CN109136239A - A kind of artificial synthesized Bt anti insect gene JBT-AD and its application - Google Patents
A kind of artificial synthesized Bt anti insect gene JBT-AD and its application Download PDFInfo
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- CN109136239A CN109136239A CN201811060393.XA CN201811060393A CN109136239A CN 109136239 A CN109136239 A CN 109136239A CN 201811060393 A CN201811060393 A CN 201811060393A CN 109136239 A CN109136239 A CN 109136239A
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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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- 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-AD and its application, the nucleotide sequence of the Bt anti insect gene JBT-AD is shown in SEQ ID No 1, use plant-preference codon, reduce in original DNA sequence and be rich in AT sequence and indefinite eukaryotic DNA sequence intron sequences, it is compared with the transgenic plant for being easier the stable expression of acquisition before its codon optimization.Gained transgenic plant insect resistant effect is good.The present invention obtains the efficiently Bt anti insect gene JBT-AD suitable for maize genetic conversion, formulates the transgenic insect-resistant corn of inheritance stability, provides New idioplasm resource for the cultivation of pest-resistant corn variety.
Description
Technical field
The invention belongs to Insect resistant gene engineers and field of molecular breeding, and in particular to a kind of artificial synthesized Bt anti insect gene
JBT-AD and its application.
Background technique
Corn is one of world's Three major grain crops, and insect pest can lead to the corn significantly underproduction.Endanger the evil of corn in the whole world
Worm has more than 400 kinds, and wherein corn borer is one of most important pest.Meanwhile maize sown area is big and is long-stalked crops, fertility
Period prevention and treatment is more difficult, and easily pest is made to develop drug resistance, and pesticide residue also will affect ecological environment and endanger human and livestock health.Cause
It is the effective ways for solving the problems, such as this that this, which cultivates pest-resistant corn variety, but due to not yet finding high pest-resistant money in corn
Source limits the feasibility by traditional breeding technology breeding pest-resistant cultivar.
Bacillus thuringiensis Bt (Bacillus thurngiensis), be 1911 by Berliner from illness ground
A kind of bacillus isolated in extra large powder snout moth's larva, since it finds in German Su Yun Jin Sheng (Thuringen) for the first time, therefore in 1915
Year is named as bacillus thuringiensis.Bt (Bacillus thurngiensis) insecticidal proteins are bacillus thuringiensis
Generated a kind of protein when forming gemma has height special the insects such as Lepidoptera, Homoptera, coleoptera, Diptera
Different cytotoxicity is widely used in preparing biological pesticide and plant genetic engineering.Cry genoid is reflected earliest in Bt gene
A genoid that is fixed, separating and announce sequence, and most new gene types are exploited out, the most thorough one kind Bt gene of research.
The three-dimensional structure of Cry protein family is typically characterised by containing three Domain that can obviously distinguish, i.e. Domain 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-AD.
A second object of the present invention is to provide artificial synthesized Bt anti insect gene JBT-AD in preparing zoophobous
Using.
Technical solution of the present invention is summarized as follows:
A kind of artificial synthesized Bt anti insect gene JBT-AD, the nucleotide sequence SEQ of the Bt anti insect gene JBT-AD
Shown in ID No 1.
Above-mentioned artificial synthesized Bt anti insect gene JBT-AD is preparing the application in zoophobous.
Advantages of the present invention: artificial synthesized Bt anti insect gene JBT-AD before its codon optimization compared with being easier to obtain high table
The transgenic plant reached.Anti insect gene JBT-AD is applied to corn transformation, shows good insect resistace.Gained transgenic corns
Insect resistace is good.New idioplasm resource is provided for the cultivation of pest-resistant corn variety.
Detailed description of the invention
Fig. 1 is JBT-AD gene design process.
Fig. 2 is the plant expression vector of JBT-AD gene.
Fig. 3 turns the corn RT-PCR testing result of JBT-AD 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-AD 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 I and Domain III is merged with the Domain II of Cry4Aa-J (see
Fig. 1), a new gene, the nucleotide sequence such as SEQ ID of Bt anti insect gene JBT-AD (abbreviation JBT-AD gene) are obtained
Shown in No:1.
The plant expression vector construction of 2 JBT-AD gene of embodiment
According to embodiment 1 scheme we design and synthesized JBT-AD gene, while being added to BamHI at 5 ' ends of gene
Restriction enzyme site, 3 ' ends are added to the restriction enzyme site of SalI.Digestion is carried out to JBT-AD gene with BamHI, SalI, and is recycled
Then JBT-AD genetic fragment is attached with the existing plant expression vector pCAMBIA3301 of BamHI, SalI digestion,
Obtain the plant expression vector pCAMBIA3301-JBT-AD containing JBT-AD gene (see Fig. 2).
3 JBT-AD genetic transformation Agrobacterium of embodiment.
Agrobacterium used in experiment is C58 bacterial strain, is built with sieve on recombinant plasmid vector pCAMBIA3301-JBT-AD
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 3 μ
LpCAMBIA3301-JBT-AD plasmid and 100 μ L of Agrobacterium are mixed, and ice bath 14min 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, 500 μ L YEP fluid nutrient mediums are added, in 28 DEG C of shaken cultivations
3h, 7000r/min, 28 DEG C, thalline were collected by centrifugation, abandon 400 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 conversion Agrobacterium single colonie of embodiment 4
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-AD 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-AD specific primer
Amplification, the clip size amplified are 1755bp (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: atgattgatatctctttatc (SEQ
NO.2)；Downstream: tgtgaccggtatgaactcaa (SEQ NO.3).Product is detected through 0.9% agarose gel electrophoresis.Such as Fig. 3
Shown, swimming lane 1 is Marker, and swimming lane 2-11 is transgenic plant, and swimming lane 12 is non-transgenic control, and swimming lane 13 is plasmid
PCAMBIA3301-JBT-AD positive control.RT-PCR testing result shows that JBT-AD gene has successfully been integrated into corn plant
In object genome, shows to have successfully been obtained and turn JBT-AD gene corn plant.
6 turns of JBT-AD 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 23 plants that 27 plants, which turn pest-resistant plant in JBT-AD gene corn plant, and anti-heavy rate is
85.19%, compared with non-transgenic corn plant, turns JBT-AD 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-AD and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1755
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgattgata tctctttatc cttgactcaa tttcttctct cagagttcgt tcctggtgct 60
ggctttgtcc tcggactggt agacataatt tgggggatct tcggtccctc gcagtgggat 120
gcctttttag tgcaaataga gcagttgatt aatcaacgta tcgaagagtt cgcacgcaac 180
caggcgataa gtcggcttga aggcctcagc aatctatatc aaatttacgc tgagtctttt 240
cgggaatggg aggccgaccc aaccaacccg gcactgagag aagagatgag catccagttc 300
aatgatatga actccgcgtt aacaacggct atacctttgt ttgccgttca aaattatcag 360
gtcccccttc tctcagtata cgtgcaagca gcgaacctac atctgtcggt tttacgtgac 420
gtcagtgtat tcggacagcg ctgggggttt gatgctgcca ctattaatag ccgatataac 480
gacttgaccc ggcttatcgg taattacaca gattatgcag tgagatggta caacacgggc 540
ctcgaaaggg tttggggacc agactctcgt gattgggtcc gctataatca attccgacgg 600
gagctaactc tgaccgtatt agacatagtg gcgttgtttc cgaactacga ttccagaagg 660
gaattcaaaa ctgaatttac ccgtgagatt tatacagctt tagttgaatc tccttcctca 720
aagtcgatcg ccgcattgga ggcggctgaa gatagtctta cgatacgcaa accccatctc 780
ttcgactacc tacaaggtat tgagtttcac actcgactcc agccaggcta tttcggaaag 840
gatagcttta attactggtc tgggaactat gtctccaccc ggccgtcaat cggttcgaat 900
gacataatta caagtccttt ctacggcaac aaaagtttag gattgcttac gcatcaaatc 960
cagctcaatt ctaacgtata taagacttcc ataaccgata catcatcgcc cagtaataga 1020
gtgacgaaaa tgctaggggt tactaaggtc gaatttagcc aatacaacga ccagaccgat 1080
gaggcctcta cacaaacgta tgactccaaa aataagaaca ttttcggtct gccaatctta 1140
aaaaggcgtg aagagcaggg caatccgact ttgtttccta ccgtaaacga ttacacacac 1200
atactttcat atgtgacgaa ttacgcagaa tgtttcctca tgcaaggatc gcgcgggact 1260
attcccgttc taacctggac acatagtagc gtggacaagc ttaataacat tatcgcttct 1320
gattccataa ctcaaattcc tgccgttaaa ggtaattttt tattcaacgg ctcagtcatc 1380
tcgggacccg ggtttaccgg tggcgacttg gtacgtctta atagtagcgg acagatatct 1440
acactccgcg tgaacattac ggcaccacta tcccaaccgt caacttcgac ccgatatcgg 1500
gttagagtca ggtacgcgag tgtaacacct atccatctga atgtgaactg ggggaatagc 1560
tctatattct ccaacacggt tcccgctact gccacctcat tagataattt gcagtcgggt 1620
agttttcgta cagtcggctt cacgactcca tttaacttca gcaatggatc ttccgtattt 1680
accctttcag cacacgtgtt caactcgggg aatgaagttt atattatcga ccgctttgag 1740
ttcataccgg tcaca 1755
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgattgata tctctttatc 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tgtgaccggt atgaactcaa 20
Claims (2)
1. a kind of artificial synthesized Bt anti insect gene JBT-AD, it is characterized in that the nucleotide sequence of the Bt anti insect gene JBT-AD
Shown in SEQ ID No 1.
2. the artificial synthesized Bt anti insect gene JBT-AD of claim 1 is preparing the application in zoophobous.
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