CN111850009A - A kind of Cry2Ab-2 insecticidal gene and its application - Google Patents

Abstract

The invention provides a mutant gene Cry2Ab-2, wherein the base sequence of the mutant gene Cry2Ab-2 is shown as a sequence table SEQ ID NO.1, and the accession number of the application NCBI is as follows: MT 273011.1. The invention provides a Cry2Ab-2 insecticidal gene, which is obtained by randomly mutating a base sequence of a Cry2Ab gene by an error-prone PCR method, has strong insecticidal activity, and can provide a candidate gene for cultivation of transgenic insect-resistant breeding and construction of engineering strains.

Description

A kind of Cry2Ab-2 insecticidal gene and its application

technical field

The invention relates to the field of biotechnology, in particular to a Cry2Ab-2 insecticidal gene and its application.

Background technique

Corn (Zeamays L.) is an important cereal crop, which can be used as food, feed and industrial raw materials. Since the introduction of high-yielding selective breeding and hybrid varieties, maize production has increased significantly, reaching nearly 20.8 and 215 million tons, respectively, in 2014. However, as China's population grows and the amount of arable land steadily declines, crop yields must increase to meet growing demand. A variety of factors limit the yield of maize, among which pests are important. The main pests of maize crops are lepidopteran insects, which cause 10% loss of spring maize yield and 20%-30% reduction in summer maize yield, causing huge economic losses every year. In addition to direct yield loss, infestation of maize by lepidopteran pests can result in production of fumonisins, mycotoxins, reduced maize quality, and possible negative effects on livestock. Various strategies have been developed to control corn pests, with the application of chemical pesticides as the main measure. However, the application of chemical pesticides has brought a series of problems, such as air, water and soil pollution, food contamination, the re-emergence of resistant herbivores, and the reduction of the number of natural enemies of crop pests. Therefore, cultivating new varieties of maize insect-resistant, reducing the use of chemical pesticides, and fundamentally controlling the impact of insect pests on crops have become an important direction for maize variety cultivation. Currently, the most widely used insect-resistant gene is Bt from Bacillus thuringiensis protein.

Bacillus thuringiensis (Bt) is a Gram-positive spore-forming bacterium with entomopathogenic characteristics. Bt produces insecticidal proteins as parasporal crystals during the sporulation stage. These crystals are mainly composed of one or more proteins (Cry and Cyt toxins), also known as delta-endotoxins. Cry proteins are accompanying inclusion body (crystal) proteins from Bacillus thuringiensis with experimentally verifiable toxic effects on target organisms or with significant sequence similarity to known Cry proteins. Similarly, Cyt proteins are paraspore-containing proteins from Bacillus thuringiensis that exhibit hemolytic (Cytolitic) activity or have significant sequence similarity to known Cyt proteins. These toxins are highly specific to their target insects, harmless to humans, vertebrates and plants, and are fully biodegradable. Therefore, Bt is a viable alternative for controlling pests and important human disease vectors in agriculture. As of May 2018, nearly 90 Cry2 insect-resistant genes have been excavated and identified, covering 9 categories from Cry2Aa to Cry2Ai, of which Cry2Ab protein accounts for the vast majority. Cry2 is the insecticidal crystal protein second only to Cry1. It has good insecticidal activity against Lepidoptera, Orthoptera and other insects. Among them, Cry2Ab, Cry2Ae and Cry2Af proteins only have insecticidal activity against Lepidoptera. , and the virulence of Cry2Ab to armyworm is higher than that of Cry1Ab and Cry1Ac, so it has a wider application prospect in pest control.

At present, the main methods of Bt protein transformation include domain switching, codon optimization, DNA Shuffling, error-prone PCR, etc. Among them, error-prone PCR technology can easily and effectively introduce mutations into known DNA sequences. The system was first established by Leung, Cadwell Error-prone PCR was performed with this system, the gene encoding Tetrahymena ribozyme was mutagenized, and the activity of the enzyme was significantly improved. Shelly Goomber et al. Evolved Bacillus lipase in vitro by error-prone PCR to make it still active at 5°C, enhancing the cold adaptability of the enzyme. In order to solve the problem of chemical pesticides polluting the environment, a Cry2Ab-2 insecticidal gene and application are provided.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Cry2Ab-2 insecticidal gene and its application in order to solve the problem of chemical pesticides polluting the environment.

The technical scheme of the present invention is realized as follows:

The present invention provides a mutant gene Cry2Ab-2, and the base sequence of the mutant gene Cry2Ab-2 is shown in SEQ ID NO. 1 of the sequence table. The registration number to apply for NCBI is: MT273011.1.

As a further improvement of the present invention, the gene is artificially synthesized.

The present invention further protects the Cry2Ab-2 protein, which is the protein expressed by the gene shown in SEQ ID NO.1 of the sequence table.

The present invention further protects the application of the above-mentioned Cry2Ab-2 protein as the preparation of pesticides.

As a further improvement of the present invention, the worms are armyworms.

The present invention further protects the application of the above mutant gene Cry2Ab-2 in cultivating armyworm-resistant transgenic plant varieties.

As a further improvement of the present invention, the plant is corn.

The present invention further protects the biological material of the above-mentioned mutant gene Cry2Ab-2, and the biological material is an expression cassette, a vector, an engineered bacteria or a cell.

As a further improvement of the present invention, the vectors include prokaryotic expression vectors and cloning vectors.

As a further improvement of the present invention, the engineered bacteria are Escherichia coli.

The invention has the following beneficial effects: the invention provides the Cry2Ab-2 insecticidal gene, which is obtained by random mutagenesis of the base sequence of the Cry2Ab gene by the error-prone PCR method. The 20th position of Thr in the amino acid sequence of -2 gene was converted into Ala, and the 582nd Gly was converted into Asp, which was 97.31% identical to the original sequence. Online analysis using the online allergen database showed that the sequence was identical to known allergens. There is no sensitization due to low origin. The results of SDS-PAGE electrophoresis analysis showed that an obvious specific band appeared at about 70.68KDa, indicating that the Cry2Ab-2 gene was correctly expressed under the induction of IPTG. The anti-insect (army worm) test was carried out with the induced expression protein, and the results showed that the expressed protein had strong insecticidal activity, and the lethality rate of army worms reached 88.98%, and the growth of the surviving pests was also severely inhibited, which was significantly different from the control. . It is preliminarily proved that this study successfully mutagenized an improved Cry2Ab gene, which has strong insecticidal activity and can provide candidate genes for the cultivation of transgenic insect-resistant breeding and the construction of engineered strains.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

Figure 1 is the electrophoresis chart of different Mg ²⁺ contents; M: DL2000 DNA marker; H: sterile water; 1-11: the Mg ²⁺ concentration in the reaction system is 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5mmol/L;

Figure 2 shows the electrophoresis of error-prone PCR; M: DL2000 DNA marker; 1-6: Error-prone PCR amplification of Cry2Ab-2;

Figure 3 is a diagram of the conserved domain of Cry2Ab-2;

Figure 4 shows the PCR verification of the recombinant plasmid; M: DL2000 DNA marker; 1-4: verification of the recombinant plasmid;

Figure 5 is the double-enzyme digestion identification diagram of the Cry2Ab-2 gene plasmid, M: DL2000 DNA marker; 1-2: enzyme digestion verification;

Figure 6 is the SDS-PAGE analysis chart of pET22b-Cry2Ab-2 protein, M: protein standard molecular weight; 1-3: Cry2Ab-induced;

Figure 7 is a diagram showing the identification of prokaryotic expression protein against insects.

Detailed ways

The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 Error-prone PCR amplification

Primers were designed using the software Primer5.0

s:CGCGATGAATAGTGTATTGAATAGCGGAAGAACTAC,

As: CGCGTTAATAAAGTGGTGAAATATTAGTTGGTAC,

Using the plasmid containing pUC57-Cry2Ab as a template, error-prone PCR was performed. The 50μl error-prone PCR reaction system contained 10.5μl of ddH ₂ O, 5μl of 10*Buffer, 3μl of Mgcl ₂ , and 4μl of dNTPs. 1ul of upstream and downstream primers, 1ul of DNA, and 0.25ul of Taq enzyme. Reaction procedure: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 40 s; annealing at 40 °C for 40 s, extension at 72 °C for 10 min; storage at 4 °C. No warm start is performed. 1% agarose gel electrophoresis was performed to purify and recover the target fragment. Using this fragment as a template, a second round of error-prone PCR was performed under the same conditions. After 1% agarose gel electrophoresis, purification and sequencing were performed. The sequencing results were compared and analyzed using the software DNAman6.0, and the allergenicity was analyzed online using the online allergen database (www.allergenonline.org) according to the national standard for allergy analysis of exogenous proteins.

Using low-fidelity Taq DNA polymerase with different magnesium ion concentrations, prolonging the time of each cycle, reducing the final concentration of the starting template, using primers with substitution sites, and not performing hot-start, error-prone PCR amplification. After preliminary exploration, it was finally determined that 4 μl of Mg ²⁺ was added (as shown in Figure 1), and 70 cycles were suitable conditions. Error-prone PCR could amplify to obtain clear and specific bands (as shown in Figure 2).

The construction of embodiment 2 cloning vector

The target fragment purified and recovered after two rounds of error-prone PCR was ligated with the vector PMD-18T overnight at 16°C. The ligation product was transformed into E. coli DH5α competent cells, and a single colony was picked for overnight culture the next day, and the plasmid was extracted for PCR verification. The PCR product was purified and recovered and sent to Jilin Province Kumei Biotechnology Co., Ltd. for sequencing verification. Named the Cry2Ab-2 gene sequence.

Cry2Ab-2 Gene Sequence and Conserved Domain Analysis and Function Prediction

Deduced from the DNA sequence, the encoded protein contains 633 amino acids, and the theoretical molecular weight is 70.68KDa. Comparison of homology by online blast (NCBI) showed that this gene was most similar to Cry2Aa. Analysis of the secondary protein structure encoded by the mutant gene using the online software SOPMA showed that α-helix=32.54%, β-turn=5.06%, Randomcoil=41.71%, Ee-strand=20.85%. The CDD (Convsed Domain Database) analysis of the amino acid sequence of the protein encoded by the gene on the website NCBI shows that the Domain I range of the protein encoded by Cry2Ab-2 is Glu165~Leu183, which are mainly involved in membrane penetration and pore formation, and Domain II corresponds to the range Ser343~Ser354 are mainly involved in the binding of receptors. The corresponding range of Domain III is Thr567~Ala585, which is related to the binding of carbohydrates and is a part of the delta endotoxin activation region, which can produce insecticidal toxins. Figure 3 shows that the 70 amino acid sequence of the protein encoded by the Cry2Ab-2 gene has less than 35% sequence homology with known allergens, that is, the protein encoded by the Cry2Ab-2 gene has no allergenicity .

Example 3 Construction and identification of prokaryotic expression vector

The plasmid DNA of pMD18T-Cry2Ab-2 was extracted for normal PCR amplification using seamless primers with XhoI and SalII restriction sites. Amplification conditions: pre-denaturation at 94°C for 5 min; 2 min extension at 72°C; 10 min post extension at 72°C; 35× cycle. After PCR, the product was subjected to 1% agarose gel electrophoresis, and the target fragment was purified and recovered. Using a seamless ligation kit, it was ligated with the prokaryotic expression vector pET-22b, and then transformed into E. coli DH5α competent cells. The next day, all single colonies were picked for overnight culture, and their plasmid DNA was extracted for PCR and enzyme digestion verification. The PCR products that were initially determined to be positive were sent to Jilin Province Kumei Biotechnology Co., Ltd. for sequencing verification.

Construction of prokaryotic expression vector of Cry2Ab-2 gene

Pick all the colonies for overnight culture, extract the plasmids for PCR verification the next day (as shown in Figure 4), screen out stable positive clones, and perform restriction digestion on the recombinant plasmid DNA to obtain 2 bands with sizes of 5.5kb and 1902bp respectively (as shown in Figure 5). ), sequencing analysis showed that the sequence was correct, indicating that the target gene fragment was successfully connected to the prokaryotic expression vector p ET22b(+).

Example 4 Expression of Cry2Ab-2 gene in Escherichia coli

Extract the plasmid DNA of pET22b-Cry2Ab-2, transform it into Escherichia coli BL21(DE3) competent cells, pick a single colony for overnight culture, and inoculate the bacterial solution at a ratio of 1:100 to a 50ml container the next day. In the LB liquid medium (containing Kan100mg/ml), shake culture at 37°C at 198rpm for 3.5h, when the OD600 reaches 0.6, add 100mM IPTG stock solution to its final concentration of 1mM, continue shaking culture for 3h. The conical flask was placed on ice for 5 min, and the cells were collected by centrifugation (4°C, 10000 r/min, 8 min), and the cells were resuspended in pre-cooled sterile water and centrifuged again, and repeated twice. Discard the supernatant and resuspend in PBS Buffer. The suspension was disrupted by ultrasonic wave (1200W, 6s on, 5s off for 10min), centrifuged (4°C, 10000r/min, 10min) to collect bacterial cells and supernatant for use. SDS-PAGE electrophoresis analysis was performed for verification.

Expression analysis of Cry2Ab-2 gene in Escherichia coli

SDS-PAGE electrophoresis analysis, the results showed (Figure 6) that after induction by IPTG, the engineering bacteria containing Cry2Ab-2 (insoluble fraction) was about 70.68KDa, and an obvious specific band was expressed, which was consistent with the expected size, indicating that The insecticidal protein encoded by the Cry2Ab-2 gene was correctly expressed in E. coli BL21.

Example 5 Biological identification of Cry2Ab-2 protein insecticidal activity

After induction and expression of the recombinant bacterial cells, ultrasonication was performed to recover the Cry2Ab-2 protein, which was used for the identification of the following insecticidal activities.

The insecticidal activity of armyworm was identified by artificial diet method: the preparation of artificial diet was based on the method and conditions of Jiang SJ. The disrupted bacterial solution containing Cry2Ab-2 and Cry2Ab vector was used as positive control, and the disrupted bacterial solution containing empty vector was used as negative control.

Biological identification of insecticidal activity of Cry2Ab-2 protein (Fig. 7)

(1) Weigh 30g of artificial feed and place it in a sterilized 9cm petri dish;

(2) Add 3 mL of the protein solution to be tested, stir well, and evenly distribute it into three 9cm petri dishes after mixing;

(3) Put it in the ultra-clean table for a period of time according to the dryness and wetness of the feed, until there are no obvious water droplets on the surface of the feed.

(4) 30 newly hatched larvae were connected to each dish, and 3 repetitions were set, and a total of 90 test worms were processed (after the worms were connected, three layers of toilet paper were placed on the petri dish and tightly tied with a rubber band to prevent the worms from escaping);

(5) The samples were cultured in a 24°C light incubator, the photoperiod was 12:12, and the humidity was about 70% to 80%. Observe the dryness and wetness of the feed every day, and make appropriate adjustments;

(6) Investigate the number of dead and live worms after culturing for 7 days, and calculate the mortality rate.

As shown in Table 1, within 24h, the three groups of armyworm larvae all took a large amount of feed. With the prolongation of the treatment time, the newly hatched armyworm larvae that fed the target protein appeared atrophy and dead worms, while the negative treatment The group ate normally. The mortality of armyworms was counted after 7 days of treatment. As shown in Table 1, the mortality rates of newly hatched armyworms fed on Cry2Ab protein- and Cry2Ab-2 protein were 87.78% and 88.83%.

Table 1 The lethality of Cry2Ab protein to armyworm

Compared with the prior art, the present invention conducts random mutagenesis research on the base sequence of the Cry2Ab gene by the error-prone PCR method, and the result of the sequence analysis after the mutagenesis shows that: the Thr at position 20 of the amino acid sequence of the Cry2Ab-2 gene is converted into Ala, Gly at position 582 is converted into Asp, and the identity with the original sequence reaches 99.05%. The online analysis using the online allergen database shows that the sequence has low homology with known allergens and does not have sensitization. By SDS The results of -PAGE electrophoresis analysis showed that an obvious specific band appeared at about 70.68KDa, indicating that the Cry2Ab-2 gene was correctly expressed under the induction of IPTG. The anti-insect (army worm) test was carried out with the induced expression protein, and the results showed that the expressed protein had strong insecticidal activity, so that the death rate of army worm larvae reached 88.98%, and the growth of the surviving pests was also severely inhibited. The difference is obvious. It is preliminarily proved that this study successfully mutagenized an improved Cry2Ab gene, which has strong insecticidal activity and can provide candidate genes for the cultivation of transgenic insect-resistant breeding and the construction of engineered strains.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention. within the scope of protection.

sequence listing

<110> Jilin Agricultural University

<120> A Cry2Ab-2 insecticidal gene and its application

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<170> SIPOSequenceListing 1.0

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<211> 1902

<212> DNA

<213> Bacillus thuringiensis

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catgatccat ttagttttca acacaaatca ttagataccg tacaaaagga atggacggag 120

tggaaaaaaa ataatcatag tttataccta gatcctattg ttggaactgt ggctagtttt 180

ctgttaaaga aagtggggag tcttgttgga aaaaggatac taagtgagtt acggaattta 240

atatttccta gtggtagtac aaatctaatg caagatattt taagagagac agaaaaattc 300

ctgaatcaaa gacttaatac agacactctt gcccgtgtaa atgcggaatt gacagggctg 360

caagcaaatg tagaagagtt taatcgacaa gtagataatt ttttgaaccc taaccgaaac 420

gctgttcctt tatcaataac ttcttcagtt aatacaatgc aacaattatt tctaaataga 480

ttaccccagt tccagatgca aggataccaa ctgttattat tacctttatt tgcacaggcg 540

gccaatttac atctttcttt tattagagat gttattctaa atgcagatga atggggaatt 600

tcagcagcaa cattacgtac gtatcgagat tacttgaaaa attatacaag agattactct 660

aactattgta taaatacgta tcaaagtgcg tttaaaggtt taaacactcg tttacacgat 720

atgttagaat ttagaacata tatgttttta aatgtatttg agtatgtatc tatctggtcg 780

ttgtttaaat atcaaagtct tctagtatct tccggtgcta atttatatgc aagtggtagt 840

ggaccacagc agacccaatc atttacttca caagactggc catttttata ttctcttttc 900

caagttaatt caaattatgt gttaaatgga tttagtggtg ctaggctttc taataccttc 960

cctaatatag ttggtttacc tggttctact acaactcacg cattgcttgc tgcaagggtt 1020

aattacagtg gaggaatttc gtctggtgat ataggtgcat ctccgtttaa tcaaaatttt 1080

aattgtagca catttctccc cccattgtta acgccatttg ttaggagttg gctagattca 1140

ggttcagatc gggagggcgt tgccaccgtt acaaattggc aaacaggatc ctttgagaca 1200

actttagggt taaggagtgg tgcttttaca gctcgcggta attcaaacta tttcccagat 1260

tattttattc gtaatatttc tggagttcct ttagttgtta gaaatgaaga tttaagaaga 1320

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aatggtaatg gagctcgttt ttcagatatt aatatcggta atgtagtagc aagtagtaat 1800

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aatattatgc ttgtaccaac taatatttca ccactttatt aa 1902

Claims (10)

1. The mutant gene Cry2Ab-2 is characterized in that the base sequence of the mutant gene Cry2Ab-2 is shown in a sequence table SEQ ID NO. 1.

2. The mutant gene Cry2Ab-2 of claim 1, wherein said gene is artificially synthesized.

Cry2Ab-2 protein, characterized in that the Cry2Ab-2 protein is expressed by a gene shown in a sequence table SEQ ID NO. 1.

4. Use of the Cry2Ab-2 protein of claim 3 as a preparation of an insecticide.

5. The use of claim 4, wherein the insect is armyworm.

6. Use of the mutant gene Cry2Ab-2 of claim 1 for breeding an anti-armyworm transgenic plant variety.

7. The use of claim 6, wherein the plant is maize.

8. Biomaterial containing the mutant gene Cry2Ab-2 of claim 1, wherein said biomaterial is an expression cassette, a vector, an engineered bacterium, or a cell.

9. The biomaterial of claim 8, wherein the vector comprises a prokaryotic expression vector, a cloning vector.

10. The biomaterial of claim 8, wherein the engineered bacterium is escherichia coli.

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