CN100562565C - The gene recombined engineering bacterium of Spinosad microbial inoculum - Google Patents

Abstract

The present invention relates to gene recombined engineering bacterium of Spinosad.Invention adopts the Red/ET homologous recombination technique that functional assembly is modified and inserted to the full gene of pleocidin (Spinosad), simultaneously with the cry1Ac gene recombination of Bt4.0718 bacterial strain, do not have in crystal mutant strain and bacillus thuringiensis (Bacillus thuringiensis) bacterial strain at luminous bacillus (Photorhabdus.luminescens), Bt respectively and efficiently express, construct new and effective disinsection engineering bacteria.Sterilant has been widened insecticidal spectrum, improved insecticidal effect, the fermentative production cycle of engineering bacteria shortens nearly 300 hours, only be 1/4 time of former S.Spinosad, greatly reduce production cost, significantly improved economic benefit, engineering bacteria is the biotic pesticide of low toxicity, efficient, low residue in addition, insecticidal properties is efficiently not only arranged, and, be fit to the production application of nuisance free crops beneficial insect and mammalian safe.

Description

The gene recombined engineering bacterium of Spinosad microbial inoculum

Technical field

The present invention relates to gene recombination, be specifically related to gene recombined engineering bacterium of Spinosad.

Background technology

Chemical pesticide has been used over half a century, has brought huge economic benefit to agricultural.Yet, in proportion of crop planting, abuse chemical pesticide, ubiquity agricultural chemicals thing is residual, threatens human beings'health, and the resistance of the harmful insect that causes simultaneously strengthens day by day, and the effect of the various insects of pesticide control is obviously descended, and brings harm to proportion of crop planting.This shows that environment friendly agricultural or biological pesticide are the inexorable trends of following agricultural chemicals development, also is basic an assurance of agricultural sustainable development.Its feature is: 1) very high biological activity is arranged, and the drug effect height, the unit surface usage quantity is few; 2) selectivity is strong, to beneficial insect with non-target organism is nontoxic or toxicity is minimum; 3) harmless to farm crop; 4), generate harmless nontoxic crude substance and incorporate the Nature fully even use the back in farm crop inside and outside, agricultural-food and soil, atmosphere, water body noresidue or there is minimal residue also can degrade in a short time.

Pleocidin be wild type strain actinomycetes thorns saccharopolyspora strains (Saccharopolyspora spinosa) in developing medium through aerobic fermentation and secondary metabolite.On structure, these compounds belong to Macrolide, are the mixtures of two kinds of active ingredient Spinosad A and D.Its mechanism of action is considered to the acting body of nicotinic acid acetylcholine receptor, also act on simultaneously gamma-aminobutyric acid receptor, can make insect benumb rapidly, paralyse, cause death at last, its desinsection speed can compare favourably with chemical pesticide, safe, and there is not cross resistance with present common insecticide, be defined as the biotic pesticide of low toxicity, efficient, low residue by U.S. environmental protection organization, existing insecticidal properties efficiently, the characteristic that pair beneficial insect and mammalian safe are arranged again, the production application of suitable nuisance free crops.But the S.Spinosa growth cycle reached for two to three weeks, and its poor growth has limited the production application of Spinosad.Because the costliness of its production prices is only used in the plantation of vegetables and fruit in western countries at present.Therefore, shorten the S.Spinosa growth cycle, the turnout that improves Spinosad is a very promising scientific research project.

Bacillus thuringiensis is the microorganism that lepidopteran (Lepidopteran), Diptera (dipteran) and Coleoptera various pests such as (coleopteran) are had toxic action, the insecticidal activity of Bt is mainly from parasporal crystal, (insecticidal crystal proteins is ICPs) by cry gene and cyt genes encoding for insecticidal crystal protein.After this kind crystal toxalbumin enters the insect digestive tube, through the specific protein enzymic hydrolysis, discharge the toxicity peptide, and combine with acceptor high-affinity on the insect digestive tube velum vesica, insert cytoplasmic membrane fast and irreversibly, form hole or focus, cause the cytolemma non-polarization, destroy the Premeabilisation of cells balance, thereby cause lysis.Simultaneously, Bt breeds rapidly in the haemocoele of insect and causes the septicemia of insect.Generally believe that at present the receptors bind on this kind crystal toxalbumin elder generation and the insect midgut film is inserted film then and formed the hole, causes insect death.The bacillus thuringiensis natural enemy that do not kill off the insect pests, with its highly effective and safe, to the specificity of target pest and extremely people's favor, and become the most deep, the most widely used biotic pesticide of research gradually, but also there are some problems in the Bt gene in application, Bt crystallin insecticidal spectrum is narrower, insect easily produces tolerance to insecticidal crystal protein, strengthen as vegetables pest small cabbage moth resistance under the Bacillus thuringiensis selective pressure, therefore improve the target that the Bt insecticidal toxicity has become scientific research person's research.Make up engineering bacteria by pleocidin gene and Cry gene recombination and solve the problem that the two problem that exists separately is worth exploration.

Summary of the invention

The present invention is intended to adopt the Red/ET homologous recombination technique, will separate the full gene of Spinosad and Bt bacterial strain Cry 1 Ac gene recombination with the growth cycle that shortens S.Spinosa with solve problems such as Bt sterilant insecticidal spectrum is narrow from S.Spinosa.

Realize that technical scheme of the present invention is:

The engineering bacteria that gene recombined engineering bacterium of Spinosad is recombinated through the Red/ET homologous recombination technique by the Cry 1 Ac gene of gene cluster gene SpnA, SpnB, SpnC, SpnD, SpnE, SpnF, SpnG, SpnH, SpnI, SpnJ, SpnK, SpnL, SpnM, SpnN, SpnO, SpnQ, SpnR, SpnS and the Bt bacterial strain of pleocidin.

The construction process of gene recombined engineering bacterium of Spinosad is:

1) the isolated gene cluster of cluster needling saccharopolyspora strain (S.Spinosa) is cloned into the BAC library and obtains recombinant plasmid PBeloBAC11-Spn;

2) will be arranged in the rhamanopyranosyl that stings saccharopolyspora strain karyomit(e) different positions and obtain recombinant plasmid pTps-BAC-Spn because of gtt, epi, gdh and kre join the Spinosad gene cluster;

3) insert the modified Spinoad gene cluster from the full gene of Cry 1 Ac that obtains from Bt 4.0718 bacterium components, make up the pTps-BAC-Spn-cry1Ac recombinant vectors;

4) the pTps-BAC-Spn-cry1Ac recombinant plasmid is modified, inserted oriT, Tn5, IR, Tpase, transfection, swivel base resistance device genes involved and resistant gene tetR, Km obtain the pTps-BAC-Spn-cry1Ac-T recombinant vectors;

5) above-mentioned recombinant plasmid is incorporated on the F-factor, imports the external source heteroplasmon;

6) by recombinant plasmid T _n5 transposons are incorporated into recombination in the heteroplasmonic karyomit(e) of external source, efficiently express in heterogeneous host.

Be described in further detail the present invention below in conjunction with accompanying drawing.

Thorn saccharopolyspora strain of the present invention, luminous bacillus are delivered Chinese typical culture collection center (CCTCC) preservation on September 4th, 2006, and deposit number is respectively 206084,206085.

Description of drawings

Fig. 1: engineering bacteria makes up schema;

Fig. 2: Spinosad family's cluster gene synoptic diagram;

Fig. 3: cry1Ac gene synoptic diagram;

Its modification of BAC library of the full gene 80kb of Fig. 4: Spinosad;

Fig. 5: the pTps-BAC-Spn-cry1Ac synoptic diagram of recombinating;

Fig. 6: pTps-BAC-Spn-T modifies synoptic diagram;

Fig. 7: pTps-BAC-Spn-cry1Ac-T recombinant modified synoptic diagram;

Fig. 8: pTps-BAC-Spn-cry1Ac-T imports different heterogeneous hosts by F-factor and is incorporated into heterogeneous host's karyomit(e) synoptic diagram;

Fig. 9: the zymotechnique schema of bacterium agent of engineering bacterium.

This research separates the full gene of Spinosad from S.Spinosa, this gene is greater than 80kb, be difficult to reach with general molecular biology method functional module is modified and inserted to its full gene, among the present invention, we have adopted Red/ET homologous recombination technique [Improved RecT or RecET cloning and subcloning method, EP:01117529.6; Methods for heterologous expressio ofsecondary metabolites: PCT/IB2005/003650; Recombination method, EP:0103276.2], the Red/ET homologous recombination technique be one of biology field than quantum jump, be a kind of brand-new Nucleotide macromole manipulation technology.Use this technology, no matter dna fragmentation has muchly, and structure how, can clone and accurately modify.In target dna molecule, for any select location, can carry out the operation of site mutation, fragment insertion or disappearance, need not the restriction of being restricted property restriction enzyme site.When carrying out dna clone, the Red/ET recombinant technology is better than any traditional DNA cloning technology.The present invention clones into BAC library with the full gene cluster of Spinosad, by the Red/ET homologous recombination technique functional assembly is modified and inserted to full gene, simultaneously with full gene of Spinosad and Bt bacterial strain cry1Ac gene recombination, be that heterogeneous host expresses with luminous bacillus (P.luminescens) and bacillus thuringiensis (Bacillus thuringiensis) bacterial strain respectively, construct new and effective, safe disinsection engineering bacteria.

After heterogeneous expression system is set up, intend foundation by re-constituted compound thing storehouse (Recombinatorial compoundlibrary), use high throughput screening system (High Thruoghput Screen) and carry out microminiaturized live test, can be simultaneously or finish the screening of a large amount of compounds in the short period of time, thereby save 3～5 times quantities, thereby improved screening efficiency greatly, obtain new more effective desinsection biological pesticide.

By setting up the heterogeneous expression system of Spinosad and Bt bacterial strain cry1Ac gene recombination, improve Spinosad production level, reduce cost, high level expression Cry 1 Ac crystal insecticidal proteins simultaneously can big area uses biological pesticide and brings benefit to the mankind. develop the novel high Spinosad derivative of better pest-resistant spectrum, anti-insect activity that has simultaneously.The using gene engineering technique that takes the lead in the world is devoted to realize Spinosad efficiently expressing in engineering bacteria, realizes cheap production.

The preparation of engineering bacterium biological pesticide comprises the structure of the heterogeneous expression engineering bacteria of pleocidin and fermentative production two portions of the heterogeneous expression engineering of pleocidin microbial inoculum:

1, Spinosad and the cry1Ac structure (Fig. 1) of heterogeneous expression engineering bacteria of recombinating:

(1) modification of the separation of .spinosad gene cluster and BAC carrier

Will (culture presevation number: the chromosomal DNA that extracts CCTCCNO:M206084) digests laggard horizontal pulse electrophoresis with Sau3A I, recovery 100kb left and right sides fragment from S.spinosa.Cut BAC with BamHI (isocaudarner of Sau3A I) enzyme, be connected with the dna fragmentation that reclaims behind the dephosphorylation, 1500V 25 μ F 200 Ω electricity Transformed E .coliYZ2005 (Introducing

Recombination:DNA Engineering for the 21st Century Gene Cloning ﹠amp; Expression Technologies), through designing two pairs of primers

F1：CCGTCGAGCCTACCGCTGATTCATA R1--：GCGGCATCTCGGAGGATTTCAGCAAC

F2：CAGGCGGCGGACACCGCGCTC R2--：TCAGCGTTGCCGAGCGGTGCGCTG

The gene at pcr amplification Spinosad gene cluster two ends, pick out contain the Spinosad gene cluster (gene order number: AY007564) (Fig. 2) positive transformant, obtain recombinant plasmid PBeloBAC11-Spn.Pcr amplification is positioned at S.spinosa karyomit(e) different positions rhamanopyranosyl because of gtt[gi:15077644], epi[gi:15077642], gdh[gi:15077646] and kre[gi:15077646], and rhamnosyl (gtt, gdh, epi and kre) gene joined in the Spinosad gene cluster by the Red/ET homologous recombination, the primer of its PCR is respectively:

Fepi：TGCCTCAGCCGGAGCCTTCGCAACTTCCTGGAGGGAAACGCCACGGGATCAACAACAACTTCACCAG

Repi：CCACGAGCGCACGCCGCAAGTACTCCCGGAGCTTCTCTTCATTCGACATTGGAGGTGGATGTGAAATCCCTCGGGC

GC

Fgtt：CCAACCGCCGCCAGGGCCAGCGCCCGAGGGATTTCACATCCACCTCCAAAGGCCACCGGCAAGGTCGTGC

AGGG

Rgtt：CCACGAGCGCACGCCGCAAGTACTCCCGGAGCTTCTCTTCATTCGACATGCACCCGCCGATGGCCGACCGC

Fgdh/kre：GGCCGGCCATGCCCTCCAGGCGGCGAATGCGGTCGGCCATCGGCGGGTGCGGATCCTGCTTCGTAGCTCGGTGTG

Rgdh/kre：CACGAGCGCACGCCGCAAGTACTCCCGGAGCTTCTCTTCATTCGACATGGATCCGCTTCCCCCACGGCGACCCG

These four genes play modification in Spinosad is synthetic, obtain recombinant plasmid pTps-BAC-Spn.(Fig. 4)

(2). with the reorganization of cry1Ac gene

Extract total DNA of Bt4.0718 bacterial strain, method reference [Diversity of locations for Bacillusthuringiensis crystal protein genes.J Bacteriol.1983 Apr; 154 (1): 419-28.], total DNA with the Bt4.0718 bacterial strain is a template, adopt round pcr from the Bt4.0718 bacterial strain, to separate the full gene of cry1Ac (Fig. 3) that obtains 4.2kb, contain promotor (promoter), read frame (CDS) and terminator (terminator) design primer:

F1AC：CCAGATCCCGGGTACCGAGCTCGAATTCGCCCTATAGTGAGTCGTATTAATGGATAACAATCCGAACATCAATG

R1AC：CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGAGATTCCTCCATAAGGAGTAATTC

And utilize the Red/ET homologous recombination technique that it is recombinated with containing the full gene BAC of Spinosad library, and the full gene of cry1Ac is inserted after the full gene cluster of Spinosad, make up pTps-BAC-Spn-cry1Ac recombinant vectors (Fig. 5).Identify positive transformant by top primer PCR.

(3). recombinant expression plasmid pTps-BAC-Spn and the pTps-BAC-Spn-cry1Ac expression in heterogeneous host bacterium and the foundation of re-constituted compound library

With (the culture presevation number: CCTCC NO:M206085) of the luminous bacillus of this project team seed selection, Bacillus thuringiensis Bt4.0718 (culture presevation number: CCTCC NO:M200016) and Bt do not have the crystal mutant strain (clone of cry1Ac gene among the bacillus thuringiensis parasporal crystal 20KbDNA, efficiently express 2004 656-661 with bioactivity research biotechnology journal Vol 30No.5 September) be heterogeneous host, by transfection and Red/ET technology fusion gene is integrated on the host bacterium karyomit(e), this operation is made up of following three experimental procedures: at first: recombinant expression plasmid is further modified, insert oriT, Tn5, IR, the Tpase transfection, swivel base resistance device genes involved and resistant gene tetR, Km, (Fig. 6 and Fig. 7) its method is with reference to top rhamnosyl (gtt, gdh, epi and kre) insertion of gene, utilize the Red/ET homologous recombination technique to insert in its carrier.Secondly, because the pTps-BAC-Spn-cry1Ac-T recombinant vectors after modifying has Kb more than 90, it is so big plasmid to be changed in the external source heteroplasmon that general electricity changes, we are incorporated into recombinant plasmid pTps-BAC-Spn-cry1Ac-T on the F-factor, in the mediation recombinant plasmid pTps-BAC-Spn-cry1Ac-T guiding external source heteroplasmon by F-factor.Final step is by recombination being incorporated into (Fig. 8) in the external source heteroplasmon at the Tn5 of recombinant plasmid pTps-BAC-Spn-cry1Ac-T transposon. modifies recombinant plasmid pTps-BAC-Spn in the same way and constitutes recombinant expression plasmid pTps-BAC-Spn-T, recombinant expression plasmid pTps-BAC-Spn-T directly expresses in Bacillus thuringiensis Bt4.0718, because in the karyomit(e) of Bacillus thuringiensis Bt4.0718, contain Cry 1 Ac gene, adopt similar reaction conditions by present technique, disposable synthetic thousands of (even up to a million synchronously, hundred million) plant the different molecule of structure, make up re-constituted compound thing storehouse (Recombinatorialcompound library), carry out high efficiency screening by compound library.The cost of each compound then is traditional 1/600 in the re-constituted compound thing storehouse.Combinatorial chemistry is based on drug development and a kind of new synthetic technology that grows up, and it has accelerated the appearance and the optimal speed of new drug lead compound.

(4). the screening of efficient insecticide engineering bacteria

After changing recombinant plasmid over to heterogenous expression host bacterium, the main high transformant of the biological virulence of high-flux directed screening of using, select 3-5 transformant from same flat board and be inoculated into same shake the bottle and cultivate (30 ℃ of 37 ℃ of Bacillus thuringiensis of 30 ℃ of P.Iuminescens of S.Spinosa), inoculate a plurality of such bottles that shake simultaneously.Nutrient solution with shake-flask culture carries out biological toxicity test, therefrom screens the high nutrient solution of biological virulence.Carry out on the efficient height of biological toxicity test screening 3-5 doubly than independent single transformant like this.

2, Spinosad and the cry1Ac fermentative production (Fig. 9) of heterogeneous expression engineering microbial inoculum of recombinating:

By optimizing fermentative medium formula, screening optimal conditions of fermentation production engineering bacteria agent

(1), bacterium agent of engineering bacterium fermentation manufacturing technique condition general introduction:

1.. actication of culture

With the high-efficiency broad spectrum disinsection engineering bacteria streak inoculation of preservation on solid seed culture medium inclined-plane, the flat bottle that substratum will be housed before the inoculation was sterilized 30 minutes under 121 ℃ of conditions, 30～48h is cultivated in the inoculation back under 28～35 ℃ of conditions, be made into spore liquid, and the inoculum size with 5% is used for the seeding tank inoculation.

2.. sterilization and culture condition

High pressure steam sterilization is adopted in above-mentioned sterilization, and promptly at 121 ℃, 30min sterilizes under the pressure 0.3-0.5kg/cm2 condition

3.. the seeding tank fermentation

With the first class seed pot sterilization, sterilization again is cooled to 30 ℃ behind the substratum of packing into, and spore liquid is inserted in the nutrient solution earlier, feeds sterile air and cultivates, and can get the first class seed pot zymocyte liquid; With secondary seed jar sterilization 30min under 121 ℃, the sterilization again behind the substratum of packing into is cooled to 30 ℃, and the first class seed pot fermented liquid is inserted the secondary seed jar by 5%～8% inoculum size, feed sterile air and stirring and cultivate, can get secondary seed jar zymocyte liquid.

4.. produce fermentor tank

Cultivate earlier fermentor tank is sterilized, sterilization again behind the substratum of packing into, pressurize is cooled to 25 ℃～30 ℃, and the inoculum size by 5%～8% inserts secondary seed jar fermented liquid in the fermentor tank and cultivates, and passes through sterile air.

5.. concentrate

Fermentation cylinder for fermentation concentrates, adds Synergistic additives, subsequent bottling with bacterium liquid by ultrafiltration after finishing.Various engineering bacterias also have the difference on each comfortable substratum and the processing condition at total fermentation basic condition:

(2), engineering bacterium fermentation processing condition

1. the fermentation manufacturing technique condition of .S.Spinosa

The S.Spinosa substratum:

MM substratum: KNO ₃0.1%NaCl 0.05%K ₂HPO ₃3H ₂O 0.05%

FeSO ₄0.001%MgSO47H ₂O 0.05% glucose 2% agar 1.2%～2%pH 6.8

The 10L fermentation tank culture medium: agar in the MM substratum and NaCl are removed, add yeast extract paste, the culture temperature of each 0.4%pH6.5 of peptone～7.0 S.Spinosa is 24～33 ℃, and the optimum temperature 28～31 of synthetic Spinosad.Keep dissolved oxygen more than 65% with air flow and stir speed (S.S.) during fermentation.Tank pressure is 0.034Mpa

2. .P.luminescens technology fermentation condition:

The P.luminescens substratum:

Zulkovsky starch 1% yeast extract paste 1% peptone 1%NaCl 0.5%K ₂HPO ₄The 0.05%pH7.0 solid medium adds the temperature of agar 1.2%～2% shake-flask culture at 37 ℃ on the basis of original substratum.The temperature of fermentor tank is at 33-37 ℃, air flow 0.8～2.5Vols/vol/min, oxygen content 40～50%.

3. .Bacillus thuringiensis technology fermentation condition

Bacillus thuringiensis substratum: extractum carnis 0.3～0.8%, peptone 0.7～1.2%, glucose 0.1～0.6%, NaCl 0.1～0.6%, MgSO ₄7H ₂O 0.01～0.06%, K ₂HPO ₄0.01%～0.06%, MnSO ₄0.02%～0.08%, pH6.8～7.2.Solid medium adds agar 1.2%～2% on the basis of original substratum.Air flow 1～2Vols/vol/min, oxygen content 30～40%.

Prove by experiment:

Independent Spinosad the results are shown in Table 1 in needed time of S.Spinosa fermentative production and Spinosad and cry1Ac needed time of heterogeneous expression of recombinating:

The time ratio that the fermentative production of table one, engineering microbial inoculum needs is:

As can be seen from Table 1, the engineering microbial inoculum fermentative production time has shortened 3/4.

The insecticidal function checking

1. checking correctness of expression

Identify Spinosad by the HPLC purifying.

2.ELISA detect the expression product of toxin gene

With the target Cry 1 Ac crystallin immune rabbit of purifying, obtain to be connected with horseradish peroxidase behind the antibody formation enzyme len antibody; With the bacterium cultivated with ultrasonication after, will change film behind the total protein electrophoresis, with antibody hybridization, wash-out, colour developing have determined whether that toxin expresses.

3. indoor insecticidal test determines that the desinsection of engineering strain tires.

The biological activity determination of table two different insecticides

From table two we as can be seen, the biological virulence of engineering bacteria is expressed strong than any one monovalent toxin protein.It is eager to excel in whatever one does last 60% than independent spinosad that engineering bacteria contains (spinosad+cry1Ac), and than independent Bt (cry1Ac) Qiang Jinyi doubly.

The present invention has adopted a kind of brand-new nucleic acid molecule operating technology--Red/ET homologous recombination technique, to 80kb's The full gene of Spinosad man bunch operates modification. Realized genetic recombination, the engineering microbial inoculum of restructuring and existing biological farming Medicine is compared and is had the following advantages:

(1). good disinsection effect: Spinosad is the biological insecticides of low toxicity, efficient, low-residual, and the Bt crystalline protein also is Soviet Union A kind of pesticide that insecticidal effect has outstanding effect in the cloud gold bacillus, the present invention combines both, makes the best use of the advantages and keeps away Lack, give full play to the advantage of two kinds of pesticides, can give full play to Spinosad speed and kill effect, also can show the Bt crystal The advantages such as the high-yield and high-efficiency of albumen, safety.

(2). insecticidal spectrum is wide: engineering bacteria is with two kinds of different efficient insecticide toxin, and Spinosad is considered to nicotinic acid acetyl The acting body of choline receptor can make insect benumb rapidly, paralyse, and causes at last death, and its desinsection speed can be agricultural with chemistry Medicine compares favourably. And the receptors bind on Bt crystalline protein and the insect midgut film is inserted then film and is formed the hole, causes insect Dead. Two kinds of pesticides of different insecticidal actions are binned in together, and its discernible scope is increased, simultaneously can be to the squama wing The multiclass insects such as order, Diptera, coleoptera are effective, and it is very wide therefore to prevent and treat in the use face, and cost is relatively low.

(3). economic benefit height: Spinosad and the Cry 1 Ac needed time ratio of heterogeneous expression of recombinating is original independent Spinosad will shorten nearly 300 hours in the needed time of S.Spinosa, and the engineering bacterium fermentation cycle only is former 1/4 time of S.Spinosa, reduced greatly production cost, remarkable in economical benefits improves.

(4). security is higher: Spinosad does not have cross resistance with present common insecticide, existing efficient insecticidal properties, again The production application of suitable nuisance free crops is arranged the characteristic of beneficial insect and mammalian safe is arranged. Equally, Su Yun gold bud Spore bacillus crystalline protein is with its highly effective and safe, be proved already the specificity of target pest. Therefore, protect at ecological environment Protect, export goods and earn foreign currency and promote the aspects such as human health that great advantage is arranged.

＜110〉Hunan Normal University

＜120〉gene recombined engineering bacterium of Spinosad microbial inoculum

<160>2

<210>1

<211>25

<212>DNA

＜213〉PCR detects Spinosad gene primer 1F

<220>

<400>1

ccgtcgagcc?taccgctgat?tcata 25

<210>2

<211>26

<212>DNA

＜213〉PCR detects Spinosad gene primer 1R

<220>

<400>1

gcggcatctc?ggaggatttc?agcaac 26

<160>2

<210>1

<211>21

<212>DNA

＜213〉PCR detects Spinosad gene primer 2F

<220>

<440>1

caggcggcgg?acaccgcgct?c 21

<210>2

<211>24

<212>DNA

＜213〉PCR detects Spinosad gene primer 2R

<220>

<400>1

tcagcgttgc?cgagcggtgc?gctg 24

<160>2

<210>1

<211>67

<212>DNA

＜213〉the pcr amplification rhamanopyranosyl is because of the epi primers F

<220>

<440>1

tgcctcagcc?ggagccttcg?caacttcctg?gagggaaacg?ccacgggatc?aacaacaact 60

tcaccag 67

<210>2

<211>78

<212>DNA

＜213〉the pcr amplification rhamanopyranosyl is because of epi primer R

<220>

<400>1

ccacgagcgc?acgccgcaag?tactcccgga?gcttctcttc?attcgacatt?ggaggtggat 60

gtgaaatccc?tcgggcgc 78

<160>2

<210>1

<211>74

<212>DNA

＜213〉the pcr amplification rhamanopyranosyl is because of the gtt primers F

<220>

<440>1

ccaaccgccg?ccagggccag?cgcccgaggg?atttcacatc?cacctccaaa?ggccaccggc 60

aaggtcgtgc?aggg 74

<210>2

<211>71

<212>DNA

＜213〉the pcr amplification rhamanopyranosyl is because of gtt primer R

<220>

<400>1

ccacgagcgc?acgccgcaag?tactcccgga?gcttctcttc?attcgacatg?cacccgccga 60

tggccgaccg?c 71

<160>2

<210>1

<211>75

<212>DNA

＜213〉the pcr amplification rhamanopyranosyl is because of the gdh/kre primers F

<220>

<440>1

ggccggccat?gccctccagg?cggcgaatgc?ggtcggccat?cggcgggtgc?ggatcctgct 60

tcgtagctcg?gtgtg 75

<210>2

<211>74

<212>DNA

＜213〉the pcr amplification rhamanopyranosyl is because of gdh/kre primer R

<220>

<400>1

cacgagcgca?cgccgcaagt?actcccggag?cttctcttca?ttcgacatgg?atccgcttcc 60

cccacggcga?cccg 74

<160>2

<210>1

<211>74

<212>DNA

＜213〉the full gene primer F of pcr amplification cry1Ac

<220>

<440>1

ccagatcccg?ggtaccgagc?tcgaattcgc?cctatagtga?gtcgtattaa?tggataacaa 60

tccgaacatc?aatg 74

<210>2

<211>74

<212>DNA

＜213〉the full gene primer R of pcr amplification cry1Ac

<220>

<400>1

cgccagggtt?ttcccagtca?cgacgttgta?aaacgacggc?cagtgaattg?agattcctcc 60

ataaggagta?attc 74

Claims (2)

1, a kind of gene recombined engineering bacterium of Spinosad is by the Cry 1Ac gene of gene cluster gene SpnA, SpnB, SpnC, SpnD, SpnE, SpnF, SpnG, SpnH, SpnI, SpnJ, SpnK, SpnL, SpnM, SpnN, SpnO, SpnQ, SpnR, SpnS and the Bt bacterium of the pleocidin engineering bacteria through the reorganization of Red/ET homologous recombination technique.

2,, it is characterized in that the construction process of engineering bacteria is according to the described gene recombined engineering bacterium of Spinosad of claim 1:

1) the isolated gene cluster of cluster needling saccharopolyspora strain (S.Spinosa) is cloned into the BAC library and obtains recombinant plasmid PBeloBAC11-Spn;

2) will be arranged in the rhamanopyranosyl that stings saccharopolyspora strain karyomit(e) different positions and obtain recombinant plasmid pTps-BAC-Spn because of gtt, epi, gdh and kre join the pleocidin gene cluster;

3) insert the modified pleocidin gene cluster from the full gene of the Cry 1Ac that obtains from Bt 4.0718 bacterium components, make up the pTps-BAC-Spn-cry1Ac recombinant vectors;

4) the pTps-BAC-Spn-cry1Ac recombinant plasmid is modified, inserted oriT, Tn5, IR, Tpase transfection, swivel base resistance device genes involved and resistant gene tetR, Km obtain the pTps-BAC-Spn-cry1Ac-T recombinant vectors;

5) above-mentioned recombinant plasmid pTps-BAC-Spn-cry1Ac-T is incorporated on the F-factor, imports the external source heteroplasmon;

6) by recombinant plasmid T _n5 transposons are incorporated into recombination in the heteroplasmonic karyomit(e) of external source, efficiently express in heterogeneous host, make up gene recombined engineering bacterium of Spinosad.

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