CN104651385B - A kind of fusion and preparation method thereof, integrative gene expression vector and its construction method, fusion gene engineering bacterium and its application - Google Patents
A kind of fusion and preparation method thereof, integrative gene expression vector and its construction method, fusion gene engineering bacterium and its application Download PDFInfo
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- CN104651385B CN104651385B CN201510015304.XA CN201510015304A CN104651385B CN 104651385 B CN104651385 B CN 104651385B CN 201510015304 A CN201510015304 A CN 201510015304A CN 104651385 B CN104651385 B CN 104651385B
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Abstract
The invention discloses a kind of fusion and preparation method thereof, integrative gene expression vector and its construction method, fusion gene engineering bacterium and its application, belong to gene engineering technology field.The fusion, protein transduction domain TAT is connected into diuresis polypeptide HKI sequences, is acted on by the efficient transmembrane transport of TAT, HKI is rapidly entered internal site through esophagus system inner membrance, ensure that HKI is stable during transporting in vivo to play a role, bollworm is excessively drained death.Integrative gene expression vector of the present invention, by Fusion gene construction in plant expression vector pB I 121, driven by 35S promoter and expressed, the high efficient expression in plant, 3 instar bollworm grub body weight amplification are significantly reduced after bollworm takes food the transfer-gen plant of integrative gene expression vector genetic transformation of the present invention, and the death rate is high.Fusion gene engineering bacterium of the present invention, contaminates plant to be germinateed, and realizes fusion genetic transformation plant, cultivates transgenic pest-resistant plant.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of integrative gene expression vector and preparation method thereof,
Also relate to a kind of construction method of integrative gene expression vector, fusion gene engineering bacterium and its application.
Background technology
Bollworm, one kind of Noctuidae Helicoverpa insect, is the major pest of cotton cotton buds and bolls phase, is widely distributed in the world each
Ground, Chinese cotton region and vegetables flake have generation, main moth food flower bud, flower, bell and tender leaf.Bollworm natural enemy is a lot, there is parasitism
Property natural enemy-parasitic wasp, tachinid etc., predator crow finch and some bacteriums, fungi, virus etc. can be to the ovum of bollworm
Inhibitory action is played with larva.
Water metabolism activity of diuresis kassinin kinin helicokinin I (HKI) to bollworm stimulates (Seinsche the strongest
Et al., 2000), after the HKI injections of denier enter the bollworm young, you can make its excessive excretion, and then cause death,
HKI is used as having a high potential that insect killing substance is applied.Because it derives from organism, to higher organism and Environmental security, because
This is carried out using with inborn environmental advantage as insect killing substance.How to enter to exercise using HKI as a kind of insecticide active substance
With, if the trophic behaviour of insect can be utilized to be taken in and to be played a role in vivo；Penetrate how active short peptide molecules
Alimentary canal enters internal site, while ensureing stabilization of the small peptide during transport, this is problem demanding prompt solution.
The content of the invention
In order to overcome the defect of prior art, an object of the present invention is that offer is a kind of has strong poison to bollworm
Kill the fusion of activity.
The second object of the present invention is to provide a kind of preparation method of fusion.
The third object of the present invention is to provide a kind of integrative gene expression vector.
The fourth object of the present invention is to provide a kind of construction method of integrative gene expression vector.
The fifth object of the present invention is to provide a kind of fusion gene engineering bacterium.
The sixth object of the present invention is to provide a kind of application of fusion gene engineering bacterium.
To achieve these goals, the technical solution adopted by the present invention is as follows：
A kind of fusion, its nucleotide sequence such as SEQ ID NO：Shown in 1.
Above-mentioned fusion is prepared from by diuresis polypeptide HKI and protein transduction domain TAT fusions.
The preparation method of above-mentioned fusion, including following operating procedure：
1) diuresis kassinin kinin nucleotides is connected on solid phase carrier, adds trichloroacetic acid reaction, obtain sloughing 5 '-hydroxyl
Blocking group diuresis kassinin kinin nucleotide fragments, be designated as fragment 1；
2) the protein transduction domain nucleotide monomer that phosphoramidite is protected is mixed with tetrazole, obtains nucleosides phosphorous acid activation
Intermediate, is designated as fragment 2；
3) fragment 1 and the condensation reaction of fragment 2, add acetic anhydride and 1- methylimidazole terminating reactions, are subsequently adding iodine, obtain
Fusion crude product；
4) by step 3) the fusion crude product for preparing cut, purified, quantitative, obtains final product described fusion.
A kind of integrative gene expression vector, comprising just like SEQ ID NO：Nucleotide sequence shown in 2.
The construction method of above-mentioned integrative gene expression vector, including following operating procedure：
1) PCR amplifications fusion, obtains fusion amplified fragments；
2) by the plasmids of pB I 121 and step 1) prepare fusion amplified fragments digestion after, be attached, must connect
Product；
3) connection product is converted into bacillus coli DH 5 alpha, selects positive colony carrier, extract plasmid, digestion and sequencing result
Correct plasmid is named as pBI121-TAT-HKI, as described integrative gene expression vector.
Step 2) in digestion be the double digestions of I/Sac of BamH I.
Step 1) in the PCR primers that use of amplification for：
P1：5’-CGCGGATCCATGTACGGCCGCAAG-3’；
P2：5’-GCGAGCTCACGCCCCAGGGGCTG-3 ' (underscore is restriction enzyme site).
Step 1) in connection linked system be：The μ L of pBI121 plasmids large fragment 1.0, the μ L of fusion 3.0,2 × reaction
The μ L of 5.0 μ L, T4DNA ligase of buffer solution 1；Condition of contact is：16 DEG C of coupled reaction 24h.
Step 3) described in digestion be the double digestions of I/Sac of BamH I.
A kind of fusion gene engineering bacterium, is transferred to host and is prepared by integrative gene expression vector；The fusion table
Included just like SEQ ID NO up to carrier：Nucleotide sequence shown in 2.
The host is Agrobacterium LBA4404.
Above-mentioned fusion gene engineering bacterium can be used to cultivate pest-resistant plant.
TAT (transactivator of transcription, TAT) is necessary to virus effectively transcription and replication
Polypeptide sequence.The large biological molecule that TAT will can be covalently attached therewith non-specifically inside transducer cell, both disobeyed by this process
Rely in acceptor and transport protein, it is also unrelated with temperature and energy, and to host cell almost without toxicity.It is this with carrying
The specific polypeptide sequence of foreign protein penetration cell film ability is referred to as protein transduction domain (Protein transduction
Domains, PTDs), also referred to as cell-penetrating peptide (cell penetratingpeptides, CPP).
Diuresis polypeptide Helicokinin is belonging to the small molecule insect neuropeptide of insect kassinin kinin family, whole family
Member has the similar peptide sequence Phe-Tyr-Pro-Trp-Gly-NH of one of carbon tip five2(FYPWGa), this peptide of core 5 for
It is required that it stimulates muscle activity and diuresis activity, and research shows that micro insect kassinin kinin and the like can be to insect physiology
Metabolism causes large effect (Smagghe et al.Antifeedant activity and high mortality in
the pea aphid Acyrthosiphon pisum(Hemiptera:Aphidae)induced by biostable
Insect kinin analogs, 2010；Nachman et al.Biostable and PEG polymer-conjugated
insect pyrokinin analogs demonstrate antifeedant activity and induce high
mortality in the pea aphid Acyrthosiphon pisum(Hemiptera:Aphidae), 2012), in elder brother
Worm children's internal injection l0-9The helicokinin I of M can largely promote its excessive excretion, and then cause insect dead
Die (Seinsche et al., 2000).
Fusion of the present invention, protein transduction domain TAT is connected into diuresis polypeptide HKI sequences, by the efficient cross-film of TAT
Transhipment is acted on, and HKI is rapidly entered internal site through esophagus system inner membrance, it is ensured that diuresis polypeptide sequence is conveyed through in vivo
Stabilization plays a role in journey, bollworm is excessively drained, and causes death.
The preparation method of fusion of the present invention synthesizes fusion using solid phase phosphoramidite triester method, is grafted success rate
Height, gene purity is high, easy to operate, is suitable to industrial application.
Integrative gene expression vector of the present invention, by Fusion gene construction in plant expression vector pB I 121, is opened by 35S
After mover drives expression, the fusion genetic transformation to enter plant, the high efficient expression in plant, bollworm takes food the present invention and melts
3 instar bollworm grub body weight amplification are significantly reduced after closing the transfer-gen plant of expression vector genetic transformation, and the death rate is high.
The construction method of integrative gene expression vector of the present invention, technical process is simple, is easy to operation to realize, is suitable to industrialization
Popularization and application.
Fusion gene engineering bacterium of the present invention, host is transformed into by integrative gene expression vector, is formed with fusion
Thalline, plant to be germinateed is contaminated using fusion gene engineering bacterium of the present invention, realizes fusion genetic transformation plant, is cultivated and is turned
Gene pest-resistant plant, fusion high efficient expression in transfer-gen plant after bollworm takes food plant, makes fusion enter cotton
In earworm body, diuresis polypeptide HKI genes therein play a role in Helicoverpa armigera, cause bollworm excessively excretion and it is dead,
The final purpose for realizing preventing and treating bollworm.
Brief description of the drawings
Fig. 1 is that Tissues of Tobacco regenerates and most suitable kanamycins screening regeneration；A is differentiation initial stage leaf dish；B is most suitable differentiation training
Support；C is reconstituted tobacco tissue culture seedling rooting；D is that tobacco differentiation is completely inhibited by kanamycins；E is the kanamycins of resistant budses
Screening；F is bloomed for transgene tobacco is potted plant；
Fig. 2 is track fusion RT-PCR analyses in different tobacco plants；Wherein 1 and 2 train for the different batches of embodiment 1
The transgenic tobacco plant educated；3 is Nicotiana gossei plant；TAT-HKI represents fusion.
Specific embodiment
The present invention is described in further detail with reference to specific embodiment, but is not constituted to any limit of the invention
System.
Embodiment 1
The present embodiment fusion, its nucleotide sequence such as SEQ ID NO：Shown in 1.
The present embodiment fusion is carried out on DNA synthesizer, and its preparation method operating procedure is as follows：
1) diuresis kassinin kinin nucleotides is connected on solid phase carrier, adds trichloroacetic acid reaction, obtain sloughing 5 '-hydroxyl
The diuresis kassinin kinin nucleotide fragments of blocking group, are designated as fragment 1；
2) the protein transduction domain nucleotide monomer that phosphoramidite is protected is mixed with tetrazole, obtains nucleosides phosphorous acid activation
Intermediate, is designated as fragment 2；
3) fragment 1 and the condensation reaction of fragment 2, add acetic anhydride and 1- methylimidazole terminating reactions, are subsequently adding iodine, obtain
Fusion crude product；
4) by step 3) prepare fusion crude product cut, using polyacrylamide gel electrophoresis purifying,
It is quantitative, obtain final product described fusion.
The present embodiment integrative gene expression vector, its nucleotide sequence such as SEQ ID NO：Shown in 2.
The construction method of the present embodiment integrative gene expression vector, concrete operation step is as follows：
1) with fusion as template, design primer is
P1：5’-CGCGGATCCATGTACGGCCGCAAG-3’；
P2：5’-GCGAGCTCACGCCCCAGGGGCTG-3 ' (underscore is restriction enzyme site).
PCR expands fusion, obtains fusion amplified fragments；
PCR reaction systems：Add 2.5 μ L 10 × buffer, 1.8 μ L 25mmol/L MgCl2,1μL 10mmol/L
DNTP, Taq archaeal dna polymerase 1.0U (above reagent is purchased from Shanghai Sheng Gong bio-engineering corporations), each 1 μ L of 10nmol/L primers, plus
Appropriate distilled water is to 25 μ L；
PCR response procedures：After 94 DEG C of 4min predegenerations, 94 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 90s, totally 30 circulations, finally
72 DEG C of extension 7min；
2) by the plasmids of pB I 121 and step 1) prepare fusion amplified fragments use the double digestions of I/Sac of BamH I
Digestion, gel reclaims pBI121 plasmids large fragment and fusion after digestion, is attached using T4-DNA ligases, is connected
Practice midwifery thing；Wherein linked system is：The μ L of pBI121 plasmids large fragment 1.0, the μ L of fusion 3.0, the μ L of 2 × reaction buffer 5.0,
The μ L of T4DNA ligases 1；Condition of contact is：16 DEG C of coupled reaction 24h；
3) connection product is converted into bacillus coli DH 5 alpha, resistance is screened on the LB flat boards of the kanamycins containing 60mg/L
Plasmid, plasmid is extracted to bacterial plaque in a small amount, and extracted vector plasmid is analyzed and identified with the double digestions of I/Sac of BamH I, is finally chosen
The correct vector plasmid of sequencing analysis is selected to be named as pBI121-TAT-HKI, as described integrative gene expression vector.
The present embodiment fusion gene engineering bacterium, Agrobacterium is transferred to by integrative gene expression vector pBI121-TAT-HKI electric shocks
LBA4404, obtains final product fusion gene engineering bacterium.
The present embodiment fusion gene engineering bacterium is used to cultivate pest-resistant plant, as shown in figure 1, K326 tobacco leafs are by fusion
Genetic engineering bacterium is contaminated, and carries out differentiation screening, and the culture medium composition for breaking up screening is：MS culture mediums, 1 ㎎/L 6- benzyl amino glands
Purine, 0.2 ㎎/L methyl α-naphthyl acetates, 50 ㎎/L kanamycins, explant starts to germinate budlet point after 10d, and bud point is gradually grown greatly not
Normal bud, green adventitious bud it is long to 3cm or so when, be transferred to root induction on root media, the composition of root media is：1/
2MS culture mediums, 0.05 ㎎/L methyl α-naphthyl acetates, 50 ㎎/L kanamycins, rooted seedling are grown up during rear bearing moves to flowerpot and are cultivated, and acquisition turns base
Because of tobacco plant, after bollworm takes food transgenic tobacco plant, fusion is expressed in Helicoverpa armigera, causes bollworm excessive
Excretion is dead, reaches the purpose of preventing and treating bollworm.
Performance detection：
1st, track fusion amount in the transgenic tobacco plant that respectively prepared by detection embodiment 1 and comparative example, as a result such as
Shown in Fig. 2, as shown in Figure 2, analyzed through RT-PCR, fusion is equal in transgene tobacco prepared by 1 and 2 embodiments 1 for representing
Transcriptional expression is obtained, transgenosis system 1 is higher compared to the expression quantity of the fusion of transgenosis system 2.
2nd, toxicity test is carried out to transgenic tobacco plant prepared by embodiment 1：
Determine packet：The transgenic tobacco plant that the first batch of embodiment 1 is cultivated is test group 1；Embodiment second lot
The transgenic tobacco plant of cultivation is test group 2；Wild-type tobacco plants are control group.
Assay method：Take respectively and consistent tobacco leaf is grown in 3 groups, distilled water is cleaned and dried, and is put into culture dish, and
Larva at the beginning of accessing the age of bollworm 3 that body weight is 15mg, is inoculated with 1 bollworm in each culture dish, every group of tobacco leaf connects
60 bollworms are planted, 20 is a small group, is placed in incubator.
Condition of culture：27 ± 1 DEG C of temperature, relative humidity 65~70%, light application ratio (L：D)16：8h, changes once raise daily
Material, keeps tobacco leaf sufficient, each bollworm body weight is weighed per 2d, and count the death rate；Dead criterion：It is light with small writing brush
Touch polypide, it is impossible to which the coordinated movement of various economic factors thinks dead；Determination data carries out variance analysis using DPS softwares.
Measurement result is as shown in table 1 below：
The death rate of table 1 and body weight testing result compare
The as shown by data of the display of above-mentioned table 1, after cotton bollworm larvae takes food the transgene tobacco that fusion of the present invention is cultivated,
Cotton bollworm larvae growth is substantially suppressed, and increased weight is smaller than other groups, and with the extension of the time that takes food, dead individuality
Gradually increase, take food the transgene tobacco of first cultivation the 6th day, the death rate reaches 65.5%, and takes food second batch cultivation and turn
After genetic tobacco, the death rate is 49.8% within the 6th day, and the test group corrected mortality for taking food wild-type tobacco is only 5.9%, is taken food
The transgene tobacco test group larval mortality that fusion of the present invention is cultivated is extremely notable with difference between wild type control group, by
This visible fusion of the present invention is transferred in plant by the expression vector heredity for building, and cultivates pest-resistant plant, fusion
The energy high efficient expression in pest-resistant plant, and there is very strong murder by poisoning activity to bollworm.
Claims (9)
1. a kind of fusion, it is characterised in that its nucleotide sequence such as SEQ ID NO：Shown in 1.
2. a kind of integrative gene expression vector, it is characterised in that comprising just like SEQ ID NO：Nucleotide sequence shown in 1.
3. a kind of construction method of integrative gene expression vector as claimed in claim 2, it is characterised in that including following operation
Step：
1）PCR expands fusion as claimed in claim 1, obtains fusion amplified fragments；
2）By the plasmids of pB I 121 and step 1）After the fusion amplified fragments digestion of preparation, it is attached, obtains connection product；
3）Connection product is converted into bacillus coli DH 5 alpha, positive colony carrier is selected, plasmid is extracted, digestion and sequencing result are correct
Plasmid be named as pBI121-TAT-HKI, as described integrative gene expression vector.
4. the construction method of integrative gene expression vector as claimed in claim 3, it is characterised in that step 2）Middle digestion is
The double digestions of I/Sac of BamH I.
5. the construction method of integrative gene expression vector as claimed in claim 4, it is characterised in that step 1）Middle PCR amplifications
The primer for using for：
P1：5’-CGCGGATCCATGTACGGCCGCAAG-3’；
P2：5’-GCGAGCTCACGCCCCAGGGGCTG-3’.
6. the construction method of integrative gene expression vector as claimed in claim 3, it is characterised in that step 3）Described in digestion
It is the double digestions of BamH/Sac I.
7. a kind of fusion gene engineering bacterium, it is characterised in that host is transferred to by integrative gene expression vector and is prepared；It is described to melt
Expression vector is closed to include just like SEQ ID NO：Nucleotide sequence shown in 1.
8. fusion gene engineering bacterium as claimed in claim 7, it is characterised in that the host is Agrobacterium LBA4404.
9. application of a kind of fusion gene engineering bacterium as claimed in claim 7 in terms of bollworm resisting tobacco plant is cultivated.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6087165A (en) * | 1999-02-26 | 2000-07-11 | The United States Of America As Represented By The Secretary Of Agriculture | Recombinant baculovirus and its use as a biocontrol agent for crop pests |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6087165A (en) * | 1999-02-26 | 2000-07-11 | The United States Of America As Represented By The Secretary Of Agriculture | Recombinant baculovirus and its use as a biocontrol agent for crop pests |
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| TAT Is Not Capable of Transcellular Delivery Across an Intact Endothelial Monolayer In Vitro;MELISSA J. SIMON et al.;《Annals of Biomedical Engineering》;20111231;第39卷(第1期);全文 * |
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