CN109136238A - A kind of artificial synthesized Bt anti insect gene JBT-FA and its application - Google Patents
A kind of artificial synthesized Bt anti insect gene JBT-FA and its application Download PDFInfo
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- CN109136238A CN109136238A CN201811060374.7A CN201811060374A CN109136238A CN 109136238 A CN109136238 A CN 109136238A CN 201811060374 A CN201811060374 A CN 201811060374A CN 109136238 A CN109136238 A CN 109136238A
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- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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- Bioinformatics & Cheminformatics (AREA)
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- Pest Control & Pesticides (AREA)
- Cell Biology (AREA)
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- Insects & Arthropods (AREA)
- Crystallography & Structural Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of artificial synthesized Bt anti insect gene JBT-FA and its application, and the nucleotide sequence of the Bt anti insect gene JBT-FA is as shown in SEQID NO.1.Bt anti insect gene JBT-FA can be used for the conversion of the plants such as corn, it shows anti-maize borer activity outstanding compared with the transgenic plant for being easier the stable expression of acquisition before its codon optimization.A kind of new approach is provided for Corn Pests prevention and treatment.
Description
Technical field
The invention belongs to genetic engineerings and field of biological control, and in particular to a kind of artificial synthesized Bt anti insect gene JBT-
FA and its application.
Background technique
Corn is the important grain in China and industrial crops, and one of main three grande cultures object in the world.The life of corn
Safety is produced concerning national economy.Currently, the factors such as Corn diseases and insect pests have become restrict its stable yields volume increase principal element, corn because
Great economic loss caused by insect pest has caused the strong interest of people, and it is very urgent how to effectively prevent Corn Pests.It is conventional
Breeding in there are the breeding period is longer, to lack the resistance trait of good Resistance resource and crops itself be mostly polygenes control
System, so conventional breeding is difficult to obtain the elite crop new varieties with pest-resistant effect.Technique for gene engineering is to solve corn borer
Harm provides important channel.But as transgenic insect-resistant corn planting scale constantly expands, transgenic insect-resistant corn is still
There are many problems.Pest-resistant range is relatively small, and the pest for endangering corn is extremely more, and Corn Pests gradually generate pest-resistant corn
Resistance.
Bt (Bacillus thurngiensis) insecticidal proteins are that bacillus thuringiensis is generated when forming gemma
A kind of protein has high special cytotoxicity to insects such as Lepidoptera, Homoptera, coleoptera, Dipteras, is widely used
In preparing biological pesticide and plant genetic engineering.Cry genoid is one for being accredited, separating and announcing sequence in Bt gene earliest
Genoid, the three-dimensional structure of Cry protein family are typically characterised by containing three Domain, i.e. Domain that can obviously distinguish
Ⅰ、DomainⅡ、DomainⅢ。
Although anti insect gene is nontoxic to people and animals, welding, the genetically modified plants of current generally existing acquisition are not pest-resistant
The problem of low efficiency.Anti insect gene has larger difference greatly mostly from the microorganisms such as bacterium, codon and plant, therefore, right
Natural B t gene is transformed, and synthesizes new anti insect gene, makes anti insect gene be suitble to express in recipient plant cell, to mention
The pesticidal of high transgenic plant is current urgent problem.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of artificial synthesized Bt anti insect gene JBT-FA.
A second object of the present invention is to provide artificial synthesized Bt anti insect gene JBT-FA in preparing zoophobous
Using.
Technical solution of the present invention is summarized as follows:
A kind of artificial synthesized Bt anti insect gene JBT-FA, the nucleotide sequence SEQ of the Bt anti insect gene JBT-FA
Shown in IDNo 1.
Above-mentioned artificial synthesized Bt anti insect gene JBT-FA is preparing the application in zoophobous.
Advantages of the present invention: artificial synthesized Bt anti insect gene JBT-FA before its codon optimization compared with being easier to obtain high table
The transgenic plant reached.Anti insect gene JBT-FA can stablize heredity and expression in transgenic corns, and expression quantity is high, and gained turns
Gene corn insect resistace is good.
Detailed description of the invention
Fig. 1 is JBT-FA gene design process.
Fig. 2 is the plant expression vector of JBT-FA gene.
Fig. 3 is the corn RT-PCR testing result for turning JBT-FA gene
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
If method therefor is conventional method without particularly illustrating in following embodiments, term used and abbreviation are equal
It is the general term of those skilled in the art and abbreviation.
No. five corn seeds of its tower are purchased from Tianjin Kerun Jingfeng Seed Industry Co., Ltd..The application by taking maize seed as an example,
It is demonstrated experimentally that other plants can be used for the present invention.
C58 Agrobacterium competence is purchased from Wuhan Miao Ling Biotechnology Co., Ltd.
Plasmid pCAMBIA3301 containing gene Bar is purchased from Wuhan Miao Ling Biotechnology Co., Ltd.
The design improvement of 1 JBT-FA gene of embodiment
The present invention is that have the desinsection of special insecticidal activity brilliant lepidoptera pest found in bacillus thuringiensis
On the basis of body protein, according to the activity analysis of bacillus thuringiensis Cry1Ac albumen and Cry4Aa albumen different zones and corn
Genome characteristics, we are optimized transformation to the corresponding codon of two kinds of albumen and encoder block, are respectively designated as Cry1Ac-
J and Cry4Aa-J.Then the Domain of Cry1Ac-J II is merged with the Domain I of Cry4Aa-J and Domain III (see
Fig. 1), a new gene, the nucleotide sequence such as SEQ ID of Bt anti insect gene JBT-FA (abbreviation JBT-FA gene) are obtained
Shown in No:1.
The plant expression vector construction of 2 JBT-FA gene of embodiment
According to embodiment 1 scheme we design and synthesized JBT-FA new gene, while being added at 5 ' ends of new gene
The restriction enzyme site of BamHI, 3 ' ends are added to the restriction enzyme site of SalI.Digestion is carried out to JBT-FA gene with BamHI, SalI, and
Recycle JBT-FA new gene segment, then with the existing plant expression vector pCAMBIA3301 of BamHI, SalI digestion into
Row connection, obtains the plant expression vector pCAMBIA3301-JBT-FA containing JBT-FA gene (see Fig. 2).
3 JBT-FA genetic transformation Agrobacterium of embodiment.
Agrobacterium used in experiment is C58 bacterial strain, is built with sieve on recombinant plasmid vector pCAMBIA3301-JBT-FA
Select gene Bar.The C58 Agrobacterium competence of preservation is taken out into 100 μ L from -80 DEG C of refrigerators, is placed on ice, takes out 4 μ
LpCAMBIA3301-JBT-FA plasmid and 100 μ L of Agrobacterium are mixed, and ice bath 15min is put into the electric shock cup being pre-chilled on ice,
1500V, it is electroporated.Bacterium solution is sucked in 1.5mL centrifuge tube, 400 μ L YEP fluid nutrient mediums are added, in 28 DEG C of shaken cultivations
3h, 6000r/min, 28 DEG C, thalline were collected by centrifugation, abandon 300 μ L supernatants, be resuspended thallus, coated plate in 28 DEG C dark culture 2 days, screening
Conversion successfully converts and uses Agrobacterium single colonie out.
The preparation of 4 Agrobacterium infected liquid of embodiment
It picks from the plate conversion and is inoculated in YEP fluid nutrient medium that (that is mould for card containing 100mg/L with Agrobacterium single colonie
Element), 28 DEG C, 180r/min shaken cultivation stay overnight, (the OD when Agrobacterium grows to logarithmic growth phase600It is 0.6,20 DEG C,
5000r/min is centrifuged 7min and collects thallus, and thallus is resuspended with culture medium is infected in equal volume, obtains Agrobacterium infected liquid.
YEP culture medium: 10g/L peptone+10g/L yeast extract+5g/L NaCl, pH 7.0.
Infect culture medium: 1/2MS+65g/L sucrose+36.5g/L+150 μm of ol/L acetosyringones of glucose, pH 5.3.
The acquisition of 5 transform insect-resistant gene JBT-FA corn of embodiment.
The conversion of 1 maize bud point.Choose neat full No. five corn seeds of day tower, 70% alcohol impregnates 1min, and 0.1%
Mercuric chloride sterilizes 12min, access germination culture medium after sterile water wash 3 times, 26 DEG C, cultivates under dark condition.It is long to 4-6cm to seedling
When, cut off spire and plumule aseptically to expose bud point growing point, and gently scratch use with sterilized scalpel
In infecting.Maize bud point is immersed in Agrobacterium infected liquid (containing 150 μm of ol/L acetosyringones), vacuum drying is placed in
It in device, is infected under 50kPa pressure 20 minutes, discards bacterium solution after having infected, suck maize bud point excess surface with aseptic filter paper
Bacterium solution, continue to be put into and be cultivated 3 days in germination culture medium, obtain corn seedling.
Germinate culture medium: 1/2MS+30g/L sucrose+7.5g/L agar, pH 5.7.
The transplanting and screening of 2 transformation seedlings.The culture medium of corn seedling root is washed away, moves into and perlite, vermiculite, nutrition is housed
The volume ratio of soil is to be put in hot-house culture in the flowerpot of 1:2:2.It is when seedling grows to 5-7cm that the phosphine oxamate of 250mg/L is water-soluble
Liquid, is sprayed at the screening that resistance seedling is carried out on blade by 10mL/ plants.
The RT-PCR Molecular Detection of 3 resistant plants.With non-transgenic control plant leaf and rotaring gene plant blade (this reality
Apply the blade that a step 2 obtains) total serum IgE is extracted, the cDNA of reverse transcription synthesis is carried out as template with JBT-FA specific primer
Amplification, the clip size amplified are 1752bp (SEQ ID No:1).Amplified reaction program are as follows: 94 DEG C of 3min；(94 DEG C of 30s,
58 DEG C of 30s, 72 DEG C of 30s, 32 circulations)；72 DEG C of extension 7min.Primer is upstream: atgttatctgcttatactat
(SEQNO.2)；Downstream: gaccgggatgaactcaattt (SEQ NO.3).Product is detected through 0.9% agarose gel electrophoresis.
As shown in figure 3, swimming lane 1 is Marker, swimming lane 2-8 is transgenic plant, and swimming lane 9 is non-transgenic control, and swimming lane 10 is plasmid
PCAMBIA3301-JBT-FA positive control.RT-PCR testing result shows that JBT-FA gene has successfully been integrated into corn plant
In object genome, shows to have successfully been obtained and turn JBT-FA gene corn plant.
6 turns of JBT-FA gene corn plant insect resistace identifications of embodiment
The transgenic seedling of acquisition is planted in greenhouse, and time of infertility no pesticide grown, 16 days observation plant are pest-resistant after spending
Property, using non-transgenic plant as negative control.When field observation, as long as occurring blade on plant has horizontally-arranged small worm channel, bud
Leaf, filigree, by moth food etc., think not pest-resistant by moth food, stalk, fringe handle, cob.Plant is recognized without obvious insect pest, robust plant
It is pest-resistant.Statistical result see the table below.
Note: "+" represents test positive；"-" representative is detected as feminine gender；" having " represents the harm disease that worm can be seen on plant
Shape, as blade has horizontally-arranged small worm channel, bract, filigree to be eaten by moth food, stalk, fringe handle, cob by moth；"None" represents plant without jade
The hazard symptoms of rice snout moth's larva.
The above results show: it is 21 plants that 26 plants, which turn pest-resistant plant in JBT-FA gene corn plant, and anti-heavy rate is
80.77%, compared with non-transgenic corn plant, turns JBT-FA gene corn plant and show as pest-resistant, and insect resistant effect is good.
Sequence table
<110>University Of Tianjin
<120>a kind of artificial synthesized Bt anti insect gene JBT-FA and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1752
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgttatctg cttatactat tgttgtcggt accgtattga caggctttgg attcacgact 60
cctcttgggc tcgccctaat cggttttggc accctgatac ccgtgttatt cccagcacaa 120
gatcagtcca atacatggtc agactttatt acgcaaacta aaaacatcat aaagaaagaa 180
attgcctcga cctacatcag taatgctaac aagatattga atcgtagctt caacgttatt 240
tctacatatc ataatcacct taaaacgtgg gagaacaatc cgaaccctca gaatactcaa 300
gatgtccgca cccagatcca actcgtacat taccactttc agaacgtgat acccgaacta 360
gttaattcct gtccaccgaa cccttcagac tgcgattatt acaatattct ggtcttatcg 420
agttatcaag ccgcaaactt gcatcttagc gtactccgag acgtgtctgt tttcggacag 480
cggtgggggt ttgatgcggc tacaatcaat tccagataca acgacctaac gaggctgata 540
ggtaattata ctgatcactg tgtcaagtgg tacaacgtag gcttagacaa attgcgtgga 600
tcatcgctta atctcattaa gaccacaccc gatagtaacc tagacgggaa tatcaactgg 660
aatacgtata acactgaatt cactgtttct caattaaccc gtgaaattta tacaaatcct 720
gtcttggaga actttgatgg ttccttccgc ggcatggctc agcgaatcga acaaaatata 780
cggcagcccc atcttatgga cattctcaac tcaatcacga tatacactga tgtacacaga 840
ggatttaatt attggtcggg gcatcaaatt accgccagtc cagtgggttt cagcggcccg 900
gagtttacat tccctctata cggaacgatg gggaacgcag cgccccagca aaggatcgtt 960
gctcagctgg gtcaaggcgt ctatcgtact ttatcttcca ccttgtaccg ccgaccattt 1020
aatataggca ttccgaacaa tcaggaactt ttcgtactcg acgggacaga gtttgcctat 1080
ggtacgtcat cgactaacct acctagtacc atctaccggc aaagaggcac agtggatagc 1140
ctggacgtta tacccccaca ggataattct gtcccgccaa gggcaggatt ctcccaccgt 1200
ttatcacatg taacgatgtt gtcgcaagcg gctggggccg tgtatactct tattatccgc 1260
gcacccatgt ttagttggat acaccgaagc gcggaaaagc ttaaaaatac tatttatacc 1320
gatgctatca cacaagttcc tgccgtcaag tctaactttt taaatgcaac ggcgaaagta 1380
ataaagggtc ccggcttcac tggaggggac ttggtgcgtc ttaactcctc aggtaccctc 1440
tcgggccgca tggaaattca gtgtaaaaca agtatcttta atgatccaac gcgaagctac 1500
ttcatacgga ttagatatgc ttctaacggg tccgccaata ctagggcagt tatctcaaac 1560
aatgactttc tagtcatata cgtaccggcg accgctacat cgctggataa cttaagtatt 1620
cctggggtgg ccgagttggg tatggcactt aatcccacgt tcagcggcaa caatatcata 1680
ccaactattg gagcggaatc ttttgtttcc aacgagaaga tctatataga caaaattgag 1740
ttcatcccgg tc 1752
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgttatctg cttatactat 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gaccgggatg aactcaattt 20
Claims (2)
1. a kind of artificial synthesized Bt anti insect gene JBT-FA, it is characterized in that the nucleotide sequence of the Bt anti insect gene JBT-FA
Shown in SEQ ID No 1.
2. the artificial synthesized Bt anti insect gene JBT-FA of claim 1 is preparing the application in zoophobous.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811060374.7A CN109136238A (en) | 2018-09-12 | 2018-09-12 | A kind of artificial synthesized Bt anti insect gene JBT-FA and its application |
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