CN1164733C - High-efficiency Bt15A3 strain with excellent gene combination, isolation and application - Google Patents

Abstract

The present invention is Bacillus thuringiensis subspecies Kommer subsp. The Bt15A3 strain screened by the PCR method of specific primers for the cry gene and the biological activity assay method; wettable powder and liquid formulations have received good field control effects on major pests beet armyworm, cotton bollworm, diamondback moth and American white moth .

Description

High-efficiency Bt15A3 strain with excellent gene combination, isolation and application

technical field

The present invention relates to Bacillus thuringiensis subsp. Kommer 15A3 strain which is highly toxic to lepidopteran major pests beet armyworm and cotton bollworm. Bacillus thuringiensis subsp.colmer 15A3, referred to as Bt15A3, the gene encoding insecticidal crystal protein (ICP) carried by this strain and its excellent combination, as well as the highly effective bacterial insecticide developed by this strain.

Background technique

Bt preparation is a bacterial insecticide widely used in the world, accounting for 95% of the output of all biological insecticides, and has a very important position in the practice of microbial control of pests (Yu Ziniu et al. Bacillus thuringiensis Beijing Science Press 1990 ). The insecticidal activity of Bt comes from the ICP encoded by the cry gene carried by itself. Each ICP has a specific insecticidal spectrum. Among the ICP genes, cry1Aa, cry1Ab, and cry1Ac have the highest insecticidal toxicity, and the Bt preparations currently on the market The gene types mainly carried by the producing bacteria, wherein cry1Ac is considered to be the gene with the highest toxicity to cotton bollworm, YBT-1520 bacterial strain (Sun Ming et al. CN1055310C) and Bt.ken-Ag bacterial strain ( Liu Chunyong etc., Nankai University Journal 2000 32 (3): 163-168) all contain above-mentioned cry1Ac class gene, but above-mentioned three kinds of gene products are nontoxic or low toxicity to several pests such as beet armyworm, spodoptera littoralis, and Cry1C and Cry1D proteins are highly toxic to such pests (Frankenhuyzen K.V. The challenge of Bacillus thuringiensis. In: Entwistle P.F. et al.ed. Bacillus thuringiensis, an environmental biopesticide: theory and practice. Chichester: John willey & sons Ltd. 9-93. 10). Insecticides produced by strains containing the cry1C gene, such as B.t.subs.aziwai, are mostly used for the control of such pests. The Bt bacteria containing cryIC and cryIA have three times higher activity on Spodoptera fruqiperda than HD-1 strain without cryIC, and two to several times higher activity on diamondback moth (CN1240002A). The Bt strain with broad-spectrum insecticidal activity for insects and beet armyworm pests should carry the combination of the above-mentioned cryIAc and cryIC genes in its cells.

In order to make Bt strains have the desired gene combination and achieve broad-spectrum insecticidal activity, researchers have invested a lot of human and financial resources in recent years to genetically improve existing Bt strains, using methods such as mutagenesis, gene recombination, and conjugation transfer to try to Construct Bt engineering strains containing both cry1A and cry1C important gene combinations. In 1994, Sandoz Corporation of the United States invented a method (Eur.pat.0582541A2) for effective conjugative transfer of plasmids with the cry gene between Bts, and for the first time the plasmid with the cry1C gene in the Bt entomocidus 6.1 strain was transferred by conjugation Methods The Bt kurstaki HD562 strain containing only cry1A was introduced to obtain the engineering strain cg92004.24, which contained cry1Aa, cry1Ab, cry1Ac, cry1C and cryIIA. The biological activity test shows that the toxicity to beet armyworm is 1.7 times larger than that of the original parent strain. Due to the inability of the desired donor and the required plasmid to conjugate effectively, the incompatibility between the new plasmid transferred into the recipient and the original plasmid, and the existence of objective factors such as selection markers for screening recombinant zygotes, the obtained Engineering bacteria must go through complex processes such as more than 5 times of conjugative hybridization, multiple electroporation transformations, and a large number of screenings. The finally obtained target engineering strains also have erythromycin and tetracycline resistance markers, and in addition to the cry1C gene, The cry1Ac gene also comes from other strains, that is, the two high-efficiency genes are not the original genes of the Bt kurstaki strain, and factors such as the stability of the foreign plasmid and selection pressure will complicate the production process. In addition, all preparations produced by genetically engineered bacteria must pass the approval of the Ministry of Agriculture for the safety assessment of agricultural biogenetic engineering before they are released in the environment before field trials can be carried out.

It is precisely because of the inconvenience of engineering bacteria for production that people have not given up screening Bt strains with new cry genes or expected good gene combinations. Oyama Kazuhiko et al. (CN1240002A) isolated a new strain of Bt, which belongs to serotype H7. The ICP gene it carries is relatively close to cry9Ca1, and its amino acid sequence has 71% homology, which proves that this strain carries a new ICP Gene, showing broad-spectrum insecticidal activity, not only has high insecticidal activity against certain Lepidoptera pests such as Spodoptera litura, but also has activity against three Coleoptera and two Diptera insects.

Studies have shown that crystals made of multiple Cry proteins often have higher insecticidal toxicity than crystals made of a single protein (Lee M.il et.al., App.Environ.Microbio.1996.62:583-585), and CryV The role of the inhibition of insect resistance helps to delay (Poncet S. et. al., J. Invertebt. Pathol. 1995, 66: 131-135). Bt kurstaki HD-1 (HD-1 for short), which has been used to control diamondback moth for many years, has made outstanding contributions to the control of diamondback moth that is resistant to chemical pesticides. However, in recent years, it has been found that diamondback moth has a significant resistance. Therefore, the selection of Bt-producing strains with a more dominant combination of genes than the HD-1 strain can not only expand the insecticidal spectrum, but also effectively suppress and delay the resistance of insects to Bt.

Contents of the invention

The object of the present invention is to provide a high-efficiency Bacillus thuringiensis subsp.colmer 15A3 strain (Bacillus thuringiensis subsp.colmer 15A3) with excellent gene combination, its separation method and its application. The present invention screens from the rich Bt resources in my country the bacterial strains that have both the cry1Ac gene with high efficiency to cotton bollworm and the cry1C gene with high efficiency to beet armyworm, as well as cryII and cryV delayed resistance genes, etc., which not only expands the antibacterial Insect spectrum delays resistance, and can save the huge cost of artificially constructing engineering bacteria, and can also overcome various deficiencies in the production of engineering bacteria.

The present invention screens out the bacterial strain Bacillus thuringiensis subsp.colmer of the present invention through soil separation, morphology observation, PCR gene detection and biological activity experiment, numbered 15A3, referred to as Bt15A3. It has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on January 5, 2001, and the culture preservation number is CGMCC NO.0528. It not only contains the expected genes cry1Ac, cry1C and cryV, but also six ICP genes cry1Aa, cry1D and cryII. Among them, the restriction fragment length polymorphism analysis (RFLP) of cry1Aa is different from the standard cry1Aa, and there is an extra pstI cutting point. The strain was identified as Bacillus thuringiensis serotype H21 subsp. Kommer. Indoor activity tests found that the toxicity of the 15A3 strain to cotton bollworm was equivalent to that of the high-efficiency cotton bollworm strain Btken-Ag developed during the "Eighth Five-Year Plan" period of our group, while the latter had almost no toxicity to beet armyworm. After shaking flask fermentation and small tests in 6 vertically opened self-controlled tanks, it was found that the 15A3 strain had excellent production performance, and then a pilot production test was carried out to obtain a broad-spectrum insecticide product - NK Bt-II, which was sold separately in Hebei The province, Tianjin City and other places have conducted community drug efficacy tests, and it is obviously better than similar domestic products in terms of broad-spectrum of tested insect species.

The outstanding substantive features and remarkable progress of the present invention can be embodied from the following examples. But they do not limit the invention in any way.

Description of drawings

Figure 1 shows the PCR detection results of the cry gene of the 15A3 strain and the H21 standard strain.

Detailed ways

Example 1 Isolation and screening of Bt15A3 bacterial strain

(1) Weigh about 1 gram of soil sample into 10 ml of sterile water, mix well, take 1 ml into NB medium mixed with polymyxin and penicillin, and incubate at 30°C for 72 hours. The colonies similar to Bacillus thuringiensis were selected, and the smears were stained and examined under the microscope, and the colonies with high synchronous rate of spore crystal formation and polymorphic crystals such as diamond and square were further purified.

(2) Design specific primers based on the conserved regions of various cry genes, detect various cry genes on the purified Bt strains, and select strains with desired gene combinations.

(3) Inoculate the strains screened and the reference strains into the M1 fermentation medium respectively, the formula of which is: fish meal 3.0-3.5%, cornstarch 1.2-1.5%, beef extract 0.2-0.5%, inorganic salt 0.1-0.2% , pH7.5-8.0. Cultivate for 26-30 hours at 30°C, 180-220rpm/min.

(4) Dilute the culture solution by 500 and 1000 times, mix it with artificial feed, feed the newly hatched cotton bollworm and beet armyworm, and screen out the strains with high toxicity to the two test insects.

(5) Prepare the spore crystal complex freeze-dried powder of the screened bacterial strain and the reference bacterial strain by freeze-drying method, prepare artificial feed with a certain concentration of freeze-dried powder, and feed the newly hatched cotton bollworm and beet armyworm. The reference strain has Two: one is the strain Bt ken-Ag that mainly contains the cry1Ac gene and is highly effective against cotton bollworm; the other is the strain 9510 that mainly contains the cry1C gene and is highly effective against the beet armyworm. The results are shown in Table 1.

Table 1 LC ₅₀ (ppm) of 15A3 strain against cotton bollworm and beet armyworm Cotton bollworm beet armyworm 15A3Btken-Ag(CK)Bta: 9510(CK) 16.4814.9035.16 15.63 Very low toxicity 26.12

The experimental results in Table 1 show that the Bt15A3 strain has high activity against cotton bollworm and beet armyworm. The toxicity to cotton bollworm is equivalent to that of Btken-Ag, while the latter has almost no toxicity to beet armyworm. Compared with 9510 strain, the toxicity of Bt15A3 strain to cotton bollworm is 2.1 times higher, and the toxicity to beet armyworm is 1.7 times higher. times.

Finally, the bacterial strain Bt15A3 of the present invention was selected.

Example 2. The biological characteristics of the Bt15A3 strain and the detection of the cry gene

After 48 hours of culture with common NB slant, more than 95% of the cells formed spores and crystals. The 15A3 strain was determined by classical serological experimental methods. The flagellar antigen of the strain belongs to H21 type, and it is Bacillus thuringiensis subsp. colmeri. spore morphology, but differs from the standard Kommer subsp.

The insecticidal crystal protein is encoded by six genes cryIAa, cryIAc, cryIC, cryID, cryII and cryV.

The spores are egg-shaped, and the bacterium flagella antigens germinated by the spores belong to the H21 type, which is Bacillus thuringiensis subspecies Kommer, and the results of antibiotic spectrum determination: amp ^r , polymy ^r , str ^s , ery ^s , tet ^s , chl ^s .

The cry genes of the 15A3 strain and the H21 standard strain were detected by the PCR method with specific primers to prove that the 15A3 strain contained cry1Ac, cry1C, cryII and cryV genes, and the sizes of the amplified fragments of the specific products were: 1.84kb, 288bp, 600bp and 700bp, while the H21 standard strain does not contain a specific amplification product of 1.84kb of cry1Ac, and other genes are the same (see Figure 1). After testing, the 15A3 strain does not contain cry1E, cryIII.cry1V and cyt genes. Figure 1 shows the PCR detection results of the cry gene of the 15A3 strain and the H21 standard strain. As shown in the figure, lane 1 is a DNA molecular weight indicator (λDNA/EcoRI+HindIII), lanes 2, 4, 6, and 8 are cryIAc, IC, II, and V gene fragments of Bt15A3 strain, and lanes 3, 5, 7, and 9 are H21 standard strain cryIAc, IC, II, V gene fragments.

Using the restriction fragment length polymorphism (RFLP) method of Kuo W.S. et al. (Appl.andEnviro.Microbi.1996 62(4):1369-1377), it was found that the 15A3 strain contained 4 cry1 genes : In addition to the cry1Ac and cry1C detected by PCR, it also contains two genes, cry1Aa and cry1D. The RFLP results of the cry1Aa gene show that, unlike the found cry1Aa gene sequence, it only contains two fragments of 726bp and 237bp and lacks The 496bp fragment has an extra pstI cutting point in this sequence, which may be a new cry1Aa gene sequence, while the other three cry1 genes all conform to the standard restriction restriction map.

Liquid fermentation production and products of embodiment 3.15A3 strain

(1) Inclined bacteria

NB medium slant: beef extract 0.5%, peptone 1.0%, NaCl 0.5%, agar powder 1.8%, pH7.2-7.4. After the culture medium is prepared, put it into test tubes or Keshiba bottles, sterilize it with high-pressure steam at 121°C for 30 minutes, take it out, place it on a slope, and place it in an incubator at 30°C for 24-48 hours, and then inoculate without the growth of miscellaneous bacteria: take preserved bacteria Inoculate a fresh slant with one loop, incubate at 30°C for 10-12 hours, observe under the microscope after staining the smear, the bacteria are thick rod-shaped, with blunt round ends, and the protoplasm is uniform without miscellaneous bacteria.

(2) Expansion culture of Kirschner flask

Put one ring of the above-mentioned activated bacteria into the liquid NB culture medium, shake and culture at 30°C 180rpm/min for 5-6 hours, take 2ml into the Keerflask, make it evenly distributed on the surface of the medium, and culture at 30°C After 72 hours, the bacterial lawn was checked to be thick and plump, the surface of the colony was off-white and dull without miscellaneous bacteria, and the microscopic examination of the spore crystal formation rate was more than 98%.

(3) Fermentation tank seed liquid preparation

Pour 100ml of sterile water into a Kirschner flask to wash off the bacterial lawn, transfer it into a sterile flask with a small amount of glass beads, oscillate fully to break up the bacterial lawn, transfer it into a special inoculation bottle, and place it in a 75°C water bath for 20 minutes.

(4) Fermentation tank culture

Fermentation medium formula: soybean flour 4.0%, corn starch 1.5%, cottonseed powder 1.5%, yeast powder 1.0%, inorganic salts 0.5%, foam enemy 0.03-0.05%, all raw materials need to reach the fineness of 180μm sieve, pH8 .5-9.0. Feed according to 60-75% of the fermentation volume, cool to about 40°C after sterilization, add seed liquid according to 0.02% of the feed volume, stir at 250 rpm, temperature 30°C±1°C, tank pressure 0.5kg, ventilate The amount is 1:1.0-1:1.3, the fermentation period is 35-45 hours, and about 30% of the spore crystals are free, and the pH of the fermentation broth reaches 8.0 or more to stop the fermentation. The post-processing of liquid and high-content powder products is carried out according to the Btken-Ag pilot test method (Liang Fenglai et al. Journal of Nankai University 1998 31(2): 28-32). Product quality inspection is based on the agricultural industry standard of the People's Republic of China: Bacillus thuringiensis preparation (implemented on October 1, 1995).

Example 4

The insecticide NK Bt-II produced by bacterial strain 15A3 of the present invention (its liquid preparation and high-content powder product are obtained by the Btken-Ag pilot test method, Liang Fenglai, etc. Nankai University Journal 1998 31 (2): 28-32), through The field efficacy tests of Hebei Academy of Agriculture and Forestry Sciences and Tianjin Academy of Agricultural Sciences on cotton bollworm, beet armyworm and diamondback moth have proved that they can achieve good control effects on the three test insects, and the broad-spectrum is obviously better than that of the same kind in China. product.

(1) The powder is diluted 500 times, and the control effect on the 1-2 instar beet moth is 82%, which is 1.78-2 times of the same dilution ratio of the control group (16,000IU/mg wettable powder produced by the Bt Center of Hubei Academy of Agricultural Sciences). . See Table 2:

Table 2 Results of field efficacy experiments of NK Bt-II powder against 1-2 instar beet armyworm Dilution factor The average control effect of medication for 4 days (%) NK Bt-II 5001000 8253 CK 5001000 4626

(2) 500 times release of powder, the effect of preventing and treating three generations of cotton bollworm reaches more than 90%, which is suitable with the matched group (samples are the same as Table 2), see Table 3:

Table 3 Results of field efficacy experiments of NKBt-II against the third generation of cotton bollworm Dilution factor Average control effect (%) 2 days 4 days NK Bt-II 5001000 72.165.5 91.581.9 CK 5001000 69.360.2 88.970.8

(3) The powder was diluted 500 times, and the average effect of preventing and controlling 2-year-old Plutella xylostella was 82%, which was comparable to that of the control group BtA powder (produced by Fujian Green Cross Bioengineering Joint Development Center, a new Bt product added with abamectin) , see Table 4:

Table 4: Field efficacy experiment results of NK Bt-II against 2-year-old Plutella xylostella Dilution factor Average control effect (%) KN Bt-II 500 82.1 CK 500 85.9

(4) The liquid preparation is diluted 100 times, and the control effect on the 4-5 age white moth after the field is broken is more than 95%.

Claims (7)

1, a kind of high-efficiency broad spectrum bacterial strain of producing germ insecticide, it is characterized in that described bacterial strain is the Gadamer subspecies 15A3 of bacillus thuringiensis section bacterial strain (Bacillus thuringiensis subsp.colmer 15A3, Bt 15A3), preserving number is CGMCC NO.0528, comprising:

A. this bacterial strain flagellar antigen belongs to the H21 type, is bacillus thuringiensis section Gadamer subspecies, and have water chestnut, side, inlay, irregular many types of crystal and normal gemma form, but variant with the genomic constitution of section's Gadamer subspecies of standard;

B. the gene of this bacterial strain coded insect-killing crystallin (ICP) has: cryIAa, cryIAc, cryIC, cryID, cryII and cryV;

C. this bacterial strain cryIAa gene is different with the sequence of the cryIAa gene of having found, and the enzyme section that only contains 726bp and 237bp is disconnected and lack the segment of 496bp, has more a pstI point of contact in this section sequence.

2, the separating screening method of the described Bt 15A3 of claim 1 bacterial strain is characterized in that it comprises the steps:

A. take by weighing 1 gram pedotheque and place the 10ml sterilized water, fully getting the 1ml adding behind the mixing is mixed with in the NB substratum of polymyxin and penicillin, cultivated 72 hours for 30 ℃, select the bacterium colony of similar bacillus thuringiensis, microscopy behind the smear staining, with the crystal formation sync rates height of gemma, and contain rhombus simultaneously, square, many types of crystalline bacterium colony is further purified;

B. according to all kinds of cry gene conservative district design special primer, the Bt bacterial strain good to purifying carries out the detection of all kinds of cry genes, and the bacterial strain that will have the assortment of genes of expectation is selected;

C. the bacterial strain and the reference bacterial strain that are screened are inoculated the No.1 fermention medium respectively, its prescription is: fish meal 3.0-3.5%, W-Gum 1.2-1.5%, extractum carnis 0.2-0.5%, inorganic salt 0.1-0.2%, pH7.5-8.0,30 ℃, 180-220rpm/ divides cultivation 26-30 hour;

D. nutrient solution is diluted to 500,1000 times, sneaks into artificial diet, feed and just incubate bollworm and beet armyworm, filter out two kinds of all high bacterial strains of examination worm virulence.

3, a kind of method of utilizing the described Bt 15A3 of claim 1 bacterial strain to produce gemma is characterized in that comprising the steps:

A. produce with bacterial classification NB medium slant, its prescription is: extractum carnis 0.5%, peptone 1.0%, NaCl0.5%, agar powder 1.8%, pH7.2-7.4, medium preparation is back packing test tube or Kolle flask well, 121 ℃ of autoclavings 30 minutes are put into the inclined-plane after the taking-up, place 30 ℃ of incubators to cultivate 24-48 hour; No varied bacteria growing just can be inoculated: goes bail for and hides the fresh inclined-plane of bacterial classification one articulating kind, cultivated 10-12 hour for 30 ℃, and microscopic examination behind the smear staining, thalline is for slightly shaft-like, and the blunt circle in two ends, protoplasma evenly do not have assorted bacterium;

B. Kolle flask enlarged culturing, above-mentioned activatory bacterial classification is got an articulating goes in the liquid NB substratum, 30 ℃ of 180rpm/min shaking culture 5-6 hour, get 2ml to Kolle flask, make it be uniformly distributed in media surface, put 30 ℃ and cultivated 72 hours, check that lawn is thick and plentiful, bacterium colony surface canescence, tarnish, the assorted bacterium of nothing, the crystal formation rate of microscopy gemma is more than 98%;

C. fermentor tank seed liquor preparation is poured the 100ml sterilized water into Kolle flask and is washed lawn, moves into and is equipped with in the sterile flask of a small amount of granulated glass sphere, and fully lawn is broken up in vibration, moves into special-purpose inoculation bottle, puts 75 ℃ of water-baths 20 minutes.

4, a kind of method of utilizing the described Bt 15A3 of claim 1 bacterial strain to produce ICP is characterized in that comprising the steps:

A. produce with bacterial classification NB medium slant, its prescription is: extractum carnis 0.5%, peptone 1.0%, NaCl0.5%, agar powder 1.8%, pH7.2-7.4, medium preparation is back packing test tube or Kolle flask well, 121 ℃ of autoclavings 30 minutes are put into the inclined-plane after the taking-up, place 30 ℃ of incubators to cultivate 24-48 hour; No varied bacteria growing just can be inoculated: goes bail for and hides the fresh inclined-plane of bacterial classification one articulating kind, cultivated 10-12 hour for 30 ℃, and microscopic examination behind the smear staining, thalline is for slightly shaft-like, and the blunt circle in two ends, protoplasma evenly do not have assorted bacterium;

B. Kolle flask enlarged culturing, above-mentioned activatory bacterial classification is got an articulating goes in the liquid NB substratum, 30 ℃ of 180rpm/min shaking culture 5-6 hour, get 2ml to Kolle flask, make it be uniformly distributed in media surface, put 30 ℃ and cultivated 72 hours, check that lawn is thick and plentiful, bacterium colony surface canescence, tarnish, the assorted bacterium of nothing, the crystal formation rate of microscopy gemma is more than 98%;

C. fermentor tank seed liquor preparation is poured the 100ml sterilized water into Kolle flask and is washed lawn, moves into and is equipped with in the sterile flask of a small amount of granulated glass sphere, and fully lawn is broken up in vibration, moves into special-purpose inoculation bottle, puts 75 ℃ of water-baths 20 minutes;

D. fermentor cultivation

Fermentative medium formula: soybean cake powder 4.0%, W-Gum 1.5%, cottonseed meal 1.5%, yeast powder 1.0%, inorganic salts 0.5%, bubble enemy 0.03-0.05%, all raw material need reach the fineness of 180 μ m sieve, pH8.5-9.0.70-75% feeds intake by the fermentation volumetrical, about sterilization postcooling to 40 ℃, insert seed liquor by 0.02% of the volume that feeds intake, stirring revolution is 250 commentaries on classics, 30 ℃ ± 1 ℃ of temperature, tank pressure 0.5kg, air flow 1: 1.0-1: 1.3, fermentation period 35-48 hour, the spore of looking about 30% was brilliant free, and fermented liquid pH reaches and stops fermentation more than 8.0.

5, the gemma of the described method production of claim 3 is characterized in that described gemma is an ovum garden shape, and the thalline flagellar antigen that its gemma is sprouted belongs to the H21 type, is bacillus thuringiensis section Gadamer subspecies, antibioticogram measurement result: amp ^r, polymy ^r, str ^s, ery ^s, tet ^s, chl ^s

6, the insecticidal crystal protein of the described method production of claim 4 is characterized in that described insecticidal crystal protein by cryIAa, cryIAc, cryIC, cryID, six kinds of genes encodings of cryII and cryV.

7, the described insecticidal crystal protein of claim 6 is as the germ insecticide of control lepidopteran noctuidae pests beet armyworm, bollworm, small cabbage moth and fall webworms.