CN102659933B - Bacillus thuringiensis gene cry8like and cry8G combination and application thereof - Google Patents

Abstract

The invention relates to "bacillus thuringiensis gene cry8like2, combination with cry8G and application thereof", belonging to the technical field of biological control. The present invention isolates the cry 8lik1e gene from HBF-18, its nucleotide sequence is shown in SEQ ID NO1, and the cry8like2 synthetic gene is obtained after being connected with cry 8E, and its nucleotide sequence is shown in EQ ID NO2. The gene is highly virulent to coleopteran pests, especially to grubs. At the same time, the present invention provides a protein combination, Cry8like2 and Cry8G have a synergistic effect on coleopteran pests, which is of great significance to the biological control of major underground pests grubs and the sustainable development of my country's agricultural green, further for the development of grub-resistant Bt Engineering strains and transgenic crops provide gene sources.

Description

Bacillus thuringiensis gene cry8like, combination with cry8G and its application

technical field

The invention relates to the field of biotechnology prevention and control, in particular to a new combination of cry8like and cry8G genes of bacillus thuringiensis and its application.

Background technique

Grubs are the general term for the larvae of the superfamily Scarabaeoidea, belonging to the order Coleoptera and the family Scarabae. There are many kinds of scarab superfamily, there are more than 30,000 kinds recorded in the world, and more than 1,800 kinds have been recorded in my country at present. This kind of pest has miscellaneous feeding habits, wide distribution, hidden life, strong adaptability, and different life history. Among them, it is harmful to agriculture, forestry and pasture. There are more than 100 species of it, and it is very difficult to prevent and treat it. The most important ones are Holotrichia parallela, Holotrichia parallela, North China giant beetle (Holotrichia beita) and Anomala carpulenta (Xu Jianguo, Fan Hui, Zhang Mingkao, Yang Enhua, and Guo Lin. (2002) Life of the dark beetle Habit observation and control technology research, plant protection technology and extension, 9-10; Li Encai, Chen Jinhuan. (1998) Farmland grub population distribution and control technology, Shaanxi Agricultural Sciences, 48-49; Jiang Changsong, Jiang Fujun, and Bao Xinping. (2008) ) Peanut grub sowing stage one-time application of the whole growth period control technology, Modern Agricultural Science and Technology, 121-123). The living habits of grubs are complex and diverse, and the harmful insect states are also different. Some are harmful by adults, and some are harmful by larvae (Hu Qiongbo, 2004). Grub adults like to eat crops, forest fruit trees (poplar, elm, willow, apple, pear, etc.) leaves, tender stems, flower buds, fruit ears, etc., causing harm. The damage caused by grub larvae is even more serious. The larvae live underground, eat the germinated seeds, bite off the rhizomes of the seedlings, and eat the roots and tubers, etc., resulting in ranging from lack of seedlings and broken ridges, to severe cases of crop failure. Larvae not only have a wide range of damage, but also do damage for a long time, from spring to autumn, from sowing to harvesting. Grubs gnaw on underground roots and tubers, weakening crop growth and directly affecting yield and quality. For example, peanuts, sweet potatoes, potatoes, and radishes are severely damaged in spring and autumn. Grubs not only affect the appearance and quality of the product after eating, but also easily lead to the invasion of pathogenic bacteria from the wound, which brings great inconvenience to the storage of winter vegetables. In recent years, with the development of cities, the area of turf greening has increased year by year, and the maintenance of green vegetation has become an important task in urban development. Grub larvae bite turf rhizomes, and further cause disease invasion to become one of the main hazards of urban afforestation turf (Xie Mufa. (1999) Grass subterranean pests and their control, Guangdong Garden, 46-47.). In addition, grubs are also the intermediate host and transmission medium of the parasitic disease (Acanthocephalus porcini) that harms pigs. Acanthocephalus parasitizes in the small intestine of pigs and manure is applied to the field, and grubs are its intermediate hosts. Therefore, grubs are important pests that endanger agriculture, forestry, and urban greening.

Grubs are recognized as difficult-to-control underground pests at home and abroad. 86% of the loss of underground parts of crops is caused by grubs. According to statistics, the annual occurrence area of grubs in the country is about 7 million hectares, and the yield is generally reduced by 20% to 30%. In serious years, the occurrence area of grubs in China has reached 20 million hectares, and the yield reduction has reached more than 50% (Jiang Changsong, Jiang Fujun, and Bao Xinping. (2008) Control technology of peanut grubs with one-time application at planting stage and whole growth period, Modern Agricultural Science and Technology, 121-123) , or even become extinct, becoming the most important subterranean pest at present. Peanuts caused by grubs usually reduce production by 20% to 40%, and in severe cases can reduce production by 70% to 80%, or even stop production, and the quality of peanuts has greatly dampened the enthusiasm of the masses to plant peanuts (Liu Shusen, Li Kebin, Yin Jiao, Cao Yazhong (2008) Research progress on grub biological control. China Biological Control, 168-173; Xu Tingting, Jiang Chen, Gong Qingxuan, Chen Jinfeng, and Qu Mingjing. (2010) Research on the occurrence and pollution-free control of grubs in peanut fields in Qingdao, Peanut Journal , 42-44; Wang Zhenjun, Guo Zhigang, Lei Xiaotian, and Ru Deping. (2010) The causes of grub occurrence and control technology in peanut fields, Agricultural Science and Technology Communication, 198-200, 2010;). In mid-to-late August 2007, in the Heze area of Shandong Province, the average population density of grubs was 8.7 individuals/m ² , and the highest was 22 individuals/m ² (Wang Aidong. (2008) Farmland grub occurrence and comprehensive control technology, modern agricultural science and technology) . From 2003 to 2007, in Wudi County, a combination of black light trapping and soil excavation survey in spring, summer and autumn was adopted. A random survey was conducted on field crops, and the scarabs in soybean fields and corn fields reached 5.63 and 3.00 heads/m2 respectively. There are 7 types of beetle chafers in soybean field. Among them, the Chinese beetle is the most, accounting for 50.2%, followed by the verdigris beetle, accounting for 25.1%. North China giant black gill beetle ranks third, accounting for 13.0%, and other species are less. In the cornfield, North China giant beetles accounted for the most, accounting for 38.7%, followed by aeruginous beetles, accounting for 29.5%, and black beetles were third, accounting for 14.6%. Other species were less (Tian Fangwen, Peng Yanming, Zhang Weihua, and Cui Shaoying.( 2008) Investigation on the Species and Occurrence of Scarabs in Wudi County, Shandong Province, Anhui Agricultural Sciences, 12322-12323). At present, due to the promotion of mechanized operations and changes in the farming system of agricultural production, such as the promotion of technologies such as no-till farming and straw returning to the field, grubs have brought a comfortable living environment and abundant food, increasing the number of insects per unit area. The number of populations makes the damage of grubs more and more serious, especially in Northeast China, North China and Central China, which brings difficulties to the control of grubs (Pei Guiying, Ma Saifei, Liu Jian, Liu Baocai. (2010) Effects of different farming modes on the occurrence and yield of grubs in soybean fields , scientific cultivation).

Screening new Bt strains with specific insecticidal spectrum has always been the most active field of Bt research. Since the 1990s, when reviewing the history of Bt insecticides to control pests, people have found that the existing Bt insecticides are mainly used to control sensitive Lepidoptera pests on the ground, and are used to control Coleoptera leaf beetle pests. rare. However, parasporal crystal proteins and spores, which play the main role in insecticidal activity, are easily inactivated under ultraviolet radiation in the sun and lose their insecticidal activity (Chu Liliang, Zheng Guiling, Zhou Hongxu, Li Changyou, Li Guoxun. Two strains of Bt that kill Coleoptera pests Bioactivity of strains and identification of insecticidal protein genes, Journal of North China Agricultural Science, 2010 (3)). The application of Bt preparations to control grubs has certain advantages, because the living environment of grubs is concealed, which avoids the irradiation of paracellular crystals and spores by ultraviolet rays, prolongs the insecticidal duration and improves the insecticidal effect. Therefore, some Bt researchers have shifted their research focus to screening strains with insecticidal activity against the main underground pests, scarab larvae, and have made breakthroughs. At present, there have been many reports about strains and genes effective against grubs. In 1992, Ohba et al. screened for the first time in the world a new Bt strain (Bt .subsp.japonensis BuiBui), this is the first time that Bt has been used in research on the control of grubs. Sato et al. cloned a new insecticidal gene—cry8Ca1 from this strain in 1994. In 1994, cry8Aa1 and cry8Ba1 isolated from the Bt strain PS50C by Mycogen Company of the United States had obvious insecticidal activity on the flower beetle Cotinis sp. The Bt strain SDS-502 containing cry8Da discovered in 2003 by Shin-ichiro Asano et al. has high activity on the I. aeruginosa beetle.

The excavation of related genes in our country started after 2002. The Institute of Plant Protection, Hebei Academy of Agricultural Sciences screened several Bt strains with specific insecticidal activity against the larvae of Anomala.exoleta and Anomala..corpulenta. In 2004, our laboratory cloned the cry8Ca2 gene with high efficiency against C. aeruginosa from strain HBF-1, and then carried out mutation research to improve the insecticidal activity against C. aeruginosa. In 2006, our laboratory also found that the Bt strain Bt185 has good insecticidal activity against the black beetle. This is the first gene found to have insecticidal activity against the black beetle. In 2008, the model was cloned from this strain. The gene cry8Fa1 and the model gene cry8Ea1 with high activity against the black beetle. In 2009, our laboratory discovered a Bt strain HBF-18 with high insecticidal activity against both the black beetle and the large black beetle. A new gene cry8Ga1 of the second taxonomic level that is effective for both large and dark beetles. The research results show that there are strains and genes with specific insecticidal activity against grubs in nature. By screening highly effective insecticidal strains and genes, Bt can be used to effectively control grubs. At present, the Bt genes found to have insecticidal activity against grubs include cry3, cry8, cry23, cry18, cry37, cry43 and other genes. The cry8 gene is more researched, and the cry8 gene is composed of 1160-1210 amino acids, and the molecular weight is between 128-137kDa. The cloning of these strains and genes provides high-quality strain resources for the control of grubs in my country, and also provides genetic resources for the further development of grub-resistant transgenic crops.

After the 1990s, with the large-scale application of chemical pesticides, problems became more and more prominent. my country gradually restricted the use of related highly toxic pesticides. Since January 1, 2007, methamidophos has been completely banned. Application of highly toxic chemical pesticides in agricultural production. The gradual withdrawal of highly toxic chemical pesticides has undoubtedly freed up a huge market space for biological pesticides. According to the "Twelfth Five-Year Plan" determined by my country's pesticide industry, the pesticide industry will continue to deepen the substitution of highly toxic pesticides. It is estimated that by 2015, the proportion of highly toxic pesticides will be reduced from the current 20% to about 5%. The share of all pesticides will increase from less than 10% at present to more than 30%. In order to obtain the best social, economic and ecological benefits, improve the agricultural ecological environment, realize the sustainable development of my country's agricultural economy, ensure food and food safety production, and enhance the international competitiveness of my country's agricultural products, develop and promote grubs with biological control as the core Comprehensive prevention and control technology is the current mainstream trend.

Strain HBF-18 has good insecticidal activity against beetle and black beetle, from which the project team isolated the cry8Ga1 gene that is active against both beetle and beetle. The cry8Ga gene is also the only cry gene type reported so far that is active against the large black beetle. However, the expression product of the cry8Ga gene is quite different from that of the starting strain (HBF-18), and the results show that the activity of the amorphic mutant strain containing the cry8Ga gene against the large black beetle and the dark beetle is lower than that of the starting strain , and their LC ₅₀ are 50 times and 70 times higher respectively (Shu et al.2009) (Table 1-1). Other grub-active genes cloned by our research group did not have this phenomenon. The Bt amorphous mutant strains transferred with cry8Ca, cry8Ea, cry8Ha, and cry8Ia genes had comparable insecticidal effects to the original strains HBF-1, Bt185, and BtSU4 (Table 1-2, 1-3). These phenomena indicated that besides the cry8Ga gene, strain HBF-18 also contained some other new insecticidal genes.

Table 1-1. Comparison of the insecticidal activity of the strains HBF-18 and HD8G (only the expression strain of cry8Ga gene) against the large black beetle and the dark beetle

^a 95% confidence interval in parentheses

^bMean ± standard error

Table 1-2. Comparison of the insecticidal activity of strains HBF-1 and HD8C (strains that only express the cry8Ca gene) against C. aeruginosa

^a 95% confidence interval in parentheses ^b mean ± standard error

Table 1-3. Comparison of the insecticidal activity of strains Bt185 and HD8E (strains that only express the cry8Ea gene) against black beetles

^a 95% confidence interval in parentheses ^b mean ± standard error

The project team also found that the synergistic use of multiple genes can improve the control effect of Bt on grubs. The research results of Yan Guixin et al. showed that the co-expression of cry3Aa gene and cry8Ca gene can improve the insecticidal effect on A. aeruginosa (Yan et al.2009) (Table 1-4); the research results of Liu Jingjing et al. Can significantly improve the control effect (Liu et al.2010) (Table 1-5) of bacterial strain to Diablo beetle. The results of these studies indicated that the co-expression of multiple genes can improve the insecticidal effect. However, the activity of the amorphous mutant strain containing the cry8Ga gene was 50-fold and 70-fold lower than that of the original strain, indicating that in addition to the cry8Ga gene, the HBF-18 strain may also contain some other novel Insecticidal genes. These new insecticidal genes cannot be identified by traditional identification methods, so we need to use new methods to identify new genes to identify these genes. Solexa sequencing is a new and efficient method for identifying new genes.

Table 1-4. Comparison of insecticidal activity of strains HBF-1, Bt22 and 3A-HBF against potato leaf beetle, great ape beetle, and aeruginosa

Table 1-5. Comparison of the insecticidal activity of strains HBF-1, Bt185 and BIOT185 against Beetle aeruginosa and Beetle diablo

Contents of the invention

The invention separates the cry 81ike1 gene from HBF-18 and connects it with cry 8E to obtain the cry 8like2 synthetic gene. The gene has high toxicity to coleopteran pests, especially to grubs.

At the same time, the invention provides a protein combination, Cry8like2 and Cry8G have a synergistic effect on coleopteran pests.

Cry 8like1 protein, which has the amino acid sequence shown in SEQ ID NO3.

The protein is a Cry8like2 protein, and its amino acid sequence is as shown in SEQ ID NO4.

The gene encoding Cry 8like1 protein has the nucleotide sequence shown in SEQ ID NO1.

The nucleotide sequence of the gene encoding the Cry 8like2 protein is shown in SEQ ID NO2.

A vector containing the above-mentioned genes.

The vector is pSTK 8like2, as described in Figure 13.

A protein composition consisting of Cry8like2 and Cry8Ga1 proteins.

Application of the above-mentioned protein or protein composition in resisting coleopteran pests.

Said application is to make protein or protein composition into insecticide for killing coleopteran pests.

The application is to transfer the gene encoding the protein into plants or microorganisms to express the characteristics of resisting coleopteran pests.

The coleopteran pests are grubs.

In this study, the isolated Bt strain HBF-18 (cry8Ga1) containing the cry8Ga gene was studied. The goal is to isolate new insecticidal genes other than the cry8Ga gene from the HBF-18 strain that is highly active against the large black beetle, and analyze the function of the new protein. First, use Solexa high-throughput sequencing technology to determine the genome sequence of the strain, predict all coding genes, obtain the possible coding amino acid sequence of the strain, and use the existing reported insecticidal protein database for sequence comparison, and isolate HBF-18 from the HBF-18 strain. In addition to the cry8Ga gene, the new insecticidal gene 8like1 was analyzed, and the function of the new protein was analyzed. At the same time, the cry81ike2 gene was synthesized, which has high activity against the black beetle. Experiments have proved that cry8like2 and 8G gene combination have the same synergistic effect on the main underground pest grub. It is of great significance for the biological control and the green and sustainable development of agriculture in China. It will further provide a gene source for the development of grub-resistant Bt engineering strains and transgenic crops.

Description of drawings

Figure 1 sequence analysis of cry8like1

Figure 2 Cry8like1BlastP results

Figure 3 Transcription analysis of candidate genes in HBF-18 strain, A is the result of Cry8like1 RT-PCR, B is the result of Cry8Ga RT-PCR, M: BM2000 Marker; 1: HBF 18 3h cDNA; 2: HBF 18 3h RNA; 3 : HBF 18 5h cDNA; 4: HBF 18 5h RNA; 5: HBF-18 8h cDNA; 6: HBF-18 8h RNA; 7: HBF-18 12h cDNA; 8: HBF-18 12h RNA; 9: HBF-18 24h cDNA; 10: HBF-18 24h RNA; 11: HBF-18 genome positive control; 12: no template negative control

Figure 4 cry8like1 PCR product

M: λDNA/Eco130Ⅰ; 1: cry8like1 PCR product, 2: cry8like1 negative control

Figure 5 PCR amplification of fragments at both ends of PCR with overlapping primers

1: cry8like1 N-terminal PCR product; 2: cry8like1 N-terminal PCR negative control; 3: cry8EaC-terminal PCR product; 4: cry8EaC-terminal PCR negative control; M: λDNA/Eco130Ⅰ

Figure 6 Overlapping PCR products

M: λDNA/Eco130Ⅰ; 1: full-length cry8like2 gene obtained by overlapping PCR

Figure 7 Flow chart of recombinant plasmid construction

Figure 8 PCR identification of cry8 bacteria solution

M: λDNA/Eco130Ⅰ; 1, 2: PCR product of cry8like1 bacterial liquid; 3: positive control of cry8like1; 4: negative control of cry8like1; 5: PCR product of cry8like2 bacterial liquid; 6: positive control of cry8like2; 7: negative control of cry8like2

Figure 9 Plasmid enzyme digestion identification

M-BM10000; 4-cry8like1-pEB-JM109 plasmid digestion identification; 5-cry8like2-pEB-JM109 plasmid digestion identification;

Figure 10 Insoluble components of cry8like1 expression products

1-Marker, 2-Cry8like1

Figure 11cry8like2 plus restriction site PCR

M-λDNA/Eco130Ⅰ, 1-cry8like2 PCR product

Figure 12 Shuttle vector pSTK double restriction product

M-λDNA/Eco130Ⅰ, 1-pSTK double digestion product

Figure 13 Construction of expression vector pSTK

Figure 14 PCR identification of cry8like2 expression vector construction

M: BM5000, 1: cry8like2-pSTK-HD73-PCR identification

Figure 15 Plasmid enzyme digestion identification

Identification of M-BM10000, 1: cry8like2-pSTK-JM109 plasmid digestion

Figure 16cry8 expression protein

1-cry8like2 expression protein, M-BM10000

Figure 17 Mean and Standard Deviation

Detailed ways

1 Experimental materials

1.1 Strains and plasmids

The strains and plasmids used are shown in Table 21. The following strains and plasmids are kept by applicants and can be distributed to the public.

Table 21 Strains and plasmids

1.2 Reagents

1.2.1 Medium

Liquid LB medium: 0.5% yeast extract, 1.0% trytone, 1.0% NaCl, pH 7.0, sterilized at 121°C for 20mim.

Solid LB medium: add 1.3% agar powder to liquid LB medium, and sterilize at 121°C for 20mim.

BH medium: 3.7% Brain and heart infusion broth, sterilized at 121°C for 20mim.

Beef extract peptone medium: 3g beef extract, 5g soybean peptone, dissolve in distilled water, adjust the pH to 7.2 with NaOH, adjust the volume to 1000mL, and sterilize at 121°C for 20min.

1.2.2 Antibiotics

Ampicillin aqueous solution 100mg/ml, stored at -20°C, diluted 1000 times when used; chloramphenicol absolute ethanol solution 34mg/mL, stored at -20°C, diluted 1000 times when used. Kanamycin aqueous solution 50mg/ml, stored at -20°C, diluted 1000 times when used.

1.2.3 Enzymes and biochemical reagents

1) PCR amplification reagents were purchased from Beijing Bomaide Technology Development Co., Ltd. and TaKaRa Company;

2) Restriction enzymes and 2x ligation kits were purchased from TaKaRa;

3) Plasmid extraction and DNA recovery kits were purchased from Axygen;

4) The M-MLV first-strand synthesis system used for qRT-PCR was purchased from Invitrogen;

5) DNA molecular weight markers were purchased from Beijing Bomaide Technology Development Co., Ltd.;

6) Antibiotics were purchased from Beijing Dingsheng Weiye Biotechnology Co., Ltd. Other reagents are commercially available domestic or imported analytically pure or electrophoretic grade pure chemical reagents.

7) The PCR primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the sequence determination was completed by the Open Laboratory of Crop Gene Resources and Gene Improvement National Major Scientific Projects of the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

8) Protein molecular weight standards: Precision Plus Protein ^TM Standards (kDa): 250, 150, 100, 75, 50, 37, 25, 20, 15, 10.

9) DNA standard molecular weight:

λDNA/Eco130I (λ/E): 19329, 7743, 6223, 4254, 3472, 2690, 1882, 1489, 925, 421 (bp).

BM2000: 2000, 1000, 750, 500, 250, 100 (bp)

BM5000: 5000, 3000, 2000, 1000, 750, 500, 250, 100 (bp)

1.2.4 Commonly used solutions and buffers

1) Bt total DNA extraction solution

Solution I: 0.3% sucrose, 25mmol/L Tris·Cl (pH 8.0), 25mmol/L EDTA (pH 8.0), 50mg/mL Lysozyme.

Solution II: 0.1mol/L NaCl, 0.1% SDS, 0.1mol/L Tris·Cl (pH 8.0).

2) Bt protein extraction reagent:

Lysis solution: 50 mM Na ₂ CO ₃ , 50 mM EDTA. Preparation method: 5.30g of anhydrous sodium carbonate, 18.61g of EDTA ultrapure water to 1000ml, after sterilizing at 121℃/20mim, adjust the pH to 9.5, and add β-mercaptoethanol with a final concentration of 3% before use.

Sodium Acetate Buffer: 4M anhydrous NaAc. Preparation method: Dissolve 65.61g of anhydrous sodium acetate in a small amount of double-distilled water, adjust the pH to 4.5 with HAc, and then adjust the volume to 200ml.

Protein lysis buffer: 50 mM Na ₂ CO ₃ . Preparation method: 5.30g of anhydrous Na ₂ CO ₃ , dilute to 1000ml with ultrapure water, pH9.51M NaCl: 58.44g of NaCl, dilute to 1000ml with ultrapure water.

3) Agarose gel electrophoresis buffer:

50×TAE Buffer (pH8.5): 242g Tris, Na ₂ EDTA·2H ₂ O 37.2g, add 800ml deionized water, stir well to dissolve, then add 57.1ml glacial acetic acid, stir well and set the volume to 1000ml. Dilute 50 times.

4) Protein electrophoresis reagents and buffers:

3× Loading buffer: Tris 3.63g, bromophenol blue 0.3g, SDS 6.0g, glycerin 30.0ml, β-mercaptoethanol 15ml, pH 6.8, ultrapure water to 100ml;

30% acrylamide (Acr)/methylene bisacrylamide (Bis): 29.2g acrylamide, 0.8g methylene acrylamide, dilute to 100ml with ultrapure water;

10× electrode buffer: Tris 30.3g, glycine 144.1g, SDS 10.0g, distilled water to 1000ml, pH 8.3, dilute 10 times when used;

Separating gel buffer: Tris 27.23g, 0.4% SDS, dissolved in 100ml ultrapure water, adjusted to pH 8.8 with HCl, and dilute to 150ml with ultrapure water.

Stacking gel buffer: Tris 6.0g, 0.4% SDS, dissolved in 100ml ultrapure water, HCl to adjust pH to 6.8, and ultrapure water to 150ml.

Coomassie brilliant blue R250 staining method: SI: 50% ethanol, 10% acetic acid

SⅡ: 5% ethanol, 7.5% acetic acid

SⅢ: 0.25% Coomassie brilliant blue R250 in 95% ethanol

Note: 50ml solution two is added to 200μl solution three.

Other reagents: TEMED; 10% Ammonium persulfate (new).

1.2.5 Other reagents

1) 1M Tris-HCl (PH8.0): Dissolve 121.1g Tris in 800ml deionized water, stir and dissolve, adjust the pH to 8.0, set the volume to 1L, and sterilize under high temperature and high pressure.

2) 0.5M EDTA (PH8.0): 186.1g Na ₂ EDTA·2H ₂ 0, add 800ml deionized water, stir well, adjust the pH to 8.0 with NaOH (about 20gNaOH), dilute the volume to 1L with deionized water, High temperature and high pressure sterilization.

3) TE buffer: 1M Tris-HCl (PH8.0) 10ml, 0.5M EDTA (PH8.0) 2ml, deionized water to 1L.

4) PBS buffer: 8.006g NaCl, 0.201g KCl, 1.540g Na ₂ HPO ₄ , 0.191g KH ₂ PO ₄ , dissolved in 800ml double distilled water, adjust pH to 8.0, dilute to 1000mL, sterilize under high temperature and high pressure, store at 4°C.

5) Carbolic fuchsin staining solution: Basic Fuchsine (Basic Fuchsine,) ethanol saturated solution (about 10%) 10mL, carbolic acid aqueous solution 5%, 100mL, mix the two solutions, dilute 10 times for staining.

6) 20mg/ml X-Gal: 1g X-Gal, dilute to 50ml with DMF (dimethylformamide), aliquot into 1.5ml Ep tubes and store at -20°C in the dark.

7) 24mg/ml IPTG: IPTG1.2g, dilute to 50ml with sterilized water, filter and sterilize with a 0.22μm filter, aliquot into 1.5ml Ep tubes and store at -20°C.

8) 0.1M CaCl ₂ : 2.94g CaCl ₂ ·2H ₂ O, distilled water to 200ml, high temperature and high pressure sterilization.

1.3 Insects to be tested

Anomala corpulenta, Holotrichia oblita, and Holotrichiaparallela were provided by the Institute of Plant Protection, Cangzhou Academy of Agriculture and Forestry, Hebei Province; sensitive populations of diamondback moth (Plutella xvlostella) were bred in our laboratory standardized test insects.

1.4 Instruments and equipment

1) Shaker D250: American NBS Company;

3) High-speed vertical centrifuge: DuPont, USA, RC-5 type;

4) Desktop centrifuge: Germany Eppenddorf, 5415C;

5) Sonicator: Noise Isolating Tamber, Ningbo Scientz Biotechnology

6) PCR instrument: American AmpGene 4800.

6) Protein electrophoresis instrument: Mini protein III from Bio-Rad, USA.

7) Gel imaging system: American STRAGENE Company, Eagle EyeII System.

8) Nucleic acid electrophoresis instrument: DYY-5 type, Beijing Liuyi Factory.

9) Optical microscope: Japan OLYMPUS CX21.

10) Electric shock conversion instrument: Bio-Rad Gene Pulser;

11) Beckman high-speed centrifuge Avanti J-26xp;

12) Water bath: Yuyao Changjiang Temperature Instrument Factory, DHW-420;

13) High pressure steam sterilizer: Japan SANYO company;

14) Electronic balance: Germany Sartorius Sartorius BP 310S;

15) Ultraviolet spectrophotometer UV-2100: Unico (Shanghai) Instrument Co., Ltd.;

16:) Ultra-clean workbench: Suzhou Purification Equipment Factory;

17) DNA concentration measuring instrument: Nanodrop spectrophotometer, American Thermo Finnigan;

18) Biochemical incubator: Guangding Medical Instrument Factory, LRH-150B type.

19) Constant temperature incubator: Shanghai Experimental Instrument General Factory, DHP120 type.

20) DNA Dryer: American Disco Company;

2 Research Methods

2.1 Extraction of Bt genome

1) Pour 5mL culture solution into EP tube and centrifuge at 12,000r/min for 2min.

2) Add 200μL solution I, add 20~25mg/10mL lysozyme, mix well, and place at 37°C for 10min.

3) Add 300 μL of solution II, add 100 μL of phenol (store at 4°C), and mix well. Water bath at 70°C for one hour, with gentle shaking every 30 minutes.

4) After cooling down to room temperature, add 100 μL of chloroform, mix well, and centrifuge at 12,000 r/min for 5 min. Divided into three layers, take the supernatant from the top layer. The supernatant was transferred and precipitated with an equal volume of isopropanol for 20 min. Centrifuge at 12,000r/min for 8min to collect the precipitate.

5) Rinse twice with absolute ethanol, dry in air, and dissolve in 50 μL ddH ₂ O (containing RNase).

2.2 Extraction of Escherichia coli plasmid DNA (Axygen plasmid extraction kit)

1) Take 1-4ml of bacterial solution cultured overnight in LB liquid medium, centrifuge at 12,000rpm for 1min, and discard the supernatant;

2) Add 250μL buffer S1 containing 50mg/ml RNase A to suspend the bacterial pellet;

3) Add 250 μL of bacterial lysate S2 to the above suspension, gently and fully turn it up and down 46 times until a uniform and transparent solution is formed;

4) Add 350 μL neutralizing solution S3, gently and fully turn up and down 6-8 times. Centrifuge at 12,000rpm for 10min;

5) Aspirate the supernatant in step 4 and transfer it to a preparation tube (placed in a 2ml centrifuge tube), centrifuge at 12,000rpm for 1min, and discard the filtrate;

6) Put the preparation tube back into the centrifuge tube, add 500 μL of washing solution W ₁ , centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

7) Put the prepared tube back into the centrifuge tube, add 700 μL desalted solution W ₂ , centrifuge at 12,000 rpm for 1 min, and discard the filtrate. repeat;

8) Put the preparation tube back into the 2ml centrifuge tube and centrifuge at 12,000r/min for 1min.

9) Transfer the preparation tube into a new 1.5ml centrifuge tube, add 6080 μL deionized water to the center of the preparation membrane (heating to 65°C can improve the elution efficiency), let stand for 1 min, and centrifuge at 12,000 rpm for 1 min.

2.3 Obtaining HBF-18 Genomic DNA Sequence

The genome of the HBF 18 strain was extracted, the genome was sequenced using the Solexa high-throughput sequencing system, and the sequence was spliced using the random software of the sequencing instrument, and finally the genome information of the strain was obtained.

2.4 Acquisition of new insecticidal protein genes - gene speculation

The genome sequence of HBF-18 was deduced by using the Genemark software package to predict the coding genes, and all the protein sequences encoded by the above strains were obtained.

Through the BlastX comparison of the genome sequence and the local database of insecticidal genes of the project team (including information on all Bt insecticidal genes reported so far), the program will compare all sequences of the genome obtained (including coding and non-coding sequences) with the local database of insecticidal genes Perform protein-level comparisons. The comparison will obtain all the DNA sequences in the strains that are similar to the protein encoded by the insecticidal gene, and these sequences may contain silent genes and gene fragments produced during the evolution process.

The protein sequence encoded by the strain genome of the gene predicted by Genemark is compared with the local database of insecticidal genes of the project team (including the amino acid sequence information of all Bt insecticidal proteins currently reported). Insect proteins have similar protein sequences, and further obtain the corresponding coding genes. These sequences may contain silent genes.

2.5 Sequence analysis of new genes

The gene and its two-terminal sequence were analyzed with the software Vector NTI Suite 9 (Invitr (Yan, Song et al. 2009) gene, Carlsbad, CA, USA) and online bioinformatics software.

2.6 Extraction and purification of RNA

The HBF 18 strain was cultured in LB liquid based on 30°C and 230rpm culture for 3.5h, 5h, 8h, 12h, and 24h, and samples were taken respectively, and the total RNA was extracted with Trizol reagent. The RNA extraction process was as follows:

1) Take 2ml of the bacterial solution and place it in an RNase free 2ml Eppendorf (EP) tube, and centrifuge at 12,000rpm for 1min to collect the bacterial cells;

2) Add 500 μl Trizol reagent and a spoonful of quartz sand to the bacteria, and crush it with a cell disruptor for 1 min;

3) Add 500 μl Trizol reagent to the crushed sample, vortex and mix, and place at room temperature for 5 minutes;

4) Add 1/5 volume of chloroform (about 200 μl), shake vigorously, leave at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 10 minutes at 4°C;

5) Take the uppermost supernatant, add an equal volume of isopropanol to mix, and precipitate at room temperature for 10 minutes;

6) Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant;

7) Wash the tube wall and precipitate with 75% pre-cooled ethanol, and discard the supernatant;

8) After the precipitate is naturally dried, add 20 μl of DEPC water and store it at -70°C for later use.

DNA removal from RNA samples:

1) The residual DNA in the extracted RNA was treated with RNase-free DNase I from the Ferment kit.

DNA removal system (50μl):

2) After treating at 37°C for 30 minutes, add 2 μl EDTA (provided in the kit) to the above system, and treat at 70°C for 10 minutes.

3) The purity and concentration of RNA were measured with a spectrophotometer.

2.7 RT-PCR

According to the method provided in the instructions of SuperscriptШreverse transcriptase, 1ng-5μg RNA was used as a template, oligo (dT) was used as a primer, and cDNA chain was synthesized by M-MLV reverse transcriptase.

1) cDNA first-strand synthesis reaction system (20 μl) is as follows:

Take 1ng-5μg of purified RNA, add 1μl oligo (dT), mix well, keep at 70°C for 4min, and then add the reactants according to the following system

After mixing, place on ice for 2 min. Incubate at 42°C for 1 hour; store at -20°C for later use.

2) RT-PCR: Take 2 μl of the cDNA as a template, and perform PCR amplification with cry8Gal, cry8like1 gene-specific primers and 16S rRNA-specific primers 16S rRNA-F/16S rRNA-R. The PCR amplification cycle was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 65°C for 1 min, extension at 72°C for 40 s, 30 cycles; final extension at 72°C for 10 min. The 16S rDNA amplified by 16S rRNA-F and 16S rRNA-R was used as the internal standard, the genomic DNA of HBF-18 strain was used as the positive control, and the RNA without reverse transcription was used as the negative control to ensure that the RT-PCR reaction was not caused by DNA If it is caused by contamination, set up a negative control without template.

2.8 Cloning of new genes in Escherichia coli

2.8.1PCR primers and sequences

Based on the known full-length sequence of the new gene, the following full-length primer pair (Table 22) was designed to amplify the candidate gene from the HBF 18 wild strain.

Table 22 Primer Sequence

2.8.2 PCR reaction system and conditions

1) Reaction system:

The PCR reaction conditions were: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 1 minute, 56°C annealing for 1 minute, 72°C extension for 3 minutes, 30 cycles, and finally 72°C final extension for 10 minutes.

2) Because the HBF 18cry8like1 sequence we cloned is a half-length sequence, after comparison and analysis, it is found that its 2143-2169 bases have a very high similarity with the corresponding sequence of cry8Ea1 (as follows), so we consider based on this region Overlapping PCR primers were designed to recombine the 8like15' end sequence with the cry8Ea3' end sequence to obtain a full-length cry8like2 sequence.

Firstly, cry8l-ike1F/interchangeR and exchangeF/cry8EaR primer pairs were used to amplify cry8like1 N-terminal and cry8EaC-terminal sequences respectively, and then the two fragments were recombined by overlapping chain extension in subsequent amplification reactions.

Reaction system 1:

The PCR reaction conditions were: denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 2.5 minutes, 30 cycles, and finally extension at 72°C for 10 minutes.

In the second PCR, the mixture of two DNA fragments obtained in the first PCR was used as a template, and Cry8-like1F and cry8EaR were used as primers for PCR amplification. Since in the first PCR, there is a sequence between the overlapping primers that is reverse complementary, the two fragments can be paired and combined during the annealing process of the second PCR, and the primers at both ends are added, and through the extension process, A complete gene containing 2 gene fragments was amplified.

Reaction system 2:

The PCR reaction conditions were: denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 4 minutes, 30 cycles, and finally extension at 72°C for 10 minutes.

2.8.3 Recovery of DNA

1) Cut out the agarose gel containing the target DNA fragment under a long-wave ultraviolet lamp, wash the liquid on the surface of the gel with a paper towel, place it in a 2mL EppendorfTube, and calculate the gel weight (weigh the tube weight in advance), and use the weight as a gel Glue volume.

2) Add Buffer ED-A 3 times the volume of the gel, mix well, heat in a water bath at 75°C, and mix several times until the gel completely melts;

3) Add 0.5 Buffer ED-A volume of Buffer ED-B and mix well;

4) Transfer the mixture in step 3 to a DNA preparation tube, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

5) Place the preparation tube in a 2ml centrifuge tube, add 500μL of elution buffer, centrifuge at 12,000rpm for 30s, and discard the filtrate;

6) Put the prepared tube back into a 2ml centrifuge tube, add 700 μL of elution buffer W ₂ , centrifuge at 12,000 rpm for 30 s, and discard the filtrate. repeat;

7) Put the preparation tube back into the 2ml centrifuge tube and centrifuge at 12000rpm for 1min;

8) Place the preparation tube in a clean 1.5ml centrifuge tube, add 25-30 μL of deionized water to the center of the preparation membrane, and let stand for 1 min. Centrifuge at 12,000 rpm for 1 min to elute DNA.

2.8.4 Plasmid vector digestion system

Prepare 200 μL of pEB vector enzyme digestion system according to the following standards, and incubate at 37°C for 1 hour.

Use 0.7% agarose for electrophoresis detection analysis, 120V voltage, 1×TAE as electrophoresis buffer, electrophoresis for 30min.

2.8.5 Ligation reactions

Carrier DNA 0.1~0.2μg

Target fragment DNA 0.5~1.0μg

Ligation Kit SolutionⅠ 5μL

Make up the volume to 10 μL with ddH ₂ O, mix thoroughly, and connect at 16°C for 4 hours or overnight at 4°C.

2.8.6 Preparation of Escherichia coli Competent Cells

1) Pick a fresh single colony of E.Coli and inoculate it in 5mL LB liquid medium, culture overnight at 37°C and 220rpm with shaking;

2) Transfer 1% of the inoculum to a Erlenmeyer flask filled with 100mL liquid LB medium, culture at 37°C and 220rpm for 2~3h with shaking, so that the OD ₆₀₀ value reaches between 0.4~0.6;

3) Centrifuge at 5,000 rpm for 10 minutes at 4°C;

4) Discard the supernatant, add 1/2 volume (50 mL) ice-cold 0.1M CaCl ₂ solution to suspend the cells, and place on ice for more than 30 minutes;

5) Centrifuge at 5,000 rpm for 10 minutes at 4°C to recover cells;

6) Resuspend the cells in 2~4mL ice-cold 0.1M CaCl ₂ , add 1/2 volume of 50% glycerol, aliquot into 200μL/1.5mL centrifuge tubes, and store at -70℃ for later use.

2.8.7 Heat shock transformation of Escherichia coli

1) Add 1 μg of the plasmid DNA to be transformed or 5 μL of the ligation product into 200 μL of competent cells, and mix well;

2) After 30 minutes of ice bath, heat shock at 42°C for 90 seconds, and immediately take out the ice bath for 3 minutes;

3) Add 800 μL liquid LB medium and incubate at 37°C for 1 hour;

4) Take 200 μL of LB solid plate coated with corresponding resistance, and add 4 μL of IPTG and 40 μL of X-Gal as needed.

5) Screen positive transformants after culturing at 37°C for 16 hours.

2.8.8 Screening of positive transformants

2.8.8.1 PCR identification

Use a sterile toothpick to transfer the white spot to a new solid plate and insert it into an LB test tube, incubate at 37°C for 8 hours, and use the genome of the starting strain as a positive control to screen positive clones. PCR identification was carried out with the forward primer pEBF of the vector expression region and the reverse primer of the full-length gene, and positive transformants with the correct orientation were screened out.

reaction system:

The PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 2.5 minutes, 30 cycles, and finally extension at 72°C for 10 minutes. Take 3 μl of the amplification product for agarose electrophoresis detection (120V, 30min).

2.8.8.2 Enzyme digestion identification

Plasmid DNA was extracted from positive transformants with the correct direction (Axygen plasmid extraction kit, refer to the instructions for the operation process), and then identified by restriction enzyme digestion sites SalI and SmaI upstream and downstream of the insertion site.

reaction system:

Reaction conditions: Mix the reaction system and place it at 37°C for enzyme digestion for 2-4 hours, and take 5 μl of the enzyme digestion product for electrophoresis detection.

2.8.9 Sequence Determination and Analysis

Correctly identified positive transformants were sequenced by the Major Science and Engineering Building of the Chinese Academy of Agricultural Sciences. DNA sequence analysis was performed using DNAMAN and Invitrogen's Vector NTI Suite 9 software package.

2.9 Study on the expression of new genes in Escherichia coli

2.9.1 Construction of gene expression vector

Use high-fidelity DNA polymerase to amplify the full-length fragment of the gene, recover it and connect it with the blunt end of the expression vector pEB after Ecl136II digestion, transform Escherichia coli competent JM109, use the forward primer pEBF in the expression region of the vector and reverse the full-length gene The primers were identified by PCR, positive transformants with the correct direction were screened out, plasmids were extracted, and identified by enzyme digestion. Identify the correct positive transformants to extract plasmids and transform Escherichia coli Rosetta (DE3) strains. After colony PCR and restriction enzyme digestion, the protein was induced to express.

2.9.2 Induced expression of new genes in Escherichia coli

1) Pick a single colony of E.coli in 5ml LB liquid medium, activate at 37°C, 220rpm for 12h;

2) Inoculate in 200mL liquid LB medium according to 1% inoculum amount, and culture at 37°C and 220rpm with shaking for about 2 hours until OD ₆₀₀ =0.6;

3) Add 100μL 1M IPTG to a final concentration of 0.5mM;

4) Culture at 18°C with shaking at 150rpm, and induce protein expression at low temperature.

5) Collect the bacteria by centrifugation at 8000rpm for 10min, and suspend in 20mL 20mmol/L Tris-HCl (pH 8.0) buffer;

6) Ultrasonic disruption of the bacteria for 10 minutes;

7) Centrifuge at 12,000 rpm for 20 min at 4°C, and collect the supernatant and precipitate respectively;

8) The precipitate was dissolved with 20mmol/L Tris-HCl (pH 8.0), and the supernatant and precipitate were detected by SDS-PAGE electrophoresis respectively.

2.9.3 SDS-PAGE detection of expressed proteins

The polyacrylamide gel configuration is shown in Table 2-3.

Electrophoresis: take 20uL sample and add 10uL 3x loading buffer, boil in water for 10min, centrifuge at 12000rpm, take 5μL supernatant for loading, pre-electrophoresis at 80V for 10min, and constant voltage electrophoresis at 120V for 1h.

Staining: After electrophoresis, carefully remove the gel, add 50mL SI to microwave for 30s, shake at 60rpm for 10min, pour it off, add 50mL of SII containing 200μL of SIII, heat in microwave for 30s, shake at 60rpm for 1h.

Table 23 Preparation of SDS polyacrylamide gel

2.10 Expression of new genes in the amorphous mutant of Bacillus thuringiensis

2.10.1PCR primers and sequences

According to the known full-length sequence of the new gene, the primer pairs in Table 2-4 were designed, in which the two ends of the S8like15/S8E3 primer were respectively introduced with SacI and SalI restriction sites, and the cry8like1 gene was amplified from the HBF-18 wild strain . The cry8like2 gene was amplified from the constructed Escherichia coli expression vector pEB-cry8like2.

Table 24 Primers and sequences

2.10.2 Recovery of DNA is the same as in 2.8.3

2.10.3 Enzyme digestion system

1) pSTK vector enzyme digestion system

Prepare 50 μL of enzyme digestion system according to the following standards, and incubate at 37°C for 1 hour.

Use 0.7% agarose for electrophoresis detection analysis, voltage 6V/cm, 1×TAE, electrophoresis for 1h.

2) Enzyme digestion system for PCR products:

Prepare 30 μL of enzyme digestion system according to the following standards, and incubate at 37°C for 1 hour.

Use 2.0% agarose for electrophoresis detection and analysis, voltage 6V/cm, 1×TAE, electrophoresis for 1h.

2.10.4 Ligation reactions

Same as 2.2.8.5

2.10.5 Heat shock transformation of Escherichia coli is the same as 2.2.8.7

2.10.6 Preparation of Bt electric shock competent cells and electric shock transformation

1) Pick a single colony of Bt and place it in 5ml of LB liquid medium, culture at 30°C and shake at 230rpm overnight;

2) Transfer to 100ml of BH liquid medium according to 1% inoculum size, culture at 30°C, 230rpm for about 4h to OD ₆₀₀ 2.0;

3) Centrifuge at 8,000 rpm for 10 minutes at 4°C to collect the cells;

4) Discard the supernatant and drain the excess liquid. Add 5-10mL pre-cooled ultrapure water to rinse once, be careful not to suspend the bacteria;

5) Add 100mL of sterilized ultrapure water to suspend the bacteria;

6) 4°C, 8,000rpm, 8min centrifuge to collect the bacteria, and drain the excess liquid;

7) Add 1ml of pre-cooled 40% PEG to suspend the cells, aliquot into 100μL/1.5mL centrifuge tubes, and store at -70°C.

Electric shock transformation: add 1 μg plasmid DNA to 100 μL competent cells, mix well, place in an electric shock cup in an ice bath at 0°C, electric shock parameter settings: 2200V, 1000Ω, 25μF, transfer to a 1.5ml Ep tube after electric shock, add 800μL LB liquid medium, cultivated in a 30°C incubator for 2 hours; take 200 μL of the LB solid plate coated with kanamycin, and cultivate overnight at 30°C;

2.10.7 Construction of Bacillus thuringiensis expression vector

The full-length PCR product introduced with the restriction site and the shuttle expression vector pSTK were digested with SacI and SalI, transformed into Escherichia coli JM109 competent cells after ligation, sequenced after colony PCR and enzyme digestion identification, and the plasmid was extracted after the sequence was correct Then transfer into Escherichia coli SCS110 competent for demethylation, and finally extract the demethylated plasmid and transfer it into Bt competent cells.

2.10.8 Screening of positive transformants

Use a sterile toothpick to transfer the leukoplakia to a new plate, number and insert into LB test tubes, inoculate 5 leukoplakia in each tube, incubate at 37°C for 8 hours, take 300μl of the culture solution and centrifuge, suspend the bacteria in 200μl of ultrapure water, and cook at 100°C for 10 minutes , and the supernatant was taken by centrifugation as a PCR template, and the corresponding identification primers were used for PCR detection, and the plasmid of the starting strain was used as a positive control to screen positive clones. And the positive recombinants were identified by SDS-PAGE.

2.10.9 Observation of crystal morphology

Bt strains were inoculated in 1/2LB medium, cultured at 30°C for about 48 hours, dripped 5 μl of sterilized water on the glass slide, picked a small amount of bacteria and spread them evenly in water, dried and fixed, and stained with 10 μl carbo-fuchsin staining solution After 5 minutes, rinse the excess dye with water, drop a drop of cedar oil, and perform microscopic examination with a 100-fold oil lens. After observing the release of spore crystals under a microscope, scrape the culture and wash it 3-4 times with distilled water, suspend it in 1mL sterile water, drop the spore crystal mixture on the cover glass, spread it evenly, dry it, and spray gold (2nm) by ion sputtering , SEM observation and photographing. Electron microscopy (HITACHIS-4800) analysis was completed by the herbarium of the Institute of Botany, Chinese Academy of Sciences.

2.10.10 Extraction method of Cry protein

1) Pick a single Bt colony and place it in 5 mL of LB liquid medium containing the corresponding resistance, activate at 30°C and 230 rpm for 12 hours;

2) Transfer 1% of the inoculum into 200ml beef extract peptone medium, culture at 30°C and 230rpm for about 36 hours, and stop the culture when more than 50% of the bacteria are lysed under the microscope;

3) The fermentation broth was centrifuged at 4°C, 8000rpm for 10min, and the supernatant was discarded.

4) Wash the precipitate with pre-cooled 1M NaCl, centrifuge at 8000rpm at 4°C for 10 min, and then wash again with pre-cooled sterile water;

5) Suspend each 1L of bacterial liquid precipitate in 50ml of lysate (add 3% β-mercaptoethanol to pH 9.6 when used), adjust the pH to 9.5~10 with NaOH, and shake on ice for 4-12h;

6) Centrifuge at 12000rpm for 15min at 4°C, take the supernatant, add 1/7 volume of 3M NaAc-HAc buffer solution with pH 4.5, and adjust the pH to 4.5~5.0;

7) Precipitation at 4°C for 4 hours (precipitation on ice for 1-4 hours);

8) Centrifuge at 12000 rpm for 15 min at 4°C, wash the precipitate twice with pre-cooled sterile water, and dissolve the precipitate with 50 mM Na ₂ CO ₃ (pH 9.6).

2.10.11 SDS-PAGE detection of protein is the same as 2.9.3

Determination of insecticidal activity of 2.11Bt strain

1) Determination of indoor insecticidal activity against diamondback moth (P.xylostella)

Dilute the strain to be tested with sterile water according to the pre-designed concentration, and use 50mM Na ₂ CO ₃ (pH9.6) as a negative control. Weigh 6g of feed into a 60mm petri dish, add the corresponding protein solution into it, and press evenly to mix the protein solution and feed evenly; divide the feed into 3 petri dishes, and inoculate 30 worms per dish (30 worms per treatment) worms, repeated three times), the age of worms was 2-3 days. Put them into a light incubator at 25°C for cultivation, and investigate the number of dead and alive insects after 48 hours of cultivation, and observe the feeding situation of the larvae.

2) Determination of the biological activity of the dark beetle (H.parallela), the large black beetle (H.oblita) and the aeruginosa beetle

The strains were inoculated on LB solid medium and cultured. After the crystals were produced, the bacteria were scraped and suspended in 10mL of sterilized ddH ₂ O, and the bacterial suspension was diluted according to the 2-fold equal ratio gradient concentration, and the bacterial liquid was soaked evenly. , the remaining bacterial liquid was stirred evenly with the sterilized fine soil, and after the potato shreds were dried, the potato shreds and the soil were mixed evenly, and 57-day-old healthy and uniform individual beetle larvae and black-branched beetle larvae were used as test species. Inoculate in a 6-well plate, inoculate 1 worm per well, inoculate 30 worms for each treatment, repeat twice, take the treatment of adding water as the blank control, raise in an artificial climate box at 25°C, and inoculate on 7 days and 14 days Check the number of live and dead insects, calculate the corrected mortality and LC ₅₀ .

2.12 Data analysis and processing

Mortality, adjusted mortality, LC ₅₀ and 95% confidence intervals were calculated using the "POLO" program. The synergistic toxicity index was calculated by the formula method (BE Tabashnik, 1993). Synergistic toxicity index: LC ₅₀ value was used to evaluate the joint toxicity of the mixture. First use the following formula to calculate the expected LC ₅₀ value of the mixture.

Compare the calculated expected LC ₅₀ value of the mixture with the measured LC ₅₀ value. If the two values are equal, it indicates an additive effect. Due to experimental errors and undetected inconsistencies in the tested organisms, it is generally believed that the expected LC ₅₀ virulence ratio measured between 0.5 and _2.6 is additive, greater than 2.6 is synergistic, and less than 0.5 is synergistic. antagonism.

The GLM process of SAS 9.2 software was used for the significance test of gene and concentration differences, and the Fisher'LSD method was used for multiple comparisons. The fixed model was: y=μ+Gi+Cj+eijk, where y was the phenotype value, that is, the corrected mortality rate; μ was Overall mean; Gi is gene effect; Cj is concentration effect; eijk is random error effect.

3 Conclusion

3.1 Obtaining a new insecticidal protein gene sequence

Through the comparison of BlastX and BlastP, the following gene sequences were finally obtained (Table 3.1)

Table 31 New insecticidal gene sequences

3.2 Sequence Analysis

Using the software Vector NTI Suite 9 (Invitrogen, Carlsbad, CA, USA) and Weblab online software to analyze cry8like1 and its two-terminal sequences, the results are shown in Figure 1.

3.2.1 Sequence analysis of cry8like1 gene

The sequence analysis of cry8like1 is as follows:

The results of Cry8like1BlastP are shown in Figure 2. The results showed that there were SigA, SigD, SigE transcription factors and RBS sequences in the upstream of cry8like1 gene and downstream of ORF1; there were two inverted repeat sequences in the downstream of cry8like1 gene and upstream of ORF3.

The results of BlastP analysis on Cry8like1 showed that it was a half-length cry8 gene, but it contained three structural domains of the cry gene, but lacked a sequence of about 1.4kb at the C-terminus.

3.2.2 Transcriptional analysis of the new gene in HBF-18 strain

The RNA of HBF-18 strain was extracted by Trizol one-step extraction method, and the remaining genomic DNA in the RNA was removed by RNase-free DNase I treatment. RT-PCR was amplified by a two-step method, and the amplified products were separated on a 1.5% agarose gel (see Figure 3). The results showed that the RNA samples of HBF-18 strains at different culture times could amplify the target band with the same size as the cry8like1 positive control; while the RNA sample without reverse transcription was used as a negative control, the target band could not be amplified , indicating that the bands amplified by PCR were not caused by DNA contamination. Therefore, the results of this experiment show that the cry8like1 gene can normally transcribe the corresponding mRNA in the HBF 18 strain, indicating that the candidate gene is expressed in the wild strain.

3.3 Cloning and expression of new genes in Escherichia coli

3.3.1 PCR amplification of new genes

Using the designed full-length expression primer pair cry8like1F/cry8like1R, using HBF-18 genomic DNA as a template, a 2214bp cry8like1 gene was amplified with high-fidelity DNA polymerase (Figure 4).

Because the cloned HBF-18cry8like1 sequence is a half-length sequence, it is found that its 2143-2169 bases are very similar to the cry8Ea1 gene sequence which can be well expressed in the Bacillus thuringiensis amorphous mutant strain HD73- after comparison analysis. High similarity (see below), so we consider overlapping PCR with the 8like15' end sequence and the cry8Ea3' end sequence to obtain a full-length cry8 sequence.

In order to obtain the full-length gene of cry8like1, we carried out overlapping PCR. Firstly, using the primer pair cry8like1F/interchange R and exchange F/cry8EaR, the plasmids containing cry8like1 and cry8Ea genes were respectively used as templates to amplify 5 parts of the cry8like1 gene. ' end sequence and the 3' end sequence of cry8Ea. (Figure 5)

In the subsequent amplification reaction, the primer pair cry8like1F/cry8EaR was used as primers, and the amplified cry8like1 N-terminal and cry8EaC-terminal were used as templates to recombine the front and rear fragments by extending the overlapping chains. A full-length cry8 gene of about 3.5 kb was obtained and named cry8like2 (Fig. 6).

3.3.2 Construction of new gene heterologous expression vector

The above-mentioned amplified full-length genes were recovered and inserted into the Ecl136II site of the expression cassette of pEB to obtain the expression vectors of each gene. The construction of the expression vectors is shown in FIG. 7 . Transform Escherichia coli JM109 competent cells, use the forward primer pEBF of the vector expression region and the reverse primer of the full-length gene for PCR identification, and screen out positive transformants with the correct orientation (Figure 8). Positive clones were inoculated into LB medium, cultured overnight at 37°C and 230 rpm, plasmids were extracted, and after enzyme digestion and identification (Figure 9), positive clones were confirmed by sequencing. Transform Escherichia coli Rosetta competent cells. The expression was induced after identification by PCR and enzyme digestion.

3.3.3 Expression of new genes in E. coli

The recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) strain, and the SDS-PAGE electrophoresis results of IPTG-induced protein expression showed that in the Rosetta (DE3) strain transformed with the recombinant plasmid, cry8like1 expressed about 80kD protein in the insoluble fraction (Figure 10) , consistent with the deduced protein size. The empty plasmid pEB was transferred into the Rosetta (DE3) strain as a negative control, and the expressed 80kDa protein was not found in the soluble and insoluble fractions (see Figure 10). The above results indicate that the above-mentioned protein was successfully expressed in E. coli .

3.4 Cloning and expression of new genes in the amorphous mutant of Bacillus thuringiensis

3.4.1 Cloning of new genes in Escherichia coli

The designed primer pair S8like15/S8E3 was used to introduce SacI and SalI restriction sites at both ends of the gene respectively to prepare for the construction of their shuttle expression vectors. Using the plasmid pEB8like2 as a template and high-fidelity DNA polymerase, amplified 3528bp cry8like2 gene (Figure 11).

The full-length PCR product and the shuttle expression vector pSTK were digested with SacI and SalI (Figure 12), and the full-length gene was cloned into the vector. The vector construction process is shown in Figure 13. The positive clones were identified, and about 3.5kb was amplified by PCR. The cry8like2 fragment (Figure 14), SacI and SalI double-digested the plasmid to obtain an 8.5kb empty vector and a 3.5kb cry8like2 target gene fragment (Figure 15), indicating that the vector was constructed correctly, and the plasmid was named pSTK8like2.

The pSTK8like2 plasmid was extracted, transformed into Escherichia coli SCS110 strain after demethylation, and transformed into the Bt crystal-free mutant strain HD-73- by electric shock, and the positive transformant identified by PCR was named HD8like2.

3.4.2 Extraction and detection of Cry protein

Extract cry8like2 protein and perform SDS-PAGE (gel concentration: 10%), it can be observed that HD8like2 can produce 133kDa protein (as shown in Figure 16), while no 133kDa protein is produced in the crystal-free mutant strain, indicating that cry8like2 gene can be produced in Normal expression in recombinant Bt strains.

3.5 Sequence and analysis of new genes

The nucleic acid sequence of positive clones containing different recombinant plasmids was determined, the nucleotide sequence of the new gene was obtained, and its amino acid sequence was deduced.

The size of the cloned new gene cry8like1 is 2214bp, see SEQ ID NO1, the protein consists of 738 amino acids, see SEQ ID NO3, and the molecular weight is 83.8kD. The amino acid content of the protein from high to low is leucine (8.54%), threonine (8.40%), serine (7.99%), asparagine (7.86%), valine (6.23%), Isoleucine (6.10%), alanine (5.96%), tyrosine (5.96%), glycine (5.56%) (see Table 3-2); the isoelectric point of the protein is pH7.47, which is weak Acidic protein, as shown in Table 3-2. Amino acid sequence similarity analysis showed 51% similarity with Cry8Db protein, indicating that it is a new type (holotype) cry8 gene, which is a new gene of the second level.

Table 3-2 Amino acid composition analysis of Cry8like1 protein

The size of the cloned new gene cry8like2 is 3528bp, see SEQ ID NO2, the protein consists of 1176 amino acids, see SEQ ID NO4, and the molecular weight is 133.6kD. The amino acid content of the protein from high to low is threonine (8.33%), leucine (8.25%), asparagine (7.91%), serine (6.89%), valine (6.63%), Glutamic acid (6.55%), glycine (6.38%), tyrosine (5.87%) (see Table 3-2); the isoelectric point of the protein is pH5.02, which is a weakly acidic protein, as shown in Table 3-3 Show. Amino acid sequence similarity analysis showed 66% similarity with Cry8Ea1 protein, indicating that it is a new type (holotype) cry8 gene, which is a new gene of the second level.

Table 3-3 Amino acid composition analysis of Cry8like2 protein

3.6Bt bacterial strain insecticidal activity determination result

3.6.1 Preliminary screening results of biological activity of Cry8 on C. aeruginosa

Table 3-4 Preliminary screening of insecticidal activity of HD8like2 against C. aeruginosa

It can be seen from Table 3-4 that at a concentration of 1*10 ¹⁰ , HD8like2 has a corrected mortality rate of 96.5% against C. aeruginosa larvae, and HD8like2 has good insecticidal activity against C. aeruginosa.

3.6.2 Preliminary screening results of biological activity of Cry8like2 against Plutella xylostella

Table 3-5 Preliminary screening of insecticidal activity of Cry8like2 against diamondback moth

The results of preliminary screening of the biological activity of diamondback moth are shown in Table 3-5. When the protein concentration reaches 100ppm, the death rate of diamondback moth is 0, and the growth situation is similar to that of the control, indicating that Cry8like2 has no insecticidal activity against diamondback moth, and also No weight suppression effect.

3.6.3 Determination results and analysis of biological activity of different strains and combinations on the black beetle

3.6.3.1 Biological activity assay results

Table 3-6 Determination results of biological activity of bacterial strain HBF-18

Table 3-9 Bioactivity assay results of strain HD8G

Table 3-10 Determination results of biological activity of strain HD8like2

Table 3-12 Determination results of biological activity of strain HD8G-HD8like2

3.6.3.2 Evaluation of synergistic toxicity

Synergistic toxicity index: LC ₅₀ value was used to evaluate the joint toxicity of the mixture.

Synergistic toxicity index = experimental LC ₅₀ / expected LC ₅₀ * 100

Undetected inconsistencies due to experimental error and test organisms. It is generally believed that the virulence ratio of the expected LC ₅₀ and the measured LC ₅₀ is between 0.5-2.6, which is an additive effect, greater than 2.6 is a synergistic effect, and less than 0.5 is an antagonistic effect. (Finney, DJ, Ed. (1952). Probit Analysis.)

Table 3-14 Insecticidal effect table of different combinations

It can be seen from Table 315 that the virulence ratio of expected LC ₅₀ to measured LC ₅₀ after the combination of HD8G and Cry8like2 is additive between 0.5-2.6.

In order to test the synergistic effect between HD8G and HD8like2, SAS 9.2 software GLM process was used to test the significance of differences in genes and concentrations, and Fisher'LSD method was used for multiple comparisons, and the fixed model was: y=μ+Gi+Cj+eijk , where, y is the phenotype value, that is, the corrected mortality rate; μ is the overall mean; Gi is the gene effect; Cj is the concentration effect; eijk is the random error effect.

Table 3-15 mean and standard deviation

The results are shown in Figure 17.

Strain HBF-18 had the best insecticidal effect on black beetle, and it was significantly different from HD8G strain, HD8like2 strain and the combination of HD8G and HD8like2.

The following conclusions can be drawn from the test results:

1. Cry8like2 has insecticidal activity against C. aeruginosa and black beetle, but has no insecticidal activity against diamondback moth. Moreover, the insecticidal activity against the black beetle was significantly different from that of HBF-18, and the combination with HD8G was an additive effect.

2. There is no antagonistic effect between HD8G and HD8like2 on the dark beetle, and they can be used together for the biological control of the dark beetle. With the large-scale promotion and application of Bt preparations and Bt genetically modified crops, pests will develop resistance to Bt insecticidal crystal proteins under continuous selection pressure, and the two crystal proteins can prevent the occurrence of resistance and solve resistance question.

Claims (8)

1.Cry8like2 albumen, its aminoacid sequence is as shown in SEQ ID NO.4.

2. the encode gene of Cry8like2 albumen claimed in claim 1, its nucleotide sequence is as shown in SEQ ID NO.2.

3. a carrier, contain gene claimed in claim 2.

4. carrier according to claim 3, be pSTK8like2, and its structure as shown in Figure 13.

5. a protein composition, be comprised of Cry8like2 albumen claimed in claim 1 and Cry8Ga1 albumen.

6. the application of the described albumen of claim 1 in anti-coleopteran pest, described coleopteran pest is anomala corpulenta larva and black dull strontium cockchafer larva.

7. application according to claim 6, be that albumen is made to sterilant.

8. application according to claim 6, be by the gene transferred plant or microorganism of proteins encoded.

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