CN1609191A - Bacillus thuringiensis strains and genes highly effective against coleopteran pests - Google Patents

Abstract

The invention relates to "Bacillus thuringiensis strains and genes effective for coleopteran pests", belonging to the technical field of biological control. Further, the present invention relates to Bacillus thuringiensis bacterial strains with high toxicity to coleopteran pests, the nucleotide sequence of cry8E and cry8F genes and the amino acid sequence of the protein encoded by them, involving the use and synergistic combination of Bacillus thuringiensis bacterial strains, cry8E and cry8F gene expression product. By using the above-mentioned genes or combinations thereof, their toxicity to Coleoptera pests can be made higher than that of the starting strains and single gene expression products, and their insecticidal spectrum against Lepidoptera, Coleoptera, and Diptera pests can be expanded, and by applying It is used to transform microorganisms and plants to make them exhibit toxicity to related pests, and to overcome or delay insects' resistance to engineering bacteria and transgenic plants.

Description

To coleopteran pest bacillus thuringiensis bacterial strain and gene efficiently

Technical field of the present invention

The invention belongs to the biological control technical field.The present invention relates to a kind of coleopteran pest to be had active bacillus thuringiensis bacterial strain, relate to the nucleotide sequence of the cry8E of the high virulence of coleopteran pest and cry8F gene with respectively by the aminoacid sequence of its encoded protein matter, relate to synergistic combination and use cry8E and cry8F gene expression product.

Background technology of the present invention

Chafer belongs to Coleoptera Scarabaeoidea (Scarabaeidae), its larva (is commonly called as grub, also abbreviate " grub " below the present invention as) be the important worldwide distribution subterranean pest-insect of a class, can endanger various plants such as grain, cotton, oil crops, vegetables, sugar crop, tobacco, herbage, flowers, turfgrass, fruit tree.A large amount of investigation show that the harm of grub in subterranean pest-insect ranks first, and wherein mainly based on gill Scarabaeidae and Rutilidae larva, account for more than the 70-80% of total subterranean pest-insect amount.About 100,000,000 mu of area takes place in annual according to statistics national grub, and the serious time once reached 300,000,000 2 thousand ten thousand mu, and production loss is up to more than 20%, some plot even total crop failure.In recent years take place area maximum, generating capacity maximum be the Yellow River and Huai He River Haiti district, crops such as main harm grain, oil plant; Other geographic harm situation is also very serious, as the grub of harm sugarcane, in Guangdong, ground such as Guangxi, Yunnan, Sichuan, Fujian generally take place; In Tibet, west area such as Qinghai, Gansu, Xinjiang, the generation of grub also very serious (Wei Hongjun etc., " Chinese subterranean pest-insect ", Shanghai: Shanghai science tech publishing house, 1989,1-41; Wang Yongxiang etc., " region of no relief grub kind and integrated control technique in the Ji ", " Hebei Normal University's journal " (natural science edition), 1998,22 (2): 268-270).Being only second to rape with cultivated area in China oil crops, to occupy deputy peanut be example.The peanut yield of China accounts for about 35% of world's peanut ultimate production, occupies first place in the world, and a year outlet income reaches 20,700,000,000 dollars, calendar year 2001 whole nation peanut area (5,000,000 hectares) and gross output (1,450 ten thousand tons) all reach a record high.But grub is very serious to the harm of peanut.Be the harm of control grub, the general integrated control strategies such as agricultural, chemistry, physics that adopt though this has certain effect, are difficult to reach the effect of Sustainable Control.Therefore, seek the new method of effectively preventing and treating, become the task of top priority.

On the basis that obtains the high virulence Bt gene to grub, cultivating the transgenic plant that kill grub is new controlling ways that are worth exploration.

Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of distribution gram positive bacterium extremely widely.It is when forming gemma, can produce the parasporal crystal (parasporal crystal) of property of protein, to lepidopteran (Lepidoptera), Diptera (Diptera), Coleoptera (Coleoptera), Hymenoptera (Hymenoptera), Homoptera (Homoptera), Orthoptera (Orthoptera), Mallophaga various insects such as (Mallophaga), and nematode, mite class and protozoon have specific insecticidal activity (Schnepf, E.N.et al, Microbiol.And Molecular BiologyReview, 1998,62:3 775-806).(Insecticidal Crystal Proteins ICPs) claims delta-endotoxin (delta-endotoxin) again to this insecticidal crystal protein, and is free from environmental pollution to the person poultry harmless, thereby Bt has obtained using the most widely in the biological control of insect.

People have cloned the Bt killing gene of more than 300 kind of coded insect-killing crystallin at present, and they adhere to 141 kinds of pattern genes separately.In recent years the research trend of cry8 genoid is noticeable in the world.Studies show that this genoid has insecticidal action to multiple coleopteran pests such as Scarabaeidae, Culculionidae, Chrysomelidaes.1992, Ohba etc. filter out new bacterial strain (B.t.subsp.Japonensis the BuiBui) (Ohba that the chafer larva is had special insecticidal activity in the world first from the Bt bacterial strain, M.et al., A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activityspecific for scarabaeid beetles, Letters in Applied Microbiology, 1992.14:54-57), Sato in 1994 etc. therefrom clone a kind of new killing gene cry8C (Sato, R.et al, Cloning, heterologous expression, andlocalization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strainbuibui toxic to scarabaeid insects, Curr.Microbiol.1994.28:15-19.4).Found 9 kinds of genes at present, encoded protein is made up of 1160-1210 amino acid, and molecular weight is between 128-137kDa.Detailed information sees Table 1 (Asano, S., Yamanaka, S.and Takeuchi, K., Protein having insecticidal activity, DNA encoding the protein, and controlling agent and controlling method of noxious organisms, 2002, JP 2002045186-A andJP2002045186-A/2)).Wherein isolating Cry8Aa1 of U.S. Mycogen company and Cry8Ba1 have tangible insecticidal activity (Tracy E.Michaels to the various pests of Scarabaeidae, et al., Bacillus thuringiensis toxins active againstscarab pests, 1994, USP5554534).The U.S. has separated two kinds of gene cry8Bb1 and cry8Bc1 gene from the Bt bacterial strain, discovery has significant insecticidal effect to west corn root leaf A (Western corn rootworm) and has been used for the exploitation (Abad of transgenic insect-resistant corn, Andre, R., Duck Nicholas, B., Feng, Xiang, Flannagan Ronald, D., Kahn, Theodore, W., Sims, Lynne, E.Genes encoding novel proteins with pesticidal activity againstcoleopterans, 2002, WO 02/34774A2).In China; the screening successively in recent years of plant protection institute of Hebei province Academy of Agricultural Sciences and Agricultural University Of Hebei obtains the Bt bacterial strain that many strains have special insecticidal activity to yellowish-brown rutelian (Anomala exoleta) and anomala corpulenta (A.corpulenta) larva; the indoor biometrics mortality ratio all reaches 100% (Feng Shuliang etc.; " strain has the new strain isolated of Bacillus thuringiensis of insecticidal activity to cockchafer subclass larva "; " Chinese biological control "; 2000,16 (2): 74-78).

Table 1 bacillus thuringiensis Cry8 class insecticidal crystal protein

Title	Numbering	Molecular weight	Amino acid no	The Bt bacterial strain	Active	The document source
							CrygAa1	U04364	?131.00	?1157	kumamotoensis	?scarabs	????Tracy，et?al， ????1994
Cry8Ba1	U04365	?133.54	?1169	kumamotoensis	?scarab	????Tracy，et?al， ????1994
							Cry8Bb1	AX543924	?136.53	?1206	Bt	?Leptinotarsa ?decemlineata， ?Diabrotica?virgifera ?virgifera， ?Deiabrotica ?undecimpunctata ?howardi	????Abad，et?al， ????2002
Cry8Bc1	AX543926	?137.20	?1210	Bt	????Abad，et?al， ????2002
						Cry8Ca1	U04366	?130.42	?1160	japonensis Buibui	?Anomala?cuprea		????Sato，et?al. ????1994
Cry8Da1	AB089299	?128.05	?1144	Bt?galleriae	?Anomala?cuprea							????unpublished
						Cry8Da2	BD133574	?130.51	?1167	Bt	?scarabs		????Asano，et?al， ????2002
Cry8Da3	BD133575	?130.51	?1167	Bt	?scarabs							????Asano，et?al， ????2002

Content of the present invention

Purpose of the present invention:

The purpose of this invention is to provide a kind of bacillus thuringiensis bacterial strain and two kinds of new cry8E and the cry8F pattern gene order that Coleoptera important pests such as Holotrichia parallela, Brontispa longissima are had high virulence, and a kind of cry8E and cry8F assortment of genes that Holotrichia parallela is had the notable synergistic effect, to be applied to transform microorganism and plant, make it to show toxicity, and overcome, delay the resistance generation of insect engineering bacteria and transgenic plant to relevant insect.

Technical scheme of the present invention;

1. the screening and the evaluation of the effective Bt bacterial strain 185 of zygobranchiate cockchafer subterranean pest-insect

The present invention has separated Bt bacterial strain 185 voluntarily.Soil picks up from apple orchard, Shunping County, Baoding, Hebei province.Get the 0.1-0.2g soil sample and put into the test tube that 10ml aqua sterilisa and granulated glass sphere are housed, vibration is 3 minutes on the vortex vibrator, and grogs is smashed, be put in then on the 200rpm shaking table and vibrated 10 minutes, water-bath is 17 minutes in 75 ℃ of water-baths, fully non-sporeformer is killed, after treating to leave standstill slightly, choose 10 ^-2, 10 ^-3, 10 ^-4Three extent of dilution are drawn the 100ul bacteria suspension respectively on the BP flat board, and coating is even, and in 37 ℃ of cultivations three days, picking was like Bt bacterium colony smear for microscopic examination.Find that a strain contains the Bt bacterial strain of sphaerocrystal (seeing accompanying drawing 1).

The culture condition of this bacterial strain is 30 ℃, common LB substratum, pH7.0.This bacterial strain is bacillus (Bacillus), bacillus thuringiensis kind, and in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation date is on November 5th, 2004, and deposit number is CGMCC NO.1242.Through identifying, comprise about the information of this bacterial strain: can form gemma, can form spherical parasporal crystal simultaneously, the SDS-PAGE electrophoresis shows that its insecticidal crystal protein is about 130kDa (seeing accompanying drawing 2); Its crystallin began at 14 hours to express, and growth curve shows its 14 hours and enters lag phase (seeing accompanying drawing 3), showed that this crystallin promotor may gemma forms in order to rely on; Biology is measured and is shown that this bacterial strain has obvious insecticidal action to Scarabaeidae subterranean pest-insect Holotrichia parallela, and corrected mortality reached 90% in 7 days, and Brontispa longissima is also had certain insecticidal activity (seeing Table 2).

The insecticidal activity of table 2 Bt bacterial strain 185

	7 days corrected mortality %	14 days corrected mortality %
			Holotrichia parallela	90	?100
Brontispa longissima	22	?35

2. cry gene identification in the bacterial strain 185

According to cry8 genoid conserved regions design a pair of universal primer:

S5un8：5-’CGGCAAACTTAGTAGAATGC-3’

S3un8：5-’CTGACTGATTTCCACCATCACG-3。

Table 3 is homologous sequences of these genes and primer, and table 4 is with this cry8 gene amplification product to the primer prediction, and the endonuclease bamhi size, can identify respectively these genes by this PCR-RFLP method.

Table 3 primer and cry8 each gene conservative district pairing situation and position on gene, collochore

Primer S5un8/S3un8 and gene conservative district pairing situation

Primer S5un8: primer S3un8:

Genotype

5 '-cggcaaacttagtagaat position 5 '-the gcactaccacctttagtcagt position

gc-3′??????????????????????????????c-3

cry8Aa??cggcaaacttagtGgaatgc????2101～2120??cgtgatggtggaaatcaAtcag?????3273～3294

cry8Ba??cggcCaacttagtGgaatgc????2089～2108??cgtgatggtggaaatcaAtcag?????3261～3282

cry8Bb??cggcaaacttagtGgaatgc????2104～2123??cgtgatggtggaaacaAAcag??????3276～3297

cry8Bc??cggcaaacttagtGgaatgc????2116～2135??cgtgatggtggaaatcaAAcag?????3288～3309

cry8Ca??cggcaaacttaAtagaatgc????2092～2111??cgtgatggtgcaaatcagAcag?????3282～3303

cry8Da??TAAAaaacttagtagaatgc????2044～2063??cgtgatggtgCGaatcagTcag?????3234～3255

Annotate: " N " is unpaired base.

The pcr amplification product of table 4 cry8 and restriction enzyme digestion length polymorphism

PCR(S5un8/S3un8)

Genotype product size EcoO109I and DraI enzyme are cut the result

Size(bp)??????Digested?with?EcoO109I?and?DraI(bp)

cry8Aa????1194???????????????????????197，442，555

cry8Ba????1194???????????????????????639，555，

cry8Bb????1194???????????????????????639，555

cry8Bc????1194???????????????????????639，555

cry8Ca????1212??????????????????197，92，310，613

cry8Da????1212??????????????????197，92，310，613

(50 μ L) identified Bt bacterial strain 185 with following PCR reaction system:

10×PCR?buffer	?5μL
		MgCl 2(20mM)	6μL
dNTP(10mM)	1μL
		Primer is to (10mM)	1 μ L/
Template	1uL
		Taq polysaccharase (5U/ μ L)	0.5μL

Ultrapure water is mended to 50 μ L, and mixing is centrifugal, adds paraffin oil 30 μ L.

Amplification cycles: 94 ℃ of sex change 1 minute, 54 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, 25 circulations, last 72 ℃ were extended 10 minutes.

Result's (seeing accompanying drawing 4) shows different with the collection of illustrative plates of known cry8 genoid, shows and may contain new cry8 killing gene in the bacterial strain 185.

3. the clone of cry8E gene in the bacterial strain 185

Cut the total DNA of Bt bacterial strain with PstI and KpnI enzyme, two dna fragmentation storehouses have been set up respectively with carrier pBluescript SK (+), use primer S5un8/S3un8 (seeing Table 3) and PCR method to detect two dna libraries then, obtain two positive colony pSS3612 and pSS162, insert the pstI fragment of 11kb and the KpnI fragment of 2.0kb (seeing accompanying drawing 5) respectively.Restriction analysis pSS3612, the PstI of 7kb and KpnI double digestion fragment contain the full-length gene of cry8Ea1, with this fragment of pBluescript SK (+) subclone, obtain pSS3612-7, simultaneously subclone in the KpnI fragment of 4kb, obtain pSS3612-4 (seeing accompanying drawing 6).Insertion sequence order-checking with pSS162 and pSS3612-7.Obtain sequence SEQ ID NO 1.

4.cry8Ea1 Gene Sequence Analysis

Sequence SEQ ID NO 1 is PstI and a KpnI double digestion fragment among the pSS3612, sequence total length 7276bps, and it contains two bigger open reading frame analysis revealed, and the position of ORF1 is 3658-7152, and the position of ORF2 is 2799-3377.

The position of ORF1 is 3658-7152, and GC content is 38.03%, the albumen that 1164 amino acid of encoding are formed.After measured, its aminoacid sequence is shown in the SEQ IDNO 2.This albumen of homology analysis revealed and Cry8 proteinoid have higher homology, and table 5 is its homology data.Owing to all be lower than 78% with known Cry8 proteinoid amino acid identity, the highest have only 58.2% (Cry8Bb1), by the called after Cry8Ea1 of Bt insecticidal crystal protein NK.

Table 5 Cry8 albumen homology comparative data

	?Cry8a1	Cry8Ba1	?Cry8Bb1	?Cry8Bc1	?Cry8Da1	?Cry8Ca1	?Cry8Ea1	?Cry8Fa1
									Cry8Aa1	?100％
Cry8Ba1	?46.10％	100％
									Cry8Bb1	?47.20％	82.10％	100％
Cry8Bc1	?46.10％	83.50％	90.50％	100％
									Cry8Da1	?51.30％	26.20％	25.60％	25.50％	100％
Cry8Ca1	?36.70％	21.20％	21.30％	21.00％	63.10％	100％
									Cry8Ea1	?26.60％	55.60％	57.00％	58.20％	26.10％	21.00％	100％
Cry8Fa1	?24.40％	42.50％	42.40％	42.10％	24.60％	20.60％	64.80％	100％

The present invention has further analyzed the proteic amino acid of Cry8Ea1 and has formed (seeing Table 6, accompanying drawing 7), learns that its molecular weight is 131.56kDa, and iso-electric point is pH4.735 (seeing accompanying drawing 8).

The proteic amino acid of table 6 Cry8Ea1 is formed

Amino acid	Number	Per-cent %	Amino acid	Number	Per-cent %
						?Ala(A)：	63	?5.41	?Asn(N)：	80	?6.87
?Asx(B)：	0	?0	?Pro(P)：	52	?4.46
						?Cys(C)：	4	?.34	?Gln(Q)：	61	?5.24
?Asp(D)：	63	?5.41	?Arg(R)：	62	?5.32
						?Glu(E)：	80	?6.87	?Ser(S)：	93	?7.98
?Phe(F)：	43	?3.69	?Thr(T)：	87	?7.47
						?Gly(G)：	80	?6.87	?Val(V)：	81	?6.95
?His(H)：	9	?.77	?Trp(W)：	17	?1.46
						?Ile(I)：	62	?5.32	?Unk(X)：	0	?0
?Lys(K)：	46	?3.95	?Tyr(Y)：	69	?5.92
						?Leu(L)：	93	?7.98	?Glx(Z)：	0	?0
?Met(M)：	19	?1.63	?Total	：	?1164

5.cry8Fa1 the clone of gene

Insertion sequence in the pSS162 plasmid is analyzed, sheet segment length 2.3kb, has 3 ' complete terminal sequence, with the complete homology of cry8Ea1 sequence, but 5 ' end differs greatly, and lack complete reading frame, designed 1 couple of primer (5-185-KpnI:TTGGTATGGCGTTTCGTTG according to the special section of this sequence; And 3-185-Kpnl:TATTGCAGGTCCAGGATTCAC), be used to clone this full-length gene, plasmid DNA XbaI enzyme cutting with bacterial strain 185, be connected with carrier pBluescript SK (+), screening obtains inserting the segmental positive colony pSS266 of about 9Kb external source (seeing accompanying drawing 9), further restriction analysis shows that the ClaI enzyme cuts the 3.0kb fragment of generation and contain 5 ' end reading frame (seeing accompanying drawing 10), obtain this fragment with pBluescript SK (+) subclone, positive colony called after pSS266-3, this fragment is checked order, the insertion sequence among sequence that obtains and the pSS162 is spliced obtain the 3.9kb fragment.

6.cry8F Gene Sequence Analysis

Nucleic acid fragment to above-mentioned 3.9kb checks order, and the nucleotide sequence that obtains is shown in SEQ ID NO 3.This sequence is carried out analysis revealed: this sequence contains 1 bigger open reading frame 357-3878; GC content is 36.88%; The albumen (aminoacid sequence of its encoded protein matter is shown in SEQ ID NO 4) that 1174 amino acid of encoding are formed.Further homology analysis revealed, this protein and Cry8 proteinoid have higher homology (the homology data are seen above table 5).Owing to all be lower than 78% with known Cry8 proteinoid amino acid identity, the highest have only 64.8% (Cry8Ea1), and this albumen is by the called after Cry8Fa1 of Bt insecticidal crystal protein NK.

The present invention has further analyzed the proteic amino acid of Cry8Fa1 with bioanalysis software Bioedit and has formed, and the results are shown in Table 7 and accompanying drawing 11.The result shows that this proteic molecular weight is 133.08kDa, and iso-electric point is pH4.565 (seeing accompanying drawing 12).

The proteic amino acid of table 7 Cry8Ea1 is formed

Amino acid	Number	Per-cent %	Amino acid	Number	Per-cent %
						?Ala(A)：	68	?5.79	?Asn(N)：	91	?7.75
?Asx(B)：	0	?0	?Pro(P)：	54	?4.59
						?Cys(C)：	3	?.25	?Gln(Q)：	60	?5.11
?Asp(D)：	62	?5.28	?Arg(R)：	60	?5.11
						?Glu(E)：	87	?7.41	?Ser(S)：	82	?6.98
?Phe(F)：	47	?4	?Thr(T)：	91	?7.75
						?Gly(G)：	68	?5.79	?Val(V)：	79	?6.72
?His(H)：	12	?1.02	?Trp(W)：	15	?1.27
						?Ile(I)：	72	?6.13	?Unk(X)：	0	?0
?Lys(K)：	40	?3.4	?Tyr(Y)：	63	?5.36
						?Leu(L)：	102	?8.68	?Glx(Z)：	0	?0
?Met(M)：	18	?1.53	?Total	1174	?100

7.cry8E and cry8F expression of gene

The present invention has designed the primer that is used to express two kinds of genes according to clone's the cry8Ea1 and the full length sequence of cry8Fa1 gene, and sequence is as follows:

8E1：CGC GGATCC(BamHI)GATGAGTCCAAATAATCAAAATG

8E2：ACGC GTCGAC(SalI)CTCTACGTCAACAATCAATCAATTC

8F1：CGC GGATCC(BamHI)GATGAGTCCAAATAATCAAAATG

8F2：CATTAACTCTGCCCACGGATCT(C)TCTGGTGCAAAGAAGTCCAG

8F3：CTTGACTTCTTTGCACCAGAA(G)GATCCGTGGGCAGTTAATG

8F4：CCG CTCGAG(XhoI)CTCTACGTCAACAATCAATCAATTC

Primer 8E1 and 8E2 introduce BamHI and SalI site respectively, are template with the pSS3612 plasmid DNA that contains total length cry8Ea1, and amplification obtains full-length gene, insert among the Bt expression vector pSXY422b, transformed into escherichia coli SCS110 extracts plasmid, and electric shock transforms Bt does not have crystal mutant strain HD-73 ^-In (this mutant strain derives from Plant Protection institute, Chinese Academy of Agricultral Sciences Biotechnology Experiment chamber, can provide to the public), obtain engineering bacteria BioT8E.

Owing to there is 1 BamHI point of contact in the cry8Fa1 full-length gene, with the method for overlapping primer PCR this site is suddenlyd change, introduce point mutation (line part), primer 8F1 and 8F4 among overlapping primer 8F2 and the 8F3 and introduced BamHI and XhoI respectively.With the pSS266 plasmid DNA that contains total length cry8Fa1 gene is template, obtain 0.3kb and 3.1kb product with 8F1 and 8F2,8F3 and 8F4 amplification respectively, be template with them respectively again, utilize primer 8F1 and 8F4 amplification to obtain the full-length gene of 3.4kb respectively, insert among the Bt expression vector pSXY422b, transformed into escherichia coli SCS110 extracts plasmid, and electric shock transforms Bt does not have crystal mutant strain HD-73 ^-In, obtain engineering bacteria BioT8F.

Respectively with 30 ℃ of above-mentioned two strain engineering bacterias in extractum carnis substratum (peptone 5 grams, extractum carnis 3 grams, glucose 10 grams, water 1000mL, 121 ℃, 20 minutes high pressure steam sterilizations) the middle cultivation 30 hours, get 500 μ L bacterium liquid to the Eppendorf pipe, ultrasonic disruption 30 seconds (B.Braun U Labsonic, 230V, T _{At interval}=0.5 second); Get 100 μ L and add 25 μ L and newly join 0.5N NaOH, 25 ℃ of effects 5 minutes; Add 65 μ L, 3 * sample buffer (925 μ L sample-loading buffers+75 μ L beta-mercaptoethanols), 100 ℃ were boiled 5 minutes.The centrifugal precipitation of removing.Last sample 10uL carries out SDS-PAGE electrophoretic analysis result, and (method is referring to Sambrook, J.et al, Molecular Cloning:A Laboratory Manual, 2nd ed.Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.1989).Result's (seeing accompanying drawing 13) shows that cry8Ea1 among engineering bacteria Biot8E and the Biot8F and cry8Fa1 gene have all obtained expression, and the molecular weight of representation is about 130kDa.

8.Cry8E and the proteic determination of activity of Cry8F

The Bt engineering strain is seeded on the common bacteria agar Kolle flask substratum cultivated 3 days.With F-strain HD-73 ^-Be seeded on the common bacteria agar Kolle flask substratum and cultivated 4 days.Culture is washed, 2 times of gradient concentration dilutions, the 40ml bacteria suspension is joined 200g to be had in the fine earth (ultraviolet disinfection) of even thickness potato silk, and mixing makes soil moisture content remain on 18%-20%.Insert 20 of 15 days instar larvaes of Holotrichia parallela, adding the blank that is treated to of clear water, 28 ℃ are infected and raise, and check in 14 days to calculate LC by dead borer population ₅₀The results are shown in Table 8, show that engineering strain has high cytotoxicity to Holotrichia parallela.The Cry8E of its expression and Cry8F albumen all have the activity of Holotrichia parallela larva extremely.

Table 8 Bt engineering bacteria and 185 bacterial strains are to the toxicity test of Holotrichia parallela larva

Bacterial strain	LC 50(10 8cfu/ml)	Regression equation	The R value
				??Biot8E	????0.5250	?Y＝5.3841+1.3724X	??0.9671
??Biot8F	????0.9464	?Y＝5.0276+1.1515X	??0.9918

Culture presevation information:

Strain name: bacillus, bacillus thuringiensis, Bacillus thuringiensis

Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center

Preservation date: on November 5th, 2004

Deposit number: CGMCC NO.1242

Description of drawings:

Fig. 1: 185 bacterial strain gemma and crystal habits.

Fig. 2: 185 bacterial strain crystallin SDS-PAGE analyze.

Fig. 3: 185 strain growth curves.

Fig. 4: the PCR-RFLP collection of illustrative plates of bacterial strain 185.Wherein:

The M.DNA molecular weight standard

1.PCR product

2.PCR the product enzyme is cut

Fig. 5: the pcr amplification product of recombinant plasmid pSS162 and pSS3162 and restriction analysis.Wherein:

M.λDNA/Eco?130I

1.pSS162 PCR product

2.pSS3162 PCR product

3.pBlueScript?SK(+)/KpnI

4.pSS162/?KpnI

5.pSS3162/PstI

Fig. 6: pSS3612 inserts segmental subclone restriction analysis.Wherein:

M.λDNA/Eco?130I

1.pBlueScript?SK(+)/KpnI

2.pSS3162/PstI+KpnI

3.pSS3612/KpnI

4.pSS3162?/PstI+KpnI

The proteic amino acid composition diagram of Fig. 7: Cry8Ea1.

The proteic titration curve of Fig. 8: Cry8Ea1.

Fig. 9: the pcr amplification product of recombinant plasmid pSS266 and restriction analysis.Wherein:

M.λDNA/Eco?130I

1.pSS266 PCR product

2.pSS162/XbaI

3.pBlueScript?SK(+)/XbaI

Figure 10: the restriction analysis of recombinant plasmid pSS266.Wherein:

1.XbaI

2.NotI

3.PstI

4.NdeI

5.ClaI

6.SacI

7.KpnI

M.λDNA/Eco130I

The proteic amino acid composition diagram of Figure 11: Cry8Fa1.

The proteic titration curve of Figure 12: Cry8Fa1.

Figure 13: cry8Ea1 and cry8Fa1 gene do not have expression in the crystal mutant strain at Bt.Wherein:

M. protein molecular weight standard

1.Biot8E

2.Biot8F

3.Bt?185

4.HD-73 ^-

Specific embodiments

Below narrate embodiment according to embodiments of the present invention.Should be noted that embodiments of the invention have only illustration for the present invention, and effect without limits.

The screening and the evaluation of embodiment 1, the effective Bt bacterial strain 185 of zygobranchiate cockchafer subterranean pest-insect

Soil picks up from apple orchard, Shunping County, Baoding, Hebei province.Get the 0.1-0.2g soil sample and put into the test tube that 10ml aqua sterilisa and granulated glass sphere are housed, vibration is 3 minutes on the vortex vibrator, and grogs is smashed, be put in then on the 200rpm shaking table and vibrated 10 minutes, water-bath is 17 minutes in 75 ℃ of water-baths, fully non-sporeformer is killed, after treating to leave standstill slightly, choose 10 ^-2, 10 ^-3, 10 ^-4Three extent of dilution are drawn the 100ul bacteria suspension respectively on the BP flat board, and coating is even, and in 37 ℃ of cultivations three days, picking was like Bt bacterium colony smear for microscopic examination.Find that a strain contains the Bt bacterial strain of sphaerocrystal (seeing accompanying drawing 1).

This bacterial strain is bacillus (Bacillus), bacillus thuringiensis kind.Submit this bacterial strain to preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on November 5th, 2004, and deposit number is CGMCCNO.1242.Through identifying, information about this bacterial strain comprises: can form gemma, simultaneously can form spherical parasporal crystal, the SDS-PAGE electrophoresis shows that its insecticidal crystal protein is about 130kDa (seeing accompanying drawing 2), its crystallin began at 14 hours to express, and growth curve shows its 14 hours and enters lag phase (seeing accompanying drawing 3), shows that this crystallin promotor may gemma forms in order to rely on; Biology is measured and is shown that this bacterial strain has obvious insecticidal action to Scarabaeidae subterranean pest-insect Holotrichia parallela, and corrected mortality reached 90% in 7 days, and Brontispa longissima is also had certain insecticidal activity.

Cry gene identification in embodiment 2, the bacterial strain 185

According to cry8 genoid conserved regions design a pair of universal primer

S5un8：5-’CGGCAAACTTAGTAGAATGC-3’

S3un8：5-’CTGACTGATTTCCACCATCACG-3。

(50 μ L) identified Bt bacterial strain 185 with following PCR reaction system:

10×PCR?buffer	?5μL
		MgCl 2(20mM)	6μL
dNTP(10mM)	1μL
		Primer is to (10mM)	1 μ L/
Template	1uL
		Taq polysaccharase (5U/ μ L)	0.5μL

Ultrapure water is mended to 50 μ L, and mixing is centrifugal, adds paraffin oil 30 μ L.

Amplification cycles: 94 ℃ of sex change 1 minute, 54 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, 25 circulations, last 72 ℃ were extended 10 minutes.

Result's (seeing accompanying drawing 4) shows different with the collection of illustrative plates of known cry8 genoid, shows and may contain new cry8 killing gene in the bacterial strain 185.

The clone of cry8E gene in embodiment 3, the bacterial strain 185

Cut the total DNA of Bt bacterial strain with PstI and KpnI enzyme, two dna fragmentation storehouses have been set up respectively with carrier pBluescript SK (+), detect two dna libraries with primer S5un8/S3un8 and PCR method then, obtain two positive colony pSS3612 and pSS162, insert the pstI fragment of 11kb and the KpnI fragment of 2.0kb (seeing accompanying drawing 5) respectively.Restriction analysis pSS3612, the PstI of 7kb and KpnI double digestion fragment contain the full-length gene of cry8Ea1, with this fragment of pBluescript SK (+) subclone, obtain pSS3612-7, simultaneously subclone in the KpnI fragment of 4kb, obtain pSS3612-4 (as accompanying drawing 6).Insertion sequence order-checking with pSS162 and pSS3612-7 obtains sequence SEQ ID NO 1.This sequence is PstI and a KpnI double digestion fragment among the pSS3612, sequence total length 7276bps, and it contains two bigger open reading frame analysis revealed, and the position of ORF1 is 3658-7152, and the position of ORF2 is 2799-3377.

The position of ORF1 is 3658-7152, and GC content is 38.03%, the albumen that 1164 amino acid of encoding are formed, and after measured, its aminoacid sequence is shown in the SEQ ID NO 2.This albumen of homology analysis revealed and Cry8 proteinoid have higher homology (seeing Table 5).Owing to all be lower than 78% with known Cry8 proteinoid amino acid identity, the highest have only 58.2% (Cry8Bb1), by the called after Cry8Ea1 of Bt insecticidal crystal protein NK.

The present invention has further analyzed the proteic amino acid of Cry8Ea1 and has formed (see Table 6 and accompanying drawing 7), learns that its molecular weight is 131.56kDa, and iso-electric point is pH4.735 (seeing accompanying drawing 8).

The clone of embodiment 4, cry8Fa1 gene

Insertion sequence in the pSS162 plasmid is analyzed, sheet segment length 2.3kb, has 3 ' complete terminal sequence, with the complete homology of cry8Ea1 sequence, but 5 ' end differs greatly, and lack complete reading frame, designed 1 couple of primer (5-185-KpnI:TTGGTATGGCGTTTCGTTG according to the special section of this sequence; And 3-185-Kpnl:TATTGCAGGTCCAGGATTCAC), be used to clone this full-length gene, with 185 plasmid DNA XbaI enzyme cuttings, be connected with carrier pBluescript SK (+), screening obtains inserting the segmental positive colony pSS266 of about 9Kb external source (seeing accompanying drawing 9), further restriction analysis shows that the ClaI enzyme cuts the 3.0kb fragment of generation and contain 5 ' end reading frame (seeing accompanying drawing 10), obtain this fragment with pBluescript SK (+) subclone, positive colony called after pSS266-3, this fragment is checked order, the insertion sequence among sequence that obtains and the pSS162 is spliced obtain the 3.9kb fragment.Nucleic acid fragment is checked order, obtain the nucleotide sequence shown in SEQ ID NO 3.This sequence is carried out analysis revealed: this sequence contains 1 bigger open reading frame 357-3878; It heavily is 36.88% that GC contains; The albumen (this proteinic aminoacid sequence is shown in SEQ ID NO 4) that 1174 amino acid of encoding are formed.Further homology analysis revealed, this protein and Cry8 proteinoid have higher homology (seeing Table 5).Owing to all be lower than 78% with known Cry8 proteinoid amino acid identity, the highest have only 64.8% (Cry8Ea1), and this albumen is by the called after Cry8Fa1 of Bt insecticidal crystal protein NK.

The present invention has further analyzed the proteic amino acid of Cry8Fa1 with bioanalysis software Bioedit and has formed (see Table 7 and accompanying drawing 11).The result shows that this proteinic molecular weight is 133.08kDa, and iso-electric point is pH4.565 (seeing accompanying drawing 12).

Embodiment 5, cry8E and cry8F expression of gene

According to clone's the cry8Ea1 and the full length sequence of cry8Fa1 gene, designed the primer that is used to express two kinds of genes, sequence is as follows:

8E1：CGC GGATCC(Bam?HI)GATGAGTCCAAATAATCAAAATG

8E2：ACGC GTCGAC(Sal?I)CTCTACGTCAACAATCAATCAATTC

8F1：CGC GGATCC(Bam?HI)GATGAGTCCAAATAATCAAAATG

8F2：CATTAACTCTGCCCACGGATC T(C)TCTGGTGCAAAGAAGTCCAG

8F3：CTTGACTTCTTTGCACCAGA A(G)GATCCGTGGGCAGTTAATG

8F4：CCG CTCGAG(Xho?I)CTCTACGTCAACAATCAATCAATTC

Primer 8E1 and 8E2 introduce BamHI and SalI site respectively, are template with the pSS3612 plasmid DNA that contains total length cry8Ea1, and amplification obtains full-length gene, insert among the Bt expression vector pSXY422b, transformed into escherichia coli SCS110 extracts plasmid, and electric shock transforms Bt does not have crystal mutant strain HD-73 ^-In, obtain engineering bacteria BioT8E.

Owing to there is 1 BamHI point of contact in the cry8Fa1 full-length gene, with the method for overlapping primer PCR this site is suddenlyd change, introduce point mutation (line part), primer 8F1 and 8F4 among overlapping primer 8F2 and the 8F3 and introduced BamHI and XhoI respectively.With the pSS266 plasmid DNA that contains total length cry8Fa1 gene is template, obtain 0.3kb and 3.1kb product with 8F1 and 8F2,8F3 and 8F4 amplification respectively, be template with them respectively again, utilize primer 8F1 and 8F4 amplification to obtain the full-length gene of 3.4kb respectively, insert among the Bt expression vector pSXY422b, transformed into escherichia coli SCS110 extracts plasmid, and electric shock transforms Bt does not have crystal mutant strain HD-73 ^-In, obtain engineering bacteria BioT8F.

Respectively above-mentioned two strain engineering bacterias were cultivated 30 hours in the extractum carnis substratum for 30 ℃, got 500 μ L bacterium liquid to the Eppendorf pipe, ultrasonic disruption 30 seconds (B.Braun U Labsonic, 230V, T _{At interval}=0.5 second); Get 100 μ L and add 25 μ L and newly join 0.5NNaOH, 25 ℃ of effects 5 minutes; Add 65 μ L, 3 * sample buffer (925 μ L sample-loading buffers+75 μ L beta-mercaptoethanols), 100 ℃ were boiled 5 minutes.The centrifugal precipitation of removing.Last sample 10uL carries out SDS-PAGE electrophoretic analysis result.Result's (seeing accompanying drawing 13) shows that cry8Ea1 among engineering bacteria Biot8E and the Biot8F and cry8Fa1 gene have all obtained expression, and the molecular weight of representation is about 130kDa.

The proteic determination of activity of embodiment 6, Cry8E and Cry8F

The Bt engineering strain is seeded on the common bacteria agar Kolle flask substratum cultivated 3 days.With F-strain HD-73 ^-Be seeded on the common bacteria agar Kolle flask substratum and cultivated 4 days.Culture is washed, 2 times of gradient concentration dilutions, the 40ml bacteria suspension is joined 200g to be had in the fine earth (ultraviolet disinfection) of even thickness potato silk, and mixing makes soil moisture content remain on 18%-20%.Insert 20 of 15 days instar larvaes of Holotrichia parallela, adding the blank that is treated to of clear water, 28 ℃ are infected and raise, and check in 14 days to calculate LC by dead borer population ₅₀Result's (seeing Table 8) shows that engineering strain has high cytotoxicity to Holotrichia parallela.The Cry8E of its expression and Cry8F albumen all have the activity of Holotrichia parallela larva extremely.

Attached: dna sequence dna involved in the present invention and protein sequence

SEQ ID NO 1 (nucleotide sequence of cry8Ea1 gene):

ctgcagaata?gacacggata?cgatcgcctt?cacataaatg?ctgaaatctt?cttctagaca????60

PstI

ttcttgtgtc?acctcatttt?ttgtttttaa?actacagtat?gttatatgca?aaagaagggg????120

tagaggattg?ggccttttac?tacaaaaata?caaaaacata?cttatgattg?catatggaga????180

tgtcaaagtg?catgcattaa?aaatggatta?gaaatgattt?caaataggca?aaagcctatt????240

ccaatgaaga?aagattgaca?taggctattg?tatatagaag?aaggtaacga?ggaacatact????300

gttagggtac?acctaacata?gaagtatgct?tgtttgaagc?atgtacatct?tgaaatacca????360

gtagaaatat?gggggaacat?gttattttaa?taattggtaa?aatcttttgt?tagaaggtga????420

aggcgtatga?gacaacaaag?agtatgtgag?tgtaacaatt?gtaggaaaaa?gggtgagaag????480

aatcatcttt?gtcaatatat?aaaacgtggg?gattgtatat?gggtcagttc?ctttggaagc????540

aagttacaaa?agagtggtat?tttcctgtta?ataaaagatt?cttttttatt?atggtttgat????600

gaaaaacatc?aattaaatca?aaccagtcta?caggggatcc?atattgaaaa?aagacaataa????660

atgaagaagg?agtctcattg?ttaacgaagt?gtgtacatct?atcatgtaca?catcgtaagt????720

cgtatgttct?acctgtatct?ggtaggaaag?aattgtcgca?tgtgcaaggc?gtatatacac????780

aaacatgttt?tgttatattt?ttgaataatt?tgaaaataaa?tatgttataa?ttaatatact????840

ttcgtgtgtt?ttttttgcga?aatccctaga?aagtatcgta?aaaagtccct?aacaattttg????900

tgaactgaac?ccaaaaaatt?agacaaatat?attaagcagc?tactaaggat?tgaactctgt????960

attgcacggg?ggacaatcct?tttagttttg?ttttaattct?tttgtgattg?tagtaatgaa????1020

tataagtttc?tagttcttgc?ttaaattgtt?ccatactttc?aaactcttta?agataaagta????1080

attcagactt?taataagcca?aagaaatttt?ccatgactgc?attatctaag?caatttccct????1140

tacgggacat?actttggata?acgttatgtt?ttttaagcga?ctgatgatat?tgtcgcattt????1200

gataatgcca?accttgatcc?gagtgtaaaa?taggagtttc?cttatcattc?aaacattgaa????1260

acgccttatt?taacatttta?gaaacaaggg?aataggcagg?tctatgttct?atattgtaag????1320

ctataatttc?tccgttatat?aagtcttaaa?tgggtgatag?atatagtttt?ttaccatgta????1380

agtggaactc?cgtcacatct?gttacccatt?tctcgtttgg?ttttgatgcg?tgaaaattac????1440

gttttaaaat?attaggagcg?aatttcccga?cagtcccttt?atatgaacga?tattttttta????1500

atcgaacaag?acattttaat?cccaggatat?tcattaaacg?tcgaacggtt?ttatgattta????1560

atgcatggcc?tcgattacgt?aattccaatg?taatacgacg?ataaccatac?ctcccaaaat????1620

tctcactaaa?aatctcttta?attaattctt?taactttctt?atatttatct?ggacgtttcg????1680

cttgtttcat?ccagtaataa?tacgtactac?gagcgatatt?agcgactttc?acaaggtcaa????1740

cgaccttata?tttatgcctt?aattcataaa?tcaattgcgc?tttgtcttgg?tctgtgatgt????1800

tttcttcttt?tgaactaagg?cattcaactt?ttttaaatag?tcattttcca?tacgcagacg????1860

ttcattctct?gcttgtagcg?cttctataga?accttcaaga?aatacttcgt?tttgttttaa????1920

atgttgtagc?ttagcttttt?ctttggccat?ggttagacgc?ccctttttct?ttgattttag????1980

ggcatctaat?ccttctgttt?cataagctac?tttccatttt?cggagtgttt?cgcaagaagg????2040

aatattaaaa?aaagcagctg?tttctctcag?agatgtccca?ttttcattca?tataatgaat????2100

tacatctagt?ttatactcga?gagggtaagt?tgtatagcgt?ttttcaaacg?ccttttcccc????2160

tgaaaattca?aaccgtttaa?tccattgata?aagttctcta?ggatgaaccc?ctatagaatt????2220

agcaatggtt?tttccgcctt?ccgtaccttc?tagatatcgt?tttactgctt?gtattttatc????2280

ttttgaagaa?aatttagcca?taaaaaatgc?acctccaatt?gttaattatg?tgtctaacaa????????????????????????2340

ttggggtgca?cttcattgtt?ggggactttt?gtatgttcct?ttttagacat?tcttttaagt????????????????????????2400

tctttatata?gaaagactga?agaaagcaga?ataagaagtc?catcccctga?ttcatgagaa????????????????????????2460

ccgaaaaatt?catgatgcac?tggatgccaa?atatttagat?acatttccta?ttgatattct????????????????????????2520

acgagattga?attgatgtaa?tgttgttccc?ttttggtcaa?actgaccaat?ggcgatagct????????????????????????2580

ctccttgcat?gagtacttct?caaacttcca?ttacacattg?tattccccat?cttttttatg????????????????????????2640

tatatctttt?ggggaaaatc?gtaatttctg?cttatgatga?caagatttta?ctaaaataag????????????????????????2700

aagagtggaa?tattttactc?tatgtcaaac?aaaaaagcaa?tatatgttta?aacgcgaaaa????????????????????????2760

ORF2→

ttcgaaaagg?ctccattgat?tcgataggag?gtgcacagaa?aaaaatggaa?gaacaatatg????????????????????????2880

catcgcaaga?tcagtcagat?gtagaaggtt?tcaagcggaa?gaaaaaacat?accattccct????????????????????????2940

ttcaatgtat?ggtttctatt?ccaacagggt?ttcaaattca?aaaaccgaat?acaccaaaac????????????????????????3000

ttgtttatga?tgtaagccat?ttatctatgg?taaaagagat?gtgtaaacga?gtgattgacg????????????????????????3060

tagaggattg?tgggcaagtc?gaaatcgatt?tacatgtctt?aaaaatcaaa?ggtgtcttac????????????????????????3120

cctttattgt?gaacgtttcc?attgagccgc?ttagtatgga?acatgtgtat?accacaagtg????????????????????????3180

gtagagacac?atccctattt?ttaagttgtc?aagaaaccgt?atatgtggat?catattttaa????????????????????????3240

aatatagtgt?cgatcatgtc?ccgtattatg?tgattgatgg?tcatcatatt?ctagtacgtg????????????????????????3300

atgtcgtgat?aaagttgttg?gaagaaaacc?cgcaaacggc?tcaaatatca?ggtgtttttt????????????????????????3360

End?codon

agcatcgcca?ccttttttat?catacaatag?ttcgttctaa?gaagagccgt?aatatttttc?????????????????????????3480

tatctaacag?gaattttatc?atctacagaa?gaatattctt?atcatggtaa?tgaggagagg?????????????????????????3540

gattgaaagt?caaaagatta?cctgatttgt?catgtaagaa?aaaggaatcg?atcgtacagg?????????????????????????3600

RBS????ORF1→

agtccaaata?atcaaaatga?atatgaaatt?atagatatgg?caccttctac?atctgtatcc?????????????????????????3720

aatgattcta?acagataccc?ttttgcgagt?gatccaacaa?atgcattaca?aaatatgaat?????????????????????????3780

tataaagagt?atttaagaat?gtctgaggga?tatgatagtg?aatattctgg?ctcacctgaa?????????????????????????3840

gtgcttatta?gtgagcgaga?tgcggttaag?acagcaatca?gtttggtagg?tactatatta?????????????????????????3900

ggaaaattag?gagttccatt?ggtaggaccg?attgtgagcc?tatatagtac?acttattgat?????????????????????????3960

gttttgtggc?caggtggaaa?gagtcaatgg?gaaattttta?tggaacaagt?agaagcactt?????????????????????????4020

attaatcaaa?aaatagcaga?atacgcaagg?gctaaggcac?ttgcagaatt?agaagggtta?????????????????????????4080

ggaaataact?atcaattata?tttaacagca?cttgaagaat?ggcaggaaaa?tccaagcagt?????????????????????????4140

acaagagtct?tacgtgatgt?tcggaatcga?tttgaaatcc?ttgatagctt?atttacacaa?????????????????????????4200

tatatgcctt?cttttcgggt?aacaggttat?gaagtaccat?tactttcagt?atatgcgcaa?????????????????????????4260

gcagctaacc?ttcatttatt?gttattaaag?gacgcttcta?tttttggaga?agaatggggg?????????????????????????4320

ttctctacaa?ccgctattaa?taactattat?aatcgtcaaa?tgagtcttat?cgcgcaatat?????????????????????????4380

tctgatcatt?gtgtacaatg?gtatagaact?gggttagatc?gattaaaagg?atcgaatgct?????????????????????????4440

aaacaatggg?ttgaatataa?ccgcttccga?agagaaatga?cattatcggt?gttagatatt?????????????????????????4500

atgacattat?ttccaatgta?tgacatgcgc?acgtacccaa?tggaaacaaa?agcacaacta?????????????????????????4560

acaagggaag?tatatacaga?tccaattggt?gccataggag?cgcaaggttc?ttggtatgac?????????????????????????4620

tcagcacctt?ctttcaatac?tctggaaagt?acttttataa?gaggaaagca?tctatttgat?????????????????????????4680

tttataacta?gactctctat?atatacaggg?cgaagctcat?tcagtgctag?taattactta?????????????????????????4740

aaaaaatgga?tagggcatca?aatatcctct?caacctatag?gcggcagtat?acaaactcaa?????????????????????????4800

acctatggca?ctacgagtgg?cagttctgtt?attgctacgc?agcaaattgg?ctttacaggt?????????????????????????4860

tttgacgttt?ataagacttt?atcaacagcg?ggggttctgt?ttgcttatac?ttcgaaatat?????????????????????????????4920

tatggcgtat?ctaaagttgt?ttttgatgcg?atatatcctg?acaacaagta?taaaacaaca?????????????????????????????4980

tttacctata?atcctggatc?tgaaggtatt?ggagcgcaag?aaaaggattc?agaagttgaa?????????????????????????????5040

ttgccaccag?aaacattaga?tcaacccaat?tatgaggcgt?atagccatag?attgaattat?????????????????????????????5100

gttacattta?ttagaaatcc?agatgtacca?gtattttctt?ggacacatcg?gagtgcggat?????????????????????????????5160

cgtacgaata?cagtttattc?agataaaatc?actcaaatac?cagttgtaaa?ggccagtgac?????????????????????????????5220

ggccctaaac?cttccgctaa?cgaagttgga?cactatcttg?gtggagatcc?aatatcattt?????????????????????????????5280

aactcttctg?gtagcactgg?agtgataagg?ttaaatataa?attcaccatt?atcccaaaaa?????????????????????????????5340

taccgtgtga?gaattcgcta?ttgctcttca?gttgattttg?acttagatgt?agttcgtgga?????????????????????????????5400

ggcactactg?taaataatgg?tagatttaac?aaaagcgcgc?ctaacgtcgg?atggcaaagt?????????????????????????????5460

ttgaagtatg?aaaattttaa?atttgcaagc?ttttctacac?cttttacatt?taatcaagct?????????????????????????????5520

caagatacat?taaaaataag?tgtaaggaat?tttagttcaa?tcgtaggagg?cagcgtagtt?????????????????????????????5580

tatatagacc?gaatcgagct?catcccagta?aatgcaacat?atgaggcaga?acaagattta?????????????????????????????5640

gattcggcaa?agaaagcagt?gaataccttg?tttacgaata?caaaagatgg?tttacgacca?????????????????????????????5700

ggggtaacgg?attatgaagt?gaatcaagcg?gcaaacttag?tggaatgcct?atcggatgat?????????????????????????????5760

ttgtatccaa?atgaaaaacg?cttgttattt?gatgcagtga?aagaggcaaa?acgactcagc?????????????????????????????5820

gaggcacgta?acttactaca?agatccagat?ttccaagaga?taaatggaga?aaatggatgg?????????????????????????????5880

accgcaagta?caggaattga?ggttgtagaa?ggagatgctc?tatttaaagg?gcgttatcta?????????????????????????????5940

cgcctaccag?gtgcgagaga?aatggataca?gaaacgtatc?caacgtatct?gtatcaaaaa?????????????????????????????6000

gtagaggaag?gtgtattaaa?accatacaca?agatatagat?tgagagggtt?tgtcggaagc?????????????????????????????6060

agtcaaggct?tggaaatttc?cacaattcgt?catcagacga?accgaattgt?aaaaaatgtt?????????????????????????????6120

ccagatgatt?tattaccaga?tgtacctcct?gtaaactctg?atggtagaat?caatcgatgc?????????????????????????????6180

agcgaacaaa?agtatgtgaa?tagccgttta?gaaggagaaa?gaggattacc?aaatgggaat?????????????????????????????6240

cgttctgctg?aagcgcatga?attctctctc?cctattgata?taggagagct?ggattacaat?????????????????????????????6300

gaaaatgcag?gaatatgggt?tggatttaag?attacggacc?cagagggata?tgcaacactc?????????????????????????????6360

ggtaaccttg?aattggtaga?agagggacca?ttgtcaggag?acgcactaga?acgcctgcaa?????????????????????????????6420

agagaagaac?aacagtggaa?gcttcaaatg?acaaaaagac?gtgaagagac?ggatagaaaa?????????????????????????????6480

tatacggcag?caaaacaagc?ggtagatcgt?ttatatgcag?attaccaaga?tcaacaattg?????????????????????????????6540

aatccaaacg?tagaaattac?ggatattact?gcggcccaaa?acctgataca?gtccattcct?????????????????????????????6600

tatgtatata?atgaaatgtt?cccagaaata?caagggatga?actatacgaa?gtacacagag?????????????????????????????6660

ttaacaaatc?gactccaaca?agcgtggggt?ttgtatgatc?aacgaaacgc?cataccaaat?????????????????????????????6720

ggtgatttcc?gaaatgaatt?aagtaattgg?aatacaacat?ctggtgtaaa?tgtacaacaa?????????????????????????????6780

atcaacaata?cgtctgtctt?agtcatgcca?aactgggatg?ggcaagtttc?gcaacagttt?????????????????????????????6840

acagttcaac?cgaatcaaag?atatgtatta?cgagttactg?caagaaaaga?aggggtaggg?????????????????????????????6900

aatgggtatg?tgagtatccg?tgatggtgga?aatcaaacag?aaacgcttac?gtttagtgca?????????????????????????????6960

agcgattata?acacagatag?tgtgtataat?acgcaagtgt?cgaatacaaa?tggtttgtac?????????????????????????????7020

aatgagcaaa?caggatatac?cacaaaaaca?gtgacattca?tcccatatac?agatcaagtg?????????????????????????????7080

tggattgaga?tgagcgagac?cgaaggtatg?ttctatatag?aaagtgtcga?attgattgtt?????????????????????????????7140

End?codon

gaaaaagggc?ctttttgtag?agaagaatcc?gattatttta?ttacgattat?atattttgtg?????????????????????????????7260

gatagatcat? ggtacc???????????????????????????????????????????????????????????????????????????7276

KpnI

SEQ ID NO 2 (the proteic aminoacid sequence of Cry8Ea1):

MSPNNQNEYE?IIDMAPSTSV?SNDSNRYPFA?SDPTNALQNM?NYKEYLRMSE?GYDSEYSGSP??????????????????????????????60

EVLISERDAV?KTAISLVGTI?LGKLGVPLVG?PIVSLYSTLI?DVLWPGGKSQ?WEIFMEQVEA??????????????????????????????120

LINQKIAEYA?RAKALAELEG?LGNNYQLYLT?ALEEWQENPS?STRVLRDVRN?RFEILDSLFT??????????????????????????????180

QYMPSFRVTG?YEVPLLSVYA?QAANLHLLLL?KDASIFGEEW?GFSTTAINNY?YNRQMSLIAQ??????????????????????????????240

YSDHCVQWYR?TGLDRLKGSN?AKQWVEYNRF?RREMTLSVLD?IMTLFPMYDM?RTYPMETKAQ??????????????????????????????300

LTREVYTDPI?GAIGAQGSWY?DSAPSFNTLE?STFIRGKHLF?DFITRLSIYT?GRSSFSASNY??????????????????????????????360

LKKWIGHQIS?SQPIGGSIQT?QTYGTTSGSS?VIATQQIGFT?GFDVYKTLST?AGVLFAYTSK??????????????????????????????420

YYGVSKVVFD?AIYPDNKYKT?TFTYNPGSEG?IGAQEKDSEV?ELPPETLDQP?NYEAYSHRLN??????????????????????????????480

YVTFIRNPDV?PVFSWTHRSA?DRTNTVYSDK?ITQIPVVKAS?DGPKPSANEV?GHYLGGDPIS??????????????????????????????540

FNSSGSTGVI?RLNINSPLSQ?KYRVRIRYCS?SVDFDLDVVR?GGTTVNNGRF?NKSAPNVGWQ??????????????????????????????600

SLKYENFKFA?SFSTPFTFNQ?AQDTLKISVR?NFSSIVGGSV?VYIDRIELIP?VNATYEAEQD??????????????????????????????660

LDSAKKAVNT?LFTNTKDGLR?PGVTDYEVNQ?AANLVECLSD?DLYPNEKRLL?FDAVKEAKRL??????????????????????????????720

SEARNLLQDP?DFQEINGENG?WTASTGIEVV?EGDALFKGRY?LRLPGAREMD?TETYPTYLYQ??????????????????????????????780

KVEEGVLKPY?TRYRLRGFVG?SSQGLEISTI?RHQTNRIVKN?VPDDLLPDVP?PVNSDGRINR??????????????????????????????840

CSEQKYVNSR?LEGERGLPNG?NRSAEAHEFS?LPIDIGELDY?NENAGIWVGF?KITDPEGYAT??????????????????????????????900

LGNLELVEEG?PLSGDALERL?QREEQQWKLQ?MTKRREETDR?KYTAAKQAVD?RLYADYQDQQ??????????????????????????????960

LNPNVEITDI?TAAQNLIQSI?PYVYNEMFPE?IQGMNYTKYT?ELTNRLQQAW?GLYDQRNAIP??????????????????????????????1020

NGDFRNELSN?WNTTSGVNVQ?QINNTSVLVM?PNWDGQVSQQ?FTVQPNQRYV?LRVTARKEGV??????????????????????????????1080

GNGYVSIRDG?GNQTETLTFS?ASDYNTDSVY?NTQVSNTNGL?YNEQTGYTTK?TVTFIPYTDQ??????????????????????????????1140

VWIEMSETEG?MFYIESVELIVDVE??????????????????????????????????????????????????????????????????????1164

SEQ ID NO 3 (nucleotide sequence of cry8Fa1 gene):

atcgataaag?ggaatggaag?acaactcgca?aatggctcaa?atatcgggtg?ttttttattt??????????????????????????????60

tgattatgca?taattacaat?gaaaacaaaa?agaattcatt?tgtatagtat?tcccagaaat??????????????????????????????120

atcgtgacat?cgtttatcat?acaataattc?gttctaagaa?gagccggatt?atttttcaat??????????????????????????????180

ctaacaggaa?ttttattgtc?tacagaagaa?tattcttatc?acggtaatga?ggagagggag??????????????????????????????240

tgaaaatcaa?aagagtacct?gatttgtcat?gtaagaacaa?aagaaatcga?tcgtacagga??????????????????????????????300

RBS????ORF→

agtccaaata?atcaaaatga?atatgaaatt?atagatatgg?caccttctac?atctgtaacc???????????????????????????????420

aatgattcta?acagataccc?ttttgcgaat?gagcccacaa?atgcattaca?aaatatgaat???????????????????????????????480

tataaggatt?atttaagaat?gtctgaggga?tattctcctg?aatatttaac?aagcctaagt???????????????????????????????540

ccttacagcc?agtttggcac?agttgataag?atcatcagta?ttattagtct?attgaatagt???????????????????????????????600

gctgcaggta?ttcctggtct?tgattttttt?actggattgc?tgcaatttat?tcttgacttc???????????????????????????????660

tttgcaccag?aggatccgtg?ggcagagtta?atggaactag?tggaacaact?catagatcaa???????????????????????????????720

aaaataacag?ttgctacaag?agaaaaggcg?ctcgcagaat?taagaggact?gataaatgga???????????????????????????????780

taccttgtat?atcagcaatc?attagaaagt?tggctggaaa?atccaaatgc?tacaagagct???????????????????????????????840

agtatagttc?gagaacaata?tgtcgcttta?gaacttgatt?ttgttacttc?gatttcatct???????????????????????????????900

tttgcgatag?ctggacagga?agtaccgtta?ttagccgtgt?acgcacaagc?tgctaattta???????????????????????????????960

catttgttat?tattgagaga?tgtgtcaata?tttggagaag?aatggggatt?aacagtaaat???????????????????????????????1020

gaggttaata?ccttctatat?tcgtcaaatg?acttatacaa?ctgagtatag?tgattattgt???????????????????????????????1080

gtaagaattt?ataatactgg?cttaaataaa?ttaaaaggat?ctagtgcatc?tagttgggtt???????????????????????????????1140

gattataatc?gctttcgtag?agaaatgaac?ttactagtac?tagatattat?tgcgttattt???????????????????????????????1200

ccaaactatg?atgttcgtag?gtatccaatg?gaaacaacaa?cggaattaac?aagagtagtt???????????????????????????????1260

tacactgatc?caattgtgtt?tgacgaaagg?aagggggtgg?cgtcgactca?tagttggacg????????????????????????1320

gcgattgcac?catctttctc?aagtatagaa?tctctaactc?gacgaccagg?attatttaca????????????????????????1380

tggttagatc?aactaactat?tttttcgaaa?cgcatatcgc?aacctagtgt?atttataaat????????????????????????1440

agttgggcgg?ggcataagat?tagcaccttt?agaacacaaa?aaacagatat?actcataaat????????????????????????1500

accacccatg?gagatactaa?taatcctata?aaagaatttg?tagtagatac?caaaaaagta????????????????????????1560

gaagatattt?atcaaacgat?agcataccca?catgcagtag?caaatgaagt?attctattta????????????????????????1620

ttcggtgtcc?caaaagttga?ttttaatatg?gtacctgcag?gtggctctgc?aaactctgca????????????????????????1680

cacaccctca?ttttttctga?tagtacggga?gggagactgg?aaagtattac?gaagaactca????????????????????????1740

gaagcagaat?tacctccaac?agagtcatta?tcagatacac?ctcaaccaaa?ccaagtaact????????????????????????1800

tattctcaca?gattagatta?tgctacaata?attaaagcaa?ataaaagtta?tggaagtggg????????????????????????1860

tatattccat?tattaggttg?gacccatcgg?agtgtagatc?gtaataatac?aatttatccg????????????????????????1920

aataaaatca?ctcaaatacc?agcagtaaaa?gctttctcat?atactgaatc?atttaatgta????????????????????????1980

aatgttattg?caggtccagg?attcacagga?ggagatttaa?taagtttagg?tcatttagag????????????????????????2040

aatatttata?tgaaattaaa?cgttccaaat?cctcaaaaat?tccgtgttcg?tattcgttat????????????????????????2100

gctgctagta?caacttcgta?tttgcaaata?actgggctat?ctaatttagc?tcagtctgat????????????????????????2160

cgtttcgaac?agacgtattc?taatgaaaat?gaaaacaatt?tgatgtttga?aaattttcaa????????????????????????2220

tatgtagaac?ttagaaatat?tttttcggta?gatgctccat?tagaaaatca?tcaagtaagt????????????????????????2280

atacaaaatt?atcaaggtaa?tggttttgtt?attatagacc?gaatcgaatt?catcccagta????????????????????????2340

aatgcaacat?atgaggcaga?acaagattta?gattcggcaa?agaaagcagt?gaataccttg????????????????????????2400

tttacgaata?caaaagatgg?tttacgacca?ggggtaacgg?attatgaagt?gaatcaagcg????????????????????????2460

gcaaacttag?tggaatgcct?atcggatgat?ttgtatccaa?atgaaaaacg?cttgttattt????????????????????????2520

gatgcagtga?aagaggcaaa?acgactcagc?gaggcacgta?acttactaca?agatccagat????????????????????????2580

ttccaagaga?taaatggaga?aaatggatgg?accgcaagta?caggaattga?ggttgtagaa????????????????????????2640

ggagatgctc?tatttaaagg?gcgttatcta?cgcctaccag?gtgcgagaga?aatggataca????????????????????????2700

gaaacgtatc?caacgtatct?gtatcaaaaa?gtagaggaag?gtgtattaaa?accatacaca????????????????????????2760

agatatagat?tgagagggtt?tgtcggaagc?agtcaaggct?tggaaatttc?cacaattcgt????????????????????????2820

catcagacga?accgaattgt?aaaaaatgtt?ccagatgatt?tattaccaga?tgtacctcct????????????????????????2880

gtaaactctg?atggtagaat?caatcgatgc?agcgaacaaa?agtatgtgaa?tagccgttta????????????????????????2940

gaaggagaaa?gaggattacc?aaatgggaat?cgttctgctg?aagcgcatga?attctctctc????????????????????????3000

cctattgata?taggagagct?ggattacaat?gaaaatgcag?gaatatgggt?tggatttaag????????????????????????3060

attacggacc?cagagggata?tgcaacactc?ggtaaccttg?aattggtaga?agagggacca????????????????????????3120

ttgtcaggag?acgcactaga?acgcctgcaa?agagaagaac?aacagtggaa?gcttcaaatg????????????????????????3180

acaaaaagac?gtgaagagac?ggatagaaaa?tatacggcag?caaaacaagc?ggtagatcgt????????????????????????3240

ttatatgcag?attaccaaga?tcaacaattg?aatccaaacg?tagaaattac?ggatattact????????????????????????3300

gcggcccaaa?acctgataca?gtccattcct?tatgtatata?atgaaatgtt?cccagaaata????????????????????????3360

caagggatga?actatacgaa?gtacacagag?ttaacaaatc?gactccaaca?agcgtggggt????????????????????????3420

ttgtatgatc?aacgaaacgc?cataccaaat?ggtgatttcc?gaaatgaatt?aagtaattgg????????????????????????3480

aatacaacat?ctggtgtaaa?tgtacaacaa?atcaacaata?cgtctgtctt?agtcatgcca????????????????????????3540

aactgggatg?ggcaagtttc?gcaacagttt?acagttcaac?cgaatcaaag?atatgtatta????????????????????????3600

cgagttactg?caagaaaaga?aggggtaggg?aatgggtatg?tgagtatccg?tgatggtgga????????????????????????3660

aatcaaacag?aaacgcttac?gtttagtgca?agcgattata?acacagatag?tgtgtataat????????????????????????3720

acgcaagtgt?cgaatacaaa?tggtttgtac?aatgagcaaa?caggatatac?cacaaaaaca????????????????????????3780

gtgacattca?tcccatatac?agatcaagtg?tggattgaga?tgagcgagac?cgaaggtatg????????????????????????3840

End?codon

tacaggtttc?atctggaggg?gtttttttct?gaaaaagggc?ctttttgtag?agaagaatcc????????????????????????3960

gattatttta?ttacgattat?atattttgtg?gatagatcat?ggtacc???????????????????4006

SEQ ID NO 4 (the proteic aminoacid sequence of Cry8Fa1):

MSPNNQNEYE?IIDMAPSTSV?TNDSNRYPFA?NEPTNALQNM?NYKDYLRMSE?GYSPEYLTSL????60

SPYSQFGTVD?KIISIISLLN?SAAGIPGLDF?FTGLLQFILD?FFAPEDPWAE?LMELVEQLID????120

QKITVATREK?ALAELRGLIN?GYLVYQQSLE?SWLENPNATR?ASIVREQYVA?LELDFVTSIS????180

SFAIAGQEVP?LLAVYAQAAN?LHLLLLRDVS?IFGEEWGLTV?NEVNTFYIRQ?MTYTTEYSDY????240

CVRIYNTGLN?KLKGSSASSW?VDYNRFRREM?NLLVLDIIAL?FPNYDVRRYP?METTTELTRV????300

VYTDPIVFDE?RKGVASTHSW?TAIAPSFSSI?ESLTRRPGLF?TWLDQLTIFS?KRISQPSVFI????360

NSWAGHKIST?FRTQKTDILI?NTTHGDTNNP?IKEFVVDTKK?VEDIYQTIAY?PHAVANEVFY????420

LFGVPKVDFN?MVPAGGSANS?AHTLIFSDST?GGRLESITKN?SEAELPPTES?LSDTPQPNQV????480

TYSHRLDYAT?IIKANKSYGS?GYIPLLGWTH?RSVDRNNTIY?PNKITQIPAV?KAFSYTESFN????540

VNVIAGPGFT?GGDLISLGHL?ENIYMKLNVP?NPQKFRVRIR?YAASTTSYLQ?ITGLSNLAQS????600

DRFEQTYSNE?NENNLMFENF?QYVELRNIFS?VDAPLENHQV?SIQNYQGNGF?VIIDRIEFIP????660

VNATYEAEQD?LDSAKKAVNT?LFTNTKDGLR?PGVTDYEVNQ?AANLVECLSD?DLYPNEKRLL????720

FDAVKEAKRL?SEARNLLQDP?DFQEINGENG?WTASTGIEVV?EGDALFKGRY?LRLPGAREMD????780

TETYPTYLYQ?KVEEGVLKPY?TRYRLRGFVG?SSQGLEISTI?RHQTNRIVKN?VPDDLLPDVP????840

PVNSDGRINR?CSEQKYVNSR?LEGERGLPNG?NRSAEAHEFS?LPIDIGELDY?NENAGIWVGF????900

KITDPEGYAT?LGNLELVEEG?PLSGDALERL?QREEQQWKLQ?MTKRREETDR?KYTAAKQAVD????960

RLYADYQDQQ?LNPNVEITDI?TAAQNLIQSI?PYVYNEMFPE?IQGMNYTKYT?ELTNRLQQAW????1020

GLYDQRNAIP?NGDFRNELSN?WNTTSGVNVQ?QINNTSVLVM?PNWDGQVSQQ?FTVQPNQRYV????1080

LRVTARKEGV?GNGYVSIRDG?GNQTETLTFS?ASDYNTDSVY?NTQVSNTNGL?YNEQTGYTTK????1140

TVTFIPYTDQ?VWIEMSETEG?MFYIESVELI?VDVE????????????????????????????????1174

Claims (7)

1. a bacillus thuringiensis bacterial strain (Bacillus thuringiensis), its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC NO.1242.

2. the application of the bacterial strain of claim 1 in killing coleopteran pest.

3. an Accessory Right requires the cry8E gene of 1 described bacillus thuringiensis bacterial strain separating clone, it is characterized in that this gene has the nucleotide sequence shown in the SEQ ID NO 1.

4. a Cry8Eal albumen is characterized in that this albumen is the coded by said gene by claim 3, and has the aminoacid sequence shown in the SEQ ID NO2.

5. an Accessory Right requires the cry8F gene of 1 described bacillus thuringiensis bacterial strain separating clone, it is characterized in that this gene has the nucleotide sequence shown in the SEQ ID NO 3.

6. a Cry8Fal albumen is characterized in that this albumen is the coded by said gene by claim 5, and has the aminoacid sequence shown in the SEQ ID NO4.

7. an enhancing is characterized in that the cry8E gene of this method synergistic combination use claim 3 and the cry8F gene of claim 5 to the toxic method of coleopteran pest.

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