tyr Ile Asp Thr Val Gln Asp Arg Met Lys Gln Arg Asp

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Arg Glu Ser Lys Leu Thr Gly Lys Pro Ile Asn Met Gln Glu Gln Ile

65 70 75 80

Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp Ile Phe Lys Asp Gln Asn

85 90 95

Asn Asn His Gln Thr Asn Arg Phe Ile Ser Trp Phe Lys Asp Asn Leu

100 105 110

Ala Ser Ser Lys Gly Tyr Asp Ser Ile Ala Glu Gln Met Gly Leu Lys

115 120 125

Ile Glu Ala Leu Asn Asp Met Asp Val Thr Asn Ile Asp Tyr Thr Ser

130 135 140

Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser Glu Leu Thr Asn Tyr

145 150 155 160

Thr Gly Thr Thr Gln Lys Met Lys Thr Asp Ser Phe Gln Arg Asp Tyr

165 170 175

Thr Lys Ser Glu Ser Thr Ser Val Thr Asn Gly Leu Gln Leu Gly Phe

180 185 190

Lys Val Ala Ala Lys Gly Val Val Ala Leu Ala Gly Ala Asp Phe Glu

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Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr Thr Glu Thr Asn Thr

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Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu Val Thr Leu Ser Pro

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Gly His Lys Ala Val Val Lys His Asp Leu Arg Lys Met Val Tyr Phe

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Gly Thr Gln Asp Leu Lys Gly Asp Leu Lys Val Ser Phe Asn Asp Lys

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Glu Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr Arg Ser Ile Asp Leu

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Ser Asp Ile Arg Lys Thr Met Ile Glu Ile Asp Lys Trp Asn His Val

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Asn Thr Ile Asp Phe Tyr Gln Leu Val Gly Val Lys Asn His Ile Lys

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Asn Gly Asp Thr Leu Tyr Ile Asp Thr Pro Ala Glu Phe Thr Phe Asn

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Gly Ala Asn Pro Tyr Tyr Arg Ala Thr Phe Thr Glu Tyr Asp Glu Asn

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Gly Asn Pro Val Gln Thr Lys Ile Leu Ser Gly Asn

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<210> 2

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<212> DNA

<213> Bacillus thuringiensis (Bacillus thuringiensis)

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atgaaataca agttttcaaa agtcgttaag tgtactttac cagctttaat gattactaca 60

ttcgttactc caagtatggc agtttttgcc gcagaaacca agtcgccaaa tctaaatgca 120

tctcaacaag caataactcc atatgctgaa tcttatattg atacggttca agatagaatg 180

aaacaaagag atagggaatc aaaactaact ggtaagccaa taaatatgca agaacaaata 240

atagatggat ggtttttagc tagattctgg atatttaaag atcaaaataa caatcatcaa 300

acaaatagat ttatatcctg gtttaaagat aatcttgcta gttcgaaggg gtatgacagt 360

atagcagaac aaatgggctt aaaaatagaa gcattaaatg atatggatgt aacaaatatt 420

gattatacat ctaaaacagg tgataccata tataatggaa tttctgaact aacaaattat 480

acaggaacaa cccaaaaaat gaaaaccgat agttttcaaa gagattatac aaaatctgaa 540

tccacttcag taacaaatgg gttacaatta ggatttaaag ttgctgctaa gggagtagtt 600

gcattagcag gtgcagattt tgaaacaagt gttacctata atttatcatc tactacaact 660

gaaacaaata caatatcgga taagtttact gttccatctc aagaagttac attatcccca 720

ggtcataaag cagtagtgaa acatgatttg agaaaaatgg tgtattttgg gactcaagat 780

ttaaagggtg atttaaaagt aagttttaat gataaagaga ttgtacaaaa atttatttat 840

ccaaattata gatcaattga tttatctgat attcgtaaaa caatgattga aattgataaa 900

tggaatcatg taaataccat tgacttttat caattagttg gagttaaaaa tcatataaaa 960

aatggtgata ctttatatat agataccccg gccgaattta catttaatgg agctaatcca 1020

tattatagag caacatttac agaatacgac gagaacggaa atcctgttca aacaaagatt 1080

ttaagtggaa attaa 1095

<210> 3

<211> 364

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Lys Tyr Lys Phe Ser Lys Val Val Lys Cys Thr Leu Pro Ala Leu

1 5 10 15

Met Ile Thr Thr Phe Val Thr Pro Ser Met Ala Val Phe Ala Ala Glu

20 25 30

Thr Lys Ser Pro Asn Leu Asn Ala Ser Gln Gln Ala Ile Thr Pro Tyr

35 40 45

Ala Glu Ser Tyr Ile Asp Thr Val Gln Asp Arg Met Lys Gln Arg Asp

50 55 60

Arg Glu Ser Lys Leu Thr Gly Lys Pro Ile Asn Met Gln Glu Gln Ile

65 70 75 80

Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp Ile Phe Lys Asp Gln Asn

85 90 95

Asn Asn His Gln Thr Asn Arg Phe Ile Ser Trp Phe Lys Asp Asn Leu

100 105 110

Ala Ser Ser Lys Gly Tyr Asp Ser Ile Ala Glu Gln Met Gly Leu Lys

115 120 125

Ile Glu Ala Leu Asn Asp Met Asp Val Thr Asn Ile Asp Tyr Thr Ser

130 135 140

Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser Glu Leu Thr Asn Tyr

145 150 155 160

Thr Gly Thr Thr Gln Lys Met Lys Thr Asp Ser Phe Gln Ser Asp Tyr

165 170 175

Thr Lys Ser Glu Ser Thr Ser Val Thr Asn Gly Leu Gln Leu Gly Phe

180 185 190

Lys Val Ala Ala Lys Gly Val Val Ala Leu Ala Gly Ala Asp Phe Glu

195 200 205

Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr Thr Glu Thr Asn Thr

210 215 220

Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu Val Thr Leu Ser Pro

225 230 235 240

Gly His Lys Ala Val Val Lys His Asp Leu Arg Lys Met Val Tyr Phe

245 250 255

Gly Thr Gln Asp Leu Lys Gly Asp Leu Lys Val Ser Phe Asn Asp Lys

260 265 270

Glu Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr Arg Ser Ile Asp Leu

275 280 285

Ser Asp Ile Arg Lys Thr Met Ile Glu Ile Asp Lys Trp Asn His Val

290 295 300

Asn Thr Ile Asp Phe Tyr Gln Leu Val Gly Val Lys Asn His Ile Lys

305 310 315 320

Asn Gly Asp Thr Leu Tyr Ile Asp Thr Pro Ala Glu Phe Thr Phe Asn

325 330 335

Gly Ala Asn Pro Tyr Tyr Arg Ala Thr Phe Thr Glu Tyr Asp Glu Asn

340 345 350

Gly Asn Pro Val Gln Thr Lys Ile Leu Ser Gly Asn

355 360

<210> 4

<211> 1095

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaaataca agttttcaaa agtcgttaag tgtactttac cagctttaat gattactaca 60

ttcgttactc caagtatggc agtttttgcc gcagaaacca agtcgccaaa tctaaatgca 120

tctcaacaag caataactcc atatgctgaa tcttatattg atacggttca agatagaatg 180

aaacaaagag atagggaatc aaaactaact ggtaagccaa taaatatgca agaacaaata 240

atagatggat ggtttttagc tagattctgg atatttaaag atcaaaataa caatcatcaa 300

acaaatagat ttatatcctg gtttaaagat aatcttgcta gttcgaaggg gtatgacagt 360

atagcagaac aaatgggctt aaaaatagaa gcattaaatg atatggatgt aacaaatatt 420

gattatacat ctaaaacagg tgataccata tataatggaa tttctgaact aacaaattat 480

acaggaacaa cccaaaaaat gaaaaccgat agttttcaaa gagtttatac aaaatctgaa 540

tccacttcag taacaaatgg gttacaatta ggatttaaag ttgctgctaa gggagtagtt 600

gcattagcag gtgcagattt tgaaacaagt gttacctata atttatcatc tactacaact 660

gaaacaaata caatatcgga taagtttact gttccatctc aagaagttac attatcccca 720

ggtcataaag cagtagtgaa acatgatttg agaaaaatgg tgtattttgg gactcaagat 780

ttaaagggtg atttaaaagt aagttttaat gataaagaga ttgtacaaaa atttatttat 840

ccaaattata gatcaattga tttatctgat attcgtaaaa caatgattga aattgataaa 900

tggaatcatg taaataccat tgacttttat caattagttg gagttaaaaa tcatataaaa 960

aatggtgata ctttatatat agataccccg gccgaattta catttaatgg agctaatcca 1020

tattatagag caacatttac agaatacgac gagaacggaa atcctgttca aacaaagatt 1080

ttaagtggaa attaa 1095

<210> 5

<211> 364

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Lys Tyr Lys Phe Ser Lys Val Val Lys Cys Thr Leu Pro Ala Leu

1 5 10 15

Met Ile Thr Thr Phe Val Thr Pro Ser Met Ala Val Phe Ala Ala Glu

20 25 30

Thr Lys Ser Pro Asn Leu Asn Ala Ser Gln Gln Ala Ile Thr Pro Tyr

35 40 45

Ala Glu Ser Tyr Ile Asp Thr Val Gln Asp Arg Met Lys Gln Arg Asp

50 55 60

Arg Glu Ser Lys Leu Thr Gly Lys Pro Ile Asn Met Gln Glu Gln Ile

65 70 75 80

Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp Ile Phe Lys Asp Gln Asn

85 90 95

Asn Asn His Gln Thr Asn Arg Phe Ile Ser Trp Phe Lys Asp Asn Leu

100 105 110

Ala Ser Ser Lys Gly Tyr Asp Ser Ile Ala Glu Gln Met Gly Leu Lys

115 120 125

Ile Glu Ala Leu Asn Asp Met Asp Val Thr Asn Ile Asp Tyr Thr Ser

130 135 140

Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser Glu Leu Thr Asn Tyr

145 150 155 160

Thr Gly Thr Thr Gln Lys Met Lys Thr Asp Ser Phe Gln Arg Asp Tyr

165 170 175

Thr Lys Ser Glu Ser Thr Ser Val Thr Asn Gly Leu Gln Leu Gly Phe

180 185 190

Lys Val Ala Ala Lys Gly Val Val Ala Leu Ala Gly Ala Asp Phe Glu

195 200 205

Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr Thr Glu Thr Asn Thr

210 215 220

Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu Val Thr Leu Ser Pro

225 230 235 240

Gly His Lys Ala Val Val Lys His Asp Leu Arg Lys Met Val Tyr Phe

245 250 255

Gly Thr Gln Asp Leu Lys Gly Asp Leu Lys Val Ser Phe Asn Asp Lys

260 265 270

Glu Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr Arg Ser Ile Asp Leu

275 280 285

Ser Asp Ile Arg Lys Thr Met Ile Glu Ile Asp Lys Trp Asn His Val

290 295 300

Asn Thr Ile Asp Phe Tyr Gln Leu Val Gly Val Lys Asn His Ile Lys

305 310 315 320

Asn Gly Asp Thr Leu Tyr Ile Asp Thr Pro Ala Glu Phe Thr Phe Asn

325 330 335

Gly Ala Asn Pro Tyr Tyr Arg Ala Thr Val Thr Glu Tyr Asp Glu Asn

340 345 350

Gly Asn Pro Val Gln Thr Lys Ile Leu Ser Gly Asn

355 360

<210> 6

<211> 1095

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaaataca agttttcaaa agtcgttaag tgtactttac cagctttaat gattactaca 60

ttcgttactc caagtatggc agtttttgcc gcagaaacca agtcgccaaa tctaaatgca 120

tctcaacaag caataactcc atatgctgaa tcttatattg atacggttca agatagaatg 180

aaacaaagag atagggaatc aaaactaact ggtaagccaa taaatatgca agaacaaata 240

atagatggat ggtttttagc tagattctgg atatttaaag atcaaaataa caatcatcaa 300

acaaatagat ttatatcctg gtttaaagat aatcttgcta gttcgaaggg gtatgacagt 360

atagcagaac aaatgggctt aaaaatagaa gcattaaatg atatggatgt aacaaatatt 420

gattatacat ctaaaacagg tgataccata tataatggaa tttctgaact aacaaattat 480

acaggaacaa cccaaaaaat gaaaaccgat agttttcaaa gagattatac aaaatctgaa 540

tccacttcag taacaaatgg gttacaatta ggatttaaag ttgctgctaa gggagtagtt 600

gcattagcag gtgcagattt tgaaacaagt gttacctata atttatcatc tactacaact 660

gaaacaaata caatatcgga taagtttact gttccatctc aagaagttac attatcccca 720

ggtcataaag cagtagtgaa acatgatttg agaaaaatgg tgtattttgg gactcaagat 780

ttaaagggtg atttaaaagt aagttttaat gataaagaga ttgtacaaaa atttatttat 840

ccaaattata gatcaattga tttatctgat attcgtaaaa caatgattga aattgataaa 900

tggaatcatg taaataccat tgacttttat caattagttg gagttaaaaa tcatataaaa 960

aatggtgata ctttatatat agataccccg gccgaattta catttaatgg agctaatcca 1020

tattatagag caacagttac agaatacgac gagaacggaa atcctgttca aacaaagatt 1080

ttaagtggaa attaa 1095

Claims (10)

1. The amino acid sequence of the mutant M8 is shown in SEQ ID No. 3.

2. A gene encoding the mutant M8 of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is shown in SEQ ID No. 4.

4. Use of the mutant M8 according to claim 1 for killing a pest simian beetle.

5. The use according to claim 4, wherein the mutant M8 of claim 1 is formulated as an insecticide against bulleyaconitum apetalum.

6. The amino acid sequence of the mutant M6 is shown in SEQ ID No. 5.

7. A gene encoding the mutant M6 of claim 6.

8. The gene of claim 7, wherein the nucleotide sequence is shown in SEQ ID No. 6.

9. Use of the mutant M6 according to claim 6 for killing a pest simian beetle.

10. The use according to claim 9, wherein the mutant M6 according to claim 6 is formulated as an insecticide against bulleyana.

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