CN110229829A - A kind of Bt anti insect gene JBT-FB of engineer synthesis and its application - Google Patents
A kind of Bt anti insect gene JBT-FB of engineer synthesis and its application Download PDFInfo
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- CN110229829A CN110229829A CN201811492701.6A CN201811492701A CN110229829A CN 110229829 A CN110229829 A CN 110229829A CN 201811492701 A CN201811492701 A CN 201811492701A CN 110229829 A CN110229829 A CN 110229829A
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- jbt
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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 protein (delta-endotoxin)
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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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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The present invention provides a kind of Bt anti insect gene JBT-FB, carry out modernization overlay fusion to Cry1Ac and Cry4Aa gene, obtain the JBT-FB anti insect gene as shown in SEQ ID No:1.It is building up on the plant expression vector containing strong promoter 35S, JBT-FB gene is gone in Maize genome by agrobcterium-mediated transformation, obtains the transgenic corn plant containing JBT-FB.Research has shown that the Bt anti insect gene JBT-FB of present invention design synthesis can stablize expression in corn, and transgenic corns have significant resistance to corn borer.
Description
Technical field
The invention belongs to genetic modifications and molecular biology field, and in particular to a kind of pest-resistant base of Bt of engineer's synthesis
Because of JBT-FB and its application.
Background technique
Corn is one of Three major grain crops in the world, is the main cereal crops in China and industrial crops, to holding
The structure of agricultural production is stable or even grain security plays a significant role.Corn borer Ostrinia furnacalis Guen é e endangers corn
Each position of plant above ground, makes aggrieved some lost function, reduces grain yield, be limit that corn yield promotes it is important because
The features such as element, since it is unstable with generation, brill moth ability is strong, and medicament is not easy to reach feeding position, and drug resistance is stronger,
Chemical method is unsatisfactory to the control efficiency of corn borer.Meanwhile existing corn germplasm lacks pest-resistant resource, traditional resistance breeding
Approach is also difficult to make a breakthrough.Killing gene can be transformed into corn by technique for gene engineering genetic engineering, to obtain new resist
Worm corn variety.But as transgenic insect-resistant corn planting scale constantly expands, transgenic insect-resistant corn is still had very much
Problem.Pest-resistant range is relatively small, and the pest for endangering corn is extremely more, and Corn Pests gradually generate resistance to pest-resistant corn.
Japan biologist Shigetane Ishiwatari is separated in the silkworm larva for suffering from samping off for the first time within 1901
Go out bacillus thuringiensis, and is named as Bacillus sotto.Germany scientist Ernst Berliner is in Germany
Su Yunjin is also isolated to identical bacterium from the Mediterranean flour moth (Ephestia kuehniella) of illness, is named
For Bacillus thuringiensis.Thuringiensis is referred to as a kind of gram-positive bacteria, it is thin to belong to prokaryotes
Gammaproteobacteria Bacillaceae Bacillus, be widely present in soil, waters, plant, in insect bodies.Nineteen fifty-three is sent out for the first time
The insecticidal activity of existing thuringiensis is related with parasporal crystal, larva of the parasporal crystal to various insects especially Lepidoptera
Polypide enteron aisle can be destroyed after having toxic action, worm to eat, thus is a kind of microbial pesticide, can be used for producing biological insecticides,
Parasporal crystal eaten by insect after after the hydrolyzed under basic conditions of insect intestinal juice, activation, ultimately form active toxic peptide.
Containing three Domain that can obviously distinguish, i.e., the three-dimensional structure of Cry protein family is typically characterised by
DomainI,DomainII,DomainIII.DomainI is made of 7 α spirals, is the main region that insecticidal proteins function
Domain, taking part in insecticidal proteins oligomer and forming and be inserted into cell membrane and formed to perforate leads to the process of cell death, and DomainII is by β
3 loop rings of composition are folded, are primarily involved in insecticidal proteins in conjunction with receptor.DomainIII forms colyliform by β-pleated sheet and opens up benefit knot
Structure is primarily involved in the identification of insecticidal proteins and receptor, is the main functional areas that different Bt Cry albumen identify different pests,
DomainII and DomainIII codetermines the type of target pest.
Anti insect gene has inheritance stability, and especially effective to insect larvae, nontoxic to people and animals, welding, resistance are not held
Long.But since there is also some disadvantages to cause pest to develop drug resistance such as the plantation of long-time genetically modified plants for anti insect gene
The pest-resistant spectrum of gene is generally relatively narrow, and the pest species of nature are various.Therefore, new anti insect gene is found to improve killing rate
It is current urgent problem with pest-resistant spectrum is expanded.The present invention will create new anti insect gene, be transgenic insect-resistant corn
It cultivates and lays in useful gene, will lay a good foundation to the theoretical research of pest-resistant gene structure and function.
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-FB.
A second object of the present invention is to provide artificial synthesized Bt anti insect gene JBT-FB in preparing zoophobous
Using.
Technical solution of the present invention is summarized as follows:
A kind of artificial synthesized Bt anti insect gene JBT-FB, the nucleotide sequence SEQ of the Bt anti insect gene JBT-FB
Shown in ID No 1.
Above-mentioned artificial synthesized Bt anti insect gene JBT-FB is preparing the application in zoophobous.
Advantages of the present invention: artificial synthesized Bt anti insect gene JBT-FB before its codon optimization compared with being easier to obtain high table
The transgenic plant reached.The gene can stablize heredity and expression in transgenic corns, and expression quantity is high, gained transgenic corns
Insect resistace is good.
Detailed description of the invention
Fig. 1 .JBT-FB gene design process
The plant expression vector of Fig. 2 .JBT-FB gene
Fig. 3 turns the corn RT-PCR testing result of JBT-FB 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.
C58 Agrobacterium competence is purchased from Wuhan Miao Ling Biotechnology Co., Ltd；Plasmid containing gene Bar
PCAMBIA3301 is purchased from Wuhan Miao Ling Biotechnology Co., Ltd.
The design improvement of 1 JBT-FB 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 different zones and corn gene
Group feature, transformation is optimized to the corresponding codon of two kinds of albumen and encoder block in we, be respectively designated as Cry1Ac-J with
Cry4Aa-J.Then Domain II and the Domain III of Cry1Ac-J is merged with the Domain I of Cry4Aa-J (see
Fig. 1), a new gene is obtained, JBT-FB is named as.Gene order is as shown in SEQ ID No:1.
The plant expression vector construction of 2 JBT-FB gene of embodiment
According to embodiment 1 scheme we design and synthesized JBT-FB 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-FB gene with BamHI, SalI, and is recycled
Then JBT-FB genetic fragment is attached with the existing plant expression vector pCambia3301 of BamHI, SalI digestion,
Obtain the plant expression vector pCAMBIA3301-JBT-FB containing JBT-FB gene (see Fig. 2).
3 anti insect gene JBT-FB of embodiment converts Agrobacterium.
Agrobacterium used in experiment is C58 bacterial strain, is built with sieve on recombinant plasmid vector pCAMBIA3301-JBT-FB
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 L
PCAMBIA3301-JBT-FB 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
Successfully agrobacterium strains are used in conversion for conversion 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, when Agrobacterium grows to logarithmic growth phase (0.6,20 DEG C of OD600,
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
Base: 10g/L peptone+10g/L yeast extract+5g/L NaCl, pH 7.0.Infect culture medium: 1/2MS+65g/L sucrose+
+ 150 μm of ol/L acetosyringones of 36.5g/L glucose, pH 5.3.
The acquisition of 5 transform insect-resistant gene JBT-FB corn of embodiment.
The conversion of 1 maize bud point.Neat full corn seed is chosen, 70% alcohol impregnates 1min, the disinfection of 0.1% mercuric chloride
12min, access germination culture medium after sterile water wash 3 times, are cultivated under dark condition by 26 DEG C.When seedling it is long to 4-6cm when, in nothing
Spire and plumule are cut off under the conditions of bacterium to expose bud point growing point, and are gently scratched with sterilized scalpel for infecting.
Maize bud point is immersed in infect in culture medium and is placed in vacuum desiccator (containing 150 μm of ol/L acetosyringones),
It is infected respectively under 50kPa pressure 20 minutes, discards bacterium solution after having infected, the bacterium of maize bud point excess surface is sucked with aseptic filter paper
Liquid continues to be put into co-culturing 3 days in germination culture medium.Germinate culture medium: 1/2MS+30g/L sucrose+7.5g/L agar, pH are
5.7。
The transplanting and screening of 2 transformation seedlings.The culture medium on corn seedling surface is washed away, the flower that perlite and vermiculite are housed is moved into
In basin, it is put in hot-house culture.Phosphine oxamate spray solution is carried out to the screening of resistance seedling when seedling grows to 5-7cm on blade.
The RT-PCR Molecular Detection of 3 resistant plants.Blade total serum IgE is extracted with nontransgenic plants and transgenic plant, instead
The cDNA of synthesis is transcribed as template, is expanded with JBT-FB specific primer, the clip size amplified is 1755bp.Expand
Increase response procedures 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；Downstream: TGTGACCGGTATGAACTCAA.Product is through 0.9% agarose gel electrophoresis
Detection.As shown in figure 3, swimming lane 1 is Marker III, swimming lane 2-9 is transgenic plant, and swimming lane 10 is wild type control, swimming lane
11 be plasmid pCAMBIA3301-JBT-FB positive control.RT-PCR testing result shows that JBT-FB gene is successfully integrated
Enter in corn plant genome, shows to have successfully been obtained and turn JBT-FB gene corn plant.
6 turns of JBT-FB 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 of Hua Hou are pest-resistant
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 16 plants that 20 plants, which turn pest-resistant plant in JBT-FB gene corn plant, and anti-heavy rate is 80%, with
Non-transgenic corn plant is compared, and turns JBT-FB gene corn plant and shows as pest-resistant, and insect resistant effect is good.
Claims (2)
1. a kind of artificial synthesized Bt anti insect gene JBT-FB, it is characterized in that the nucleotide sequence of the Bt anti insect gene JBT-FB
Shown in SEQ ID No 1.
2. the artificial synthesized Bt anti insect gene JBT-FB of claim 1 is preparing the application in zoophobous.
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| CN201811492701.6A CN110229829A (en) | 2018-12-06 | 2018-12-06 | A kind of Bt anti insect gene JBT-FB of engineer synthesis and its application |
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Application publication date: 20190913 |