CN109266660A - A kind of artificial synthesized Bt anti insect gene JBT-AB and its application - Google Patents
A kind of artificial synthesized Bt anti insect gene JBT-AB and its application Download PDFInfo
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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
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- 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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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of artificial synthesized Bt anti insect gene JBT-AB and its application, and a kind of artificial synthesized Bt anti insect gene JBT-AB, the nucleotide sequence of the Bt anti insect gene JBT-AB is shown in SEQ ID No 1.The artificial synthesized Bt anti insect gene JBT-AB of the present invention can stablize heredity and expression in conversion plant, and the genetically modified plants insect resistace effect of acquisition is good.Bt anti insect gene JBT-AB can make it have corresponding insect resistance capacity with crops such as maize transformations, reduce the usage amount of pesticide, to reduce environmental pollution.The method that the present invention cultivates zoophobous is simple to operation, provides new effective selection to improve plant anti-insect.
Description
Technical field
The invention belongs to Insect resistant gene engineer technology and molecular biology fields, and in particular to a kind of artificial synthesized Bt is anti-
Worm gene JBT-AB and its application.
Background technique
Corn is one of most important cereal crops in the world, and in China, most area is planted extensively, is main grain
Food crop and industrial crops, to keeping, the structure of agricultural production is stable or even grain security plays a significant role.Corn borer Ostrinia
Furnacalis Guen é e endangers each position of plant ground, makes aggrieved some lost function, reduces grain yield,
It is an important factor for limitation corn yield is promoted, since it is unstable with generation, brill moth ability is strong, and medicament is not easy to reach
The features such as feeding position, drug resistance is stronger, chemical method are unsatisfactory to the control efficiency of corn borer.Meanwhile existing maize seed
Matter lacks pest-resistant resource, and traditional resistance breeding approach is also difficult to make a breakthrough.
Technique for gene engineering provides important channel to solve corn borer harm.Due to this method have safely, effectively, can
Many advantages, such as reducing investment and reducing environmental pollution, thus, since 1987 report insect-resistant transgenic plants for the first time, plant
The research of Insect resistant gene engineer achieves fast development.Germany scientist Ernst Berliner is in German Su Yun gold, from illness
Mediterranean flour moth (Ephestia kuehniella) in be isolated to bacillus thuringiensis, be named as Bacillus
thuringiensis(Bt)。
The toxoprotein gene (abbreviation Bt-toxin) of bacillus thuringiensis be most widely used in the world at present it is anti-
Worm gene.But in nature, due to Bt gene pairs target insect have special toxic action, cause target insect in order to
It survives and generates some mutation to resist the poisoning of Bt gene, eventually lead to and resistance is generated to Bt gene.
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-AB.
A second object of the present invention is to provide artificial synthesized Bt anti insect gene JBT-AB in preparing zoophobous
Using.
Technical solution of the present invention is summarized as follows:
A kind of artificial synthesized Bt anti insect gene JBT-AB, the nucleotide sequence SEQ of the Bt anti insect gene JBT-AB
Shown in ID No 1.
Above-mentioned artificial synthesized Bt anti insect gene JBT-AB is preparing the application in zoophobous.
Advantages of the present invention:
The artificial synthesized Bt anti insect gene JBT-AB of the present invention is compared with more easily expression is stablized in acquisition before its codon optimization
The genetically modified plants insect resistace effect of transgenic plant, acquisition is good.The artificial synthesized Bt anti insect gene JBT-AB of the present invention is anti-
The application of worm property gene lays the foundation.Bt anti insect gene JBT-AB can be such that it has corresponding anti-with crops such as maize transformations
Worm ability, reduces the usage amount of pesticide, to reduce environmental pollution.The method that the present invention cultivates zoophobous is simply easily grasped
Make, provides new effective selection to improve plant anti-insect, there is application prospect.
Detailed description of the invention
Fig. 1 is the plant expression vector of JBT-AB gene.
Fig. 2 is the corn RT-PCR testing result for turning JBT-AB 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-AB gene of embodiment
According to the activity analysis and Maize genome feature of bacillus thuringiensis Cry1Ac albumen different zones, we are right
Transformation is optimized in the corresponding codon of Cry1Ac protein amino acid sequence and encoder block, designs a new gene, life
Entitled JBT-AB.The content of G is 22.59% in gene JBT-AB；The content of C is 25%；The content of T is 2.
The nucleotide sequence of Bt anti insect gene JBT-AB (abbreviation JBT-AB gene) is shown in SEQ ID No 1.
The plant expression vector construction of 2 JBT-AB gene of embodiment
According to embodiment 1 scheme we synthesized JBT-AB gene, while being added to the enzyme of BamHI at 5 ' ends of gene
Enzyme site, 3 ' ends are added to the restriction enzyme site of SalI.Digestion is carried out to JBT-AB gene with BamHI, SalI, and recycles JBT-AB
Then genetic fragment is attached with the existing plant expression vector pCAMBIA3301 of BamHI, SalI digestion, is contained
There is the plant expression vector pCAMBIA3301-JBT-AB of JBT-AB gene (see Fig. 1).
3 JBT-AB genetic transformation Agrobacterium of embodiment
Agrobacterium used in experiment is C58 bacterial strain, is built with sieve on recombinant plasmid vector pCAMBIA3301-JBT-AB
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 L
PCAMBIA3301-JBT-AB 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 turns of Bt anti insect gene JBT-AB corns 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, write to 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-AB specific primer
Amplification, the clip size amplified are 1740bp (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: atgattgatatctctttat (SEQ
NO.2)；Downstream: tgtgaccggtatgaactcaa (SEQ NO.3).Product is detected through 0.9% agarose gel electrophoresis.Such as Fig. 2
Shown, swimming lane 1 is Marker, and swimming lane 2-4 is transgenic plant, and 5 be non-transgenic control, and 6 be plasmid pCAMBIA3301-
JBT-AB positive control.RT-PCR testing result shows that JBT-AB gene is successfully integrated into corn plant genome, table
Bright have successfully been obtained turns JBT-AB gene corn plant.
6 turns of JBT-AB 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 25 plants, which turn pest-resistant plant in JBT-AB gene corn plant, and pest-resistant rate is 84%, with
Non-transgenic corn plant is compared, and turns JBT-AB gene corn plant and shows 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-AB and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1740
<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
gaattcactg tttctcaatt aacccgtgaa atttatacaa atcctgtctt ggagaacttt 720
gatggttcct tccgcggcat ggctcagcga atcgaacaaa atatacggca gccccatctt 780
atggacattc tcaactcaat cacgatatac actgatgtac acagaggatt taattattgg 840
tcggggcatc aaattaccgc cagtccagtg ggtttcagcg gcccggagtt tacattccct 900
ctatacggaa cgatggggaa cgcagcgccc cagcaaagga tcgttgctca gctgggtcaa 960
ggcgtctatc gtactttatc ttccaccttg taccgccgac catttaatat aggcattccg 1020
aacaatcagg aacttttcgt actcgacggg acagagtttg cctatggtac gtcatcgact 1080
aacctaccta gtaccatcta ccggcaaaga ggcacagtgg atagcctgga cgttataccc 1140
ccacaggata attctgtccc gccaagggca ggattctccc accgtttatc acatgtaacg 1200
atgttgtcgc aagcggctgg ggccgtgtat actcttatta tccgcgcacc catgtttagt 1260
tggatacacc gaagcgcgga aaagcttaat aacattatcg cttctgattc cataactcaa 1320
attcctgccg ttaaaggtaa ttttttattc aacggctcag tcatctcggg acccgggttt 1380
accggtggcg acttggtacg tcttaatagt agcggacaga tatctacact ccgcgtgaac 1440
attacggcac cactatccca accgtcaact tcgacccgat atcgggttag agtcaggtac 1500
gcgagtgtaa cacctatcca tctgaatgtg aactggggga atagctctat attctccaac 1560
acggttcccg ctactgccac ctcattagat aatttgcagt cgggtagttt tcgtacagtc 1620
ggcttcacga ctccatttaa cttcagcaat ggatcttccg tatttaccct ttcagcacac 1680
gtgttcaact cggggaatga agtttatatt atcgaccgct ttgagttcat accggtcaca 1740
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgattgata tctctttat 19
<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-AB, it is characterized in that the nucleotide sequence of the Bt anti insect gene JBT-AB
Shown in SEQ ID No 1.
2. the artificial synthesized Bt anti insect gene JBT-AB of claim 1 is preparing the application in zoophobous.
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