CN106701790B - The nucleotide sequence of encoding insecticidal proteins and its application - Google Patents
The nucleotide sequence of encoding insecticidal proteins and its application Download PDFInfo
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Abstract
This application involves the anti-lepidoptera pest new gene of transformation and its applications in plant breeding.And design construction stability and high efficiency expresses the conversion carrier of the gene；By the genetic transformation into corn inbred line, transgenic insect-resistant corn is obtained.By lepidoptera pest carry out insecticidal activity bioassay control experiment, demonstrate improved gene compared with other isoformgenes insect resistace have be obviously improved.
Description
Technical field
This application involves plant genetic engineering and technical field of crop propagation.Specifically, this application involves anti-squama is transformed
The new gene of wing mesh pest and its application.
Background technique
Thuringiensis (Bacillus thuringiensis) can secrete toxalbumin, to Lepidoptera, coleoptera
Insect (such as diamondback moth) has very strong lethal effect.Research and utilization Bt bacterium kills pest to the mankind very early, has more than 100 so far
The history in year.With the appearance of the technologies such as the development of Protocols in Molecular Biology and gene cloning, DNA operation, the mankind start from Bt
Its toxoprotein gene is cloned in bacterium into engineering bacteria, makes biological pesticide.
Bt toxin is broadly divided into endotoxin and exotoxin.Exotoxin refers to the generation that bacterium excretes during vital movement
Thank object, including α-exotoxin, β-exotoxin, γ-exotoxin, unstable exotoxin and water-soluble exotoxin etc..Endotoxin is also known as
Delta-endotoxin, crystal toxin or insecticidal crystal protein parasporal crystal, are made of one or more insecticidal crystal proteins.Endotoxin exists
Bt in the lethal processes of insect to playing a major role.This application involves cry1Ca gene coding insecticidal proteins belong to endogenous toxic material
Element, i.e. insecticidal crystal protein.
There are different degrees of homologys for the amino acid sequence of many Bt insecticidal crystal proteins.1989, Hofte and
Whiteley is according to the insecticidal spectrum of Bt insecticidal crystal protein and the homology of amino acid sequence, by found at that time 42 Bt genes
It is divided into five major class, 14 subclass (Hofte H and Whiteley HR, Microbio.Rev., 53:241-255).Wherein,
Preceding four major class is the gene family cry of crystalline protein, and the 5th class is cytolytic proteins gene cyt.Cry gene can be divided into four
Group, i.e. cryI are Lepidoptera specificity, and cry II is Lepidoptera and Diptera specificity, and cry III is coleoptera specificity, cryIV
For Diptera specificity.Since the number of genes found at that time is few, there are apparent difference, therefore point of each gene each other
Class bit comparison it is clear so that the categorizing system of Hofte and Whiteley is widely accepted and uses.This application involves
Genecry1C be applicable in this categorizing system, belong to Lepidoptera specificity killing gene.
In recent years, the insecticidal crystal protein and its gene constantly discovered make amino acid sequence in original categorizing system with
Incoordination is produced between insecticidal activity, and with the discovery of new gene, this classification method be not able to satisfy increasingly this two
A condition, so that classification is difficult to determine.Crickmore etc. is in nineteen ninety-five world invertebrate pathology meeting annual meeting (SIP)
Proposing with the homology of insecticidal crystal protein amino acid sequence is unique new classification system according to difference Bt gene, by
Adopt extensively various countries.Specifically, insecticidal crystal protein amino acid identity is 45% hereinafter, being the first estate, with Arab
Digital representation；Homology is the second grade, is indicated with capitalization English letter between 45%~78%；Homology 78%~
Between 95%, it is the tertiary gradient, is indicated with small English alphabet；Homology is the fourth estate, with Arabic number 95% or more
Word indicates.Such as this application involves cry1Ca5 be the fourth estate.
CryIA (b) gene was mediated the vacation for being introduced into plant single by the scholars such as Obukowics in 1986 through transposons Tn5
In spore bacillus, make plant that there is insect resistace, to create first generation Bt transgenic anti-insect plants, this is also Bt toxoprotein gene
Plant is set to obtain the earliest report of insect resistace.Real Bt toxalbumin genetically modified plants are second generation Bt zoophobous, first
Successful example be reported by Belgian Plant Genetic Systems N. V. in June, 1987 (Vaeck etc., Nature, 328:
33-37,1987), they successfully will only be retained with agrobacterium-mediated transformation after complete cryIA (b) gene and 3 ', 5 ' end missings
CryIA (b) the channel genes tobacco of the different length of 5 ' end coding toxalbumin core spaces obtains the transgenosis of anti-maduca sexta
Tobacco plant.
Summary of the invention
On the one hand, this application provides isolated nucleic acid molecules, which is characterized in that the nucleic acid molecules include SEQ ID
Nucleotide sequence shown in NO:1 or its complementary series.Present invention also provides have at least 70% identity with SEQ ID NO:1
Sequence.In one embodiment, this application provides with the gene have at least 80%, 85%, 90%, 95%, 96%,
97%, the sequence of 98%, 99%, 99.5% or 99.9% sequence identity.In one embodiment, the nucleic acid molecules include
Nucleotide sequence shown in SEQ ID NO:1.In one embodiment, nucleic acid molecules nucleotide as shown in SEQ ID NO:1
Sequence composition.
It yet still another aspect, this application involves expression cassettes, which is characterized in that include above-mentioned isolated nucleic acid molecules.It is real one
It applies in scheme, the nucleic acid molecules are operably connected with 35S promoter and no terminator.
It yet still another aspect, it includes above-mentioned expression cassettes this application involves expression vector.In one embodiment, the table
It also include PolyA sequence and omega enhancer up to carrier.
It yet still another aspect, this application provides host cells, which is characterized in that include above-mentioned expression vector.Implement one
In scheme, the host cell is plant cell or prokaryote.In one embodiment, the host cell is large intestine
Bacillus or agrobatcerium cell.
It yet still another aspect, this application provides the methods for generating genetically modified plants, which is characterized in that with above-mentioned expression vector
Or transformation of host cells plant, obtain genetically modified plants.In one embodiment, the plant is monocotyledon.It is real one
It applies in scheme, the plant is selected from: corn, rice, wheat, oat, barley, highland barley, grain, sorghum and sugarcane.
It yet still another aspect, the application further relates to genetically modified plants comprising stable integration into its genome expression cassette,
It is characterized in that, the expression cassette includes the nucleic acid molecules for encoding anti-lepidoptera pest gene, and the nucleic acid molecules include SEQ ID
Nucleotide sequence shown in NO:1 or the nucleotide sequence with SEQ ID NO:1 at least 70% sequence identity, or above
Complementary series.In one embodiment, this application provides with the gene have at least 80%, 85%, 90%, 95%, 96%,
97%, the sequence of 98%, 99%, 99.5% or 99.9% sequence identity.In some embodiments, the nucleic acid molecules packet
Nucleotide sequence shown in the NO:1 of ID containing SEQ or the nucleic acid molecules nucleotide sequence shown in SEQ ID NO:1 form.One
In a little embodiments, the plant is monocotyledon.In some embodiments, the plant is selected from: corn, rice, small
Wheat, oat, barley, grain, sorghum and sugarcane.In one embodiment, the application further relates to the organ, tissue, cell of the plant
And pass through processed goods produced by the plant, such as food, feed.
It yet still another aspect, this application provides the methods for generating transgenic seed, which is characterized in that planted from above-mentioned transgenosis
Object generates transgenic seed.Present invention also provides the transgenic seeds generated by this method.
It yet still another aspect, this application provides the methods of control lepidopteran pest population, including make the lepidoptera pest
The genetically modified plants obtained by the above method feed in group.The plant is monocotyledon in one embodiment.One
In embodiment, the plant is selected from: corn, rice, wheat, oat, barley, grain, sorghum and sugarcane.In an embodiment
In, the plant is corn.In one embodiment, the lepidoptera pest is European corn borer (Pyrausta
) or Ostrinia furnacalis (Ostrinia furnacalis) nubilalis.Present invention also provides the sides for killing lepidoptera pest
Method, including the genetically modified plants obtained by the above method to the lepidoptera pest feeding insecticidal effective dose.
It yet still another aspect, this application provides lepidoptera pest is mitigated to the method for the injury of plant, including load will be expressed
Genome of the body stable integration into plant, which is characterized in that the expression vector includes the core for encoding anti-lepidoptera pest gene
Acid molecule, the nucleic acid molecules include nucleotide sequence shown in SEQ ID NO:1 or have at least 90% sequence with SEQ ID NO:1
The nucleotide sequence of column identity or above complementary series.In one embodiment, the nucleic acid molecules include SEQ ID
Nucleotide sequence shown in NO:1.In one embodiment, nucleic acid molecules nucleotide sequence group as shown in SEQ ID NO:1
At.
It yet still another aspect, this application provides the methods for screening anti-lepidoptera pest gene, which is characterized in that including following
Step:
Using insecticidal proteins amino acid sequence as template, it is transformed according to monocotyledon codon preference and obtains G+C content
High, stable structure gene order；
Design the carrier expression cassette of high efficiency stable expression gene, construction of expression vector；
With the expression vector transforming monocots, obtains genetically modified plants and identify transgenic event；
Low-copy transgenic plant events are selected, carry out insecticidal activity bioassay using lepidoptera pest.
In one embodiment, this method further include compare different anti insect genes insect resistant effect or verifying transformation after gene
Insect resistant effect.
In one embodiment, the insecticidal proteins are encoded by cry1Ca5.
The application has one or more of advantage: 1, provided artificial gene, the expression cassette containing the gene, packet
Expression vector containing the gene, the host cell comprising the gene have in terms of plant breeding with the plant of the genetic transformation
Extensive purposes；2, this application provides the methods of control lepidoptera pest；3, the application is that extensive cultivation insect resistace is strong
Excellent strain provides gene basis and technology platform；4, the present processes can be used for the exploitation of pest-resistant chemicals and answer
With.
Detailed description of the invention
Fig. 1 is that monocotyledon codon usage bias concludes figure
Fig. 2 is carrier P35s-cry1CgerZm-Tnos area schematic, is 35S starting the length is 2990bp, P35s
Son, T-nos are no terminator.
Fig. 3 is real-time PCR detection example schematic diagram: reference gene identifies corn internal reference IVR (single copy) amplification curve, mesh
Genetic marker cry1CgerZm gene magnification curve is marked, skeleton identifies ORI fragment amplification curve
Specific embodiment
Unless otherwise stated, nucleic acid is write from left to right with 5 ' to 3 ' directions；Amino acid sequence is with amino to carboxyl direction
It writes from left to right.Amino acid can use its generally known three letter symbols or IUPAC-IUB biological chemical name in this paper
The one-letter symbol that the committee is recommended indicates.Likewise it is possible to indicate nucleotide with the single-letter code usually received.
Digital scope includes the number for limiting the range.
As used herein, " nucleic acid " include be related to single-stranded or double-stranded form deoxyribonucleotide or ribonucleotide it is more
Polymers, and unless otherwise limitation, including the known analog (for example, peptide nucleic acid) with natural nucleotide fundamental property, institute
Analog is stated to hybridize in a manner of as naturally occurring ucleotides with single-chain nucleic acid.
As used herein, term " coding " or when " encoded " context for specific nucleic acid, refers to that the nucleic acid includes
The nucleotide sequence is instructed to translate into the required information of specific protein.Use the information of codon presentation code albumen.As herein
Used, " full length sequence " for being related to specific polynucleotides or its encoded albumen refers to natural (non-synthetic) endogenous sequence
Entire nucleic acid sequence or entire amino acid sequence.Overall length, the catalytic activity form of the overall length polynucleotide encoding specific protein.
Term " polypeptide ", " peptide " and " albumen " is interchangeably used, herein to refer to the polymer of amino acid residue.The term
For amino acid polymer, wherein one or more amino acid residues are that the artificial chemistry of corresponding naturally occurring amino acid is similar
Object.The term is also used to naturally occurring amino acid polymer.
Term " residue " or " amino acid residue " or " amino acid " are interchangeably used herein, is incorporated into albumen, more to refer to
The amino acid of peptide or peptide (being referred to as " albumen ").Amino acid can be naturally occurring amino acid, and unless otherwise limitation, can be with
Known analog including natural amino acid, the analog can be acted as in a manner of similar with naturally occurring amino acid
With.
As used herein, can be interchanged use term " separation " and " purifying ", be related to nucleic acid or polypeptide or its
Active biological moiety is substantially or essentially free of as usual adjoint or anti-found in its naturally occurring environment
It should be in the component of the nucleic acid or polypeptide.Thus, with recombinant technique generate separation or purifying nucleic acid or polypeptide when, separation or
Nucleic acid that the nucleic acid or polypeptide of purifying are separated substantially free of other cellular materials or culture medium or chemical synthesis or purifying
Or when polypeptide, substantially free of precursor or other chemicals.
" separation " nucleic acid is typically free of in the genomic DNA for the organism that the nucleic acid is derived from natural flank in this
The sequence (such as, such as sequence of coding albumen) of nucleic acid (sequence for being located at the nucleic acid 5 ' and 3 ' end).For example, in various realities
It applies in scheme, separated nucleic acid may be embodied in the genomic DNA for the cell that the nucleic acid is derived from natural flank in this
The nucleotide sequence of less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb of nucleic acid.
Through application documents, by word "include", "comprise" or its variant be understood as prompt comprising alleged element, integer or
The group of step or element, integer or step, and non-excluded any other element, integer or step or element, integer or
The group of step.
As used herein, term " influencing insect pest " refers to having for insect feed, growth and/or the behavior to any stage of development
The change of influence, including but not limited to killing insect, growth-delaying, obstruction fecundity, antifeedant activity etc..
As used herein, term " insecticidal activity " refers to the activity of organism or substance (such as, such as albumen), measurement
After can be through but not limited to Mortality of insect, pest weight loss, pest repelling and feed and the appropriate long-time of exposure
The other behaviors and physical change of pest.Thus, organism or substance with insecticidal activity negatively affect at least one can
The pest health parameters of measurement.For example, " insecticidal proteins " are itself display or show the egg of insecticidal activity with other protein combinations
It is white.
As used herein, term " insecticidal effective dose " refers to the substance or biology with insecticidal activity for being present in pest environment
The amount of body.Similarly, when pest is insect, " insecticidally effective amount " can be used for referring to " insecticidal effective dose ".
It should be understood that as used herein, term " transgenosis " includes that genotype is changed by the presence of heterologous nucleic acids
Any cell, cell line, callus, tissue, plant part or plant, including initially so change those of turn base
Those of cause, and generated from the initial transgenosis by sexual hybridization or vegetative propagation.As used herein, term " turns
Gene " it does not include by conventional plant breeding method or by the event naturally occurred to genome (chromosome or dyeing
Change in vitro), the event naturally occurred such as random allogamy, non-recombinant virus infection, non-recombinant Bacterial Transformation,
Non-recombinant swivel base or spontaneous mutation.
" subject plant " or " subject plant cell " refers to the plant or plant cell that genetic modification enters into force, Huo Zheru
The plant of this transformation or the progeny cell of cell, the progeny cell include the transformation." control " or " check plant " or " control
Plant cell " provides the reference point for measuring subject plant or plant cell phenotypic alternation.
Check plant or plant cell may include, such as: (a) wild-type plant or cell are originated with genetic modification
Material has plant or the cell of phase homogenic type, and the genetic modification generates subject plant or cell；(b) with the starting material
Expect to have phase homogenic type but with empty construct (i.e. with the construct that effect is endlessly known purpose character, such as comprising indicating
The construct of object gene) conversion plant or plant cell；(c) be subject plant or plant cell non-transformed segregant plant
Object or plant cell；(d) it genetically unanimously but is not exposed to that target gene can be induced with the subject plant or plant cell
The condition of expression or the plant of stimulant or plant cell；Or (e) subject plant or plant cell itself, it is in target gene
Under conditions of not being expressed.
Those skilled in the art can easily accept, such as site-specific mutagenesis and random mutagenesis, polymerase chain reaction
The progress of the molecular biology field of induction method and proteins engineered technology provides extensive proper implements and operating procedure,
For being transformed or being engineered the agriculturally amino acid sequence of interested albumen and potential gene order.
In some embodiments, the nucleotide sequence of the application can be changed, is replaced with carrying out conserved amino acid
It changes.The principle and example of conserved amino acid replacement are discussed further below.It in certain embodiments, can be according to Fig. 1
Disclosed in unifacial leaf codon preference do not changed the replacement of amino acid sequence to the nucleotide sequence of the application, such as
The codon with amino acid sequence can be encoded with the codon replacement of monocotyledon preference, without changing the nucleotides sequence
Arrange encoded amino acid sequence.
The application further relates to advanced optimize resulting nucleotide sequence to SEQ ID NO:1.The more details of this method
It is described in Murray et al. (1989) Nucleic Acids Res.17:477-498.Optimization nucleotide sequence, which can be used for improving, to kill
Expression of the worm albumen in plant, the plant such as monocotyledon, such as gramineae plant, such as corn (Zea
mays).As used herein, term " mutant nucleotide sequence " or " mutation " or " nucleotide sequence of mutagenesis " refer to through mutagenesis or change
Become and include the nucleotide sequence of one or more nucleotide residues (such as base-pair), the nucleotide residue is not present in phase
In the wild-type sequence answered.
In some embodiments, the part core in the application is replaced with the different codons of amino acid sequence with coding
Nucleotide sequence, to not change the amino acid sequence of its coding while changing nucleotide sequence.Conservative variant include due to
Genetic codon degeneracy and those of amino acid sequence for one of encoding the insecticidal peptide of embodiment sequence.Some
In embodiment, the partial nucleotide sequence in the application is replaced according to monocotyledon preferred codons.
In some embodiments, mutant nucleotide sequence further includes derivative nucleotide sequence, such as using for example
Site-directed mutagenesis and those of the insecticidal proteins for still encoding embodiment generated, such as Mutant toxins.In some implementations
In scheme, mutant nucleotide sequence includes addition, missing or the replacement of one or more nucleic acids.In some embodiments
In, such addition removes or replaces the addition, removal or replacement that can lead to corresponding amino acid residue.In general, real
Apply the specific nucleotide sequence of scheme variant will with the specific nucleotide sequence have at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
Higher sequence identity.Sequence identity is determined by alignment programs described below.The mutant core of embodiment
The difference of the sequence of nucleotide sequence and the application may be as few as 1-15 nucleotide, as little as 1-10 (such as 6-10), and as little as 5
It is a, as little as 4,3,2 or even 1 nucleotide.
In some embodiments, mutant nucleotide sequence can encode with improvement or reduced insecticidal activity
Mutant protein.Such insecticidal activity with respect to native protein can be different either improve or it can be
Constant, as long as remaining insecticidal activity.Mutant protein includes the polypeptide derived from native protein, and the derivative is by lacking
It loses one or more amino acid of (so-called truncation) native protein N-terminal and/or C-terminal or adds one or more amino acid
Add to the N-terminal and/or C-terminal of the native protein；Native protein one or more site deletions or addition one or
Multiple amino acid；Or replace one or more amino acid in one or more sites of native protein.
The mutant protein that embodiment includes continues have the required of native protein with biological activity, i.e. they
Biological activity, the activity are insecticidal activity as described herein.The variant can derive from such as gene pleiomorphism or the mankind
Operation.As long as any useful mutation can be added it will be appreciated by those skilled in the art that coded polypeptide retains insecticidal activity
Add to the sequence of embodiment.In some embodiments, the amino acid sequence of mutant protein is by the albumen with the application
Amino acid sequence have at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.In some embodiments
In, the difference of the albumen of mutant protein and the application may be as few as 1-15 amino acid residue, as little as 1-10 (such as 6-10
It is a), as little as 5, as little as 4,3,2 or even 1 amino acid residue.
It in some embodiments, can also be with mutant nucleotide sequence, so that proteolytic digestion of the coded polypeptide to protease
It is resistant.Mutation can protect the polypeptide from proteasome degradation, and the protection is, for example, all by removing from different zones
Such as the proteolysis sites of the presumption of the serine protease site and elastin laminin enzyme recognition site of presumption.It can remove or change
Some or all for making these presumption sites, so that the proteolysis of original site location is lowered.It can be by comparing mutation
Body polypeptide and wild-type toxin or the by comparing different Mutant toxins of amino acid sequence, to evaluate proteolysis
Variation.Proteolysis sites and the proteolysis sites of presumption include but is not limited to following sequence: positioned trypsin cleavage site, gruel
Protease site and chymotrypsin site.It, can be any by adding or lacking as long as the insecticidal activity of the polypeptide is enhanced
The amino acid residue of number and type is transformed these sites.Thus, relative to native sequences or background sequence, by including mutation
Nucleotide sequence coded polypeptide by comprising at least one amino acid change or addition or 2,3,4,5,6,7,8,9,10,
11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、32、35、38、40、45、
47、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、
240,250,260,270,280,290,300 or more amino acid changes or addition.
It will be appreciated by those skilled in the art that amino acid addition and/or substitution are typically based on the phase of amino acid side chain substituent group
To similitude, for example, the hydrophobicity of the substituent group, charge, size etc..With the exemplary of various aforementioned considered properties
Amino acid substitution groups are known to those skilled in the art, and including arginine and lysine；Glutamic acid and asparagine
Acid；Serine and threonine；Glutamine and asparagine；And valine, leucine and isoleucine.About not influencing mesh
The guide of appropriate amino acid substitution of biological activity of albumen can be in Dayhoff et al. (1978) Atlas of Protein
Sequence and Structure (protein sequence and structure atlas) (Natl.Biomed.Res.Found., Washington,
D.C it) is found in the model (being incorporated herein by reference).It can carry out such as changing an amino acid and make that there is similar quality
The conservative replaces of another amino acid.
When before being replaced, being lacked or being inserted into, it is difficult to when predicting its precise effects, it will be appreciated by those skilled in the art that institute
Stating effect will be evaluated by the conventional screening analysis of such as insect eating analysis.See, e.g., what is be incorporated herein by reference
Marrone et al. (1985) J.Econ.Entomol.78:290-293 and Czapla and Lang (1990)
J.Econ.Entomol.83:2480-2485。
It in some embodiments, can be in the nucleotide sequence and other known Cry nucleotide sequence of embodiment
Corresponding part between recombinant full-lenght coded sequence, encode target structure domain sequence motifs or embodiment nucleotides sequence
Any segment of column, to obtain the new gene with improved properties.
The property to attract attention includes but is not limited to the insecticidal activities of per unit insecticidal proteins, protein stability, to non-target
Species, particularly to the mankind, domestic animal and express embodiment insecticidal peptide plant and microorganism toxicity.Embodiment
It is not limited by specific re-arrangement strategy, rearrangement can only relate to nucleotide sequence disclosed herein, or can be additionally related to
Reset other nucleotide sequences known in the art.DNA re-arrangement strategy is known in the art.
The identification of sequence identity includes hybridization technique.For example, by all or part of known nucleotide sequence be used as with
The probe of other corresponding nucleotide sequence selectivity hybridization, other corresponding nucleotide sequences are present in from selected organism
Cloned genomic DNA fragments or cDNA segment group (i.e. genomic library or cDNA library).The hybridization probe can be
Genomic DNA fragment, cDNA segment, RNA segment or other oligonucleotides, and can be with such as32The detectable group of P or its
It can detect marker to mark.Thus, for example, can be by marking the synthetic oligonucleotide based on embodiment sequence to prepare
Hybridization probe.The method for preparing hybridization probe and construction cDNA and genomic library is usually known in the art.
In some embodiments, complete sequence disclosed herein or one or more part can be used as can
With the probe of corresponding sequence and mRNA specific hybrid.To complete specific hybrid under various conditions, the probe includes
It is unique to the sequence of embodiment sequence and usually long at least about 10 or 20 nucleotide.The probe can be used, with logical
It crosses PCR and expands corresponding Cry sequence from selected organism.The technology can be used, to separate other codings from required organism
Sequence, or the technology is used as diagnostic analysis, to determine organism with the presence or absence of coded sequence.Hybridization technique includes screening by hybridization
The DNA library being inoculated with.
The hybridization of the sequence can be carried out under stringent condition.As used herein, term " stringent condition " or " tight miscellaneous
Friendship condition " is expressed as follows condition, i.e., under this condition, hybridizes relative to other sequences, and probe will be with detectable more great Cheng
Degree (for example, at least 2 times of background, 5 times or 10 times) hybridizes with its target sequence.Stringent condition is sequence dependent and not
With different in environment.By control hybridization stringency and/or control cleaning condition, can identify and the probe 100%
Complementary target sequence (homologous probe method).Selectively, adjustable stringent condition, to allow some sequence mismatch, to examine
Survey lower similarity (heterologous probe method).In general, probe length less than about 1000 or 500 nucleotide.
In general, stringent condition is following condition, i.e., in the condition, salinity is under pH 7.0 to 8.3, less than about
1.5M Na ion, normally about 0.01M to 1.0M Na ion concentration (or other salt), and temperature condition are as follows: when for short spy
(such as 10 to 50 nucleotide), at least about 30 DEG C when needle；(50 nucleotide are greater than) when being used for long probe, at least about
60℃.Stringent condition can also be realized by adding the destabilizing agent of such as formamide.Illustratively low stringent condition includes
Hybridized at 37 DEG C using 30% to 35% formamide buffer, 1M NaCl, 1%SDS (lauryl sodium sulfate), 50 DEG C extremely
At 55 DEG C 1 × cleaning into 2 × SSC (20 × SSC=3.0M NaCl/0.3M trisodium citrate).Illustrative Moderate
Condition includes hybridizing in 40% to 45% formamide, 1.0M NaCl, 1%SDS at 37 DEG C, at 55 DEG C to 60 DEG C 0.5 × extremely
It is cleaned in 1 × SSC.Illustrative high stringent condition includes hybridizing in 50% formamide, 1M NaCl, 1%SDS at 37 DEG C, and 60
DEG C to finally being cleaned in 0.1 × SSC at least about 20 minutes at 65 DEG C.Optionally, cleaning buffer solution may include about 0.1% to
About 1%SDS.Duration of hybridization is generally less than about 24 hours, typically about 4 hours to about 12 hours.
Specificity generally relies on the cleaning after hybridization, and key factor is the ionic strength and temperature of last cleaning solution.
The T of DNA-DNA heterozygotem(thermal melting point) can be approximate from Meinkoth and Wahl (1984)
The formula of Anal.Biochem.138:267-284: Tm=81.5 DEG C of+16.6-0.61 (% formyl of (log M)+0.41 (%GC)
Amine) -500/L；Wherein M is the molar concentration of monovalent cation, and %GC is the percentage of guanosine and cytidylic acid in DNA
Number, " formamide % " is the formamide percentage of hybridization solution, and L is the base pairs length of heterozygote.TmIt is (determining ion
Under intensity and pH) temperature of 50% complementary target sequence when hybridizing with the probe of exact matching.Usually will cleaning at least carry out to
Reach balance, and reach low hybrid context level, such as carries out 2 hours, 1 hour or 30 minutes.
Every 1% mispairing correspondence makes TmReduce about 1 DEG C；Therefore, it is possible to adjust Tm, hybridization and/or cleaning condition, thus with
The sequence of required consistency hybridizes.For example, if necessary to the sequence of >=90% consistency, it can be by TmReduce by 10 DEG C.In general, will
Stringent condition is selected as the T than distinguished sequence and its complementary series under determining ionic strength and pHmLow about 5 DEG C.However, non-
It, can be than the T under conditions of often tightmHybridized and/or cleaned at low 1 DEG C, 2 DEG C, 3 DEG C or 4 DEG C；In Moderate
Under the conditions of, it can be than the TmHybridized and/or cleaned at low 6 DEG C, 7 DEG C, 8 DEG C, 9 DEG C or 10 DEG C；In low stringent condition
Under, it can be than the TmHybridized and/or cleaned at low 11 DEG C, 12 DEG C, 13 DEG C, 14 DEG C, 15 DEG C or 20 DEG C.
In some embodiments, further include nucleotide sequence and its coding amino acid sequence segment.Such as this paper institute
With term " segment " refers to the one of a part of the nucleotide sequence of the polynucleotides of embodiment or the amino acid sequence of polypeptide
Part.The segment of nucleotide sequence can encode protein fragments, and the protein fragments retain the life of natural or corresponding full-length proteins
Object activity, and thus have insecticidal activity.Mutant protein includes the bioactive fragment of native protein, and it includes retain day
Right biological activity of albumen, i.e. the continuous amino acid residue of enough numbers with insecticidal activity.
Cutting of the insecticidal activity of Bt toxin known in the art usually by various protease in insect enteral to the peptide
It is activated.Because peptide is not in insect enteral always with the cutting of full effect, the segment of overall length toxin is relative to overall length toxin itself
It can have the insecticidal activity of enhancing.It in some embodiments, can also be more to improve by truncating natural or full length sequence
The insecticidal activity of peptide.
Thus, in some embodiments, this application involves the clipped form of overall length insecticidal peptide or segments.In some realities
It applies in scheme, some naturally occurring insecticidals that will be derived from relative to it in the polypeptide fragment, variant and mutation
The activity of polypeptide has the insecticidal activity of enhancing.In some embodiments, in the polypeptide fragment, variant and mutation
Some activity by relative to its naturally occurring insecticidal polypeptide being derived from have the insecticidal activity weakened.
The nucleotide sequence fragment for encoding the embodiment of the insecticidal proteins active biological moiety of embodiment will encode
At least 15,25,30,50,100,200,300,400,500 or 600 continuous amino acid, or coding up to embodiment
Total amount of amino acid present in insecticidal peptide.As embodiment nucleotide sequence fragment nucleic acid include at least 16,
20、50、75、100、150、200、250、300、350、400、450、500、600、700、800、1,000、1,200、1,400、1,
600 or 1, the nucleotide of quantity in the presence of 800 nucleotide, or nucleotide sequence up to disclosed herein.Particular implementation
Scheme is related to the segment of the first nucleic acid derived from (such as being produced from) embodiment, wherein the fragment coding is with insecticidal activity
The truncated toxin being characterized.The encoded truncated polypeptide of the polynucleotide passage of embodiment is characterized in that of equal value or changes
Good insecticidal activity, described of equal value or improvement are the corresponding full-length polypeptides of the first encoded by nucleic acid relative to the fragment derivitization certainly
Activity.Related is that the nucleic acid fragment of embodiment can be cut at 3 ' ends of natural or corresponding complete encoding sequence
It is short.Nucleic acid fragment can also be truncated at 5 ' and 3 ' ends of natural or corresponding complete encoding sequence.
This application involves expression cassettes, which is characterized in that includes above-mentioned isolated nucleic acid molecules.The application does not limit expression cassette
In specifically used promoter and terminator, as long as it is suitable for expressing in plant.In one embodiment, the nucleic acid
Molecule is operably connected with 35S promoter and no terminator.
The application further relates to expression vector, and it includes above-mentioned isolated nucleic acid molecules.In some embodiments, expression carries
Body includes above-mentioned expression cassette.In some embodiments, the expression vector also includes PolyA sequence and omega enhancer.In
In some embodiments, expression vector also includes other tables that can be used for detecting expression vector expression in cell
Up to box.
Some embodiments further include the microorganism of conversion, such as Agrobacterium or Escherichia coli.The conversion is using at least
A kind of nucleic acid of embodiment, the expression cassette comprising the nucleic acid or the carrier comprising the expression cassette.In some embodiment party
In case, the microorganism is the microorganism by plant generation.
Some embodiments further include plant cell or the genetically modified plants of conversion, and it includes at least one embodiments
Nucleotide sequence.In some embodiments, plant is converted using expression vector, the expression vector includes at least one implements
The nucleotide sequence of scheme and what is be operably connected drive the promoter of expression in plant cell with it.The plant of conversion
Cell and genetically modified plants indicate plant cell or plant in genome comprising heterologous polynucleotide.In general, described different
Source polynucleotides are steadily integrated in the plant cell of conversion or the genome of genetically modified plants, so that by the polynucleotides
Pass to offspring.The heterologous polynucleotide can be integrated into genome either individually or as a part of expression vector.
In some embodiments, this application involves plant be monocotyledon；Optionally, the plant is selected from: beautiful
Rice, rice, wheat, oat, barley, highland barley, grain, sorghum and sugarcane.
In some embodiments, this application involves plant include plant cell, plant protoplast, can regenerate
Plant cell tissue cultures, plant callus, agglomerate and the plant cell of plant are complete plant or plant
The part of object, such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, kernel, fringe, cob, shell, stalk, root, the tip of a root,
Anther etc..The application further includes derived from the genetically modified plants of the application or its filial generation and thus at least partly comprising this Shen
Plant cell, protoplast, tissue, callus, embryo and the flower of nucleotide sequence please, stem, fruit, leaf and root.
Although embodiment does not depend on particular biological mechanism to increase the resistance of plants against plant pest, embodiment party
The nucleotides sequence of case, which is listed in the expression in plant, can lead to the insecticidal proteins for generating embodiment and improve the plant pair
The resistance of plant insect.The plant of embodiment can be used for agriculturally influencing the method for insect pest.Certain embodiments, which provide, to be turned
The crop plants of change can be used for influencing the method for the insect pest of the plant, the insect pest such as lepidoptera pest.
Lepidoptera pest includes but is not limited to the pest of Noctuidae (Noctuidae): autumn armyworm (Spodoptera
Frugiperda), beet armyworm (Spodoptera exigua), prodenia litura (Spodoptera litura), bud band noctuid
(Mamestra configurata), lopper worm (Mamestra brassicae), black cutworm (Agrotis
Ipsilon), western cutworm (Agrotis orthogonia), yellow cutworm (Agrotis segetum), kapok worm
(Alabama argillacea), cabbage looper (Trichoplusia ni), soybean noctuid (Pseudoplusia
Includens), Anticarsia (Anticarsia gemmatalis), the green noctuid of clover (Hypena scabra), tobacco budworm
(Heliothis virescens), a star mythimna separata (Pseudaletia unipuncta), rough bark noctuid (Athetis
Mindara), dark edge cutworm (Euxoa messoria), earias insulana (Earias insulana), earias fabia
(Earias vittella), heliothis zea (Helicoverpa armigera), paddy reality noctuid (Helicoverpa zea)；
The pest of Pyralidae (Pyralidae): European corn borer (Ostrinia nubilalis), Ostrinia furnacalis (Ostrinia
Furnacalis), navel orangeworm (Amyelois transitella), Mediterranean flour moth (Anagasta kuehniella), dry fruit
Phycitid (Cadra cautella), striped rice borer (Chilo suppressalis), spot dogstail snout moth's larva (Chilo partellus), rice
Moth (Corcyra cephalonica), corn root web spinner (Crambus caliginosellus), bluegrass web spinner
(Crambus teterrellus), cnaphalocrocis medinalls guenee (Cnaphalocrocis medinalis), grape leaf folder
(Desmia funeralis), sweet tea Diaphania indica (Diaphania hyalinata), Southwest Maize snout moth's larva (Diatraea
Grandiosella), small sugarcane borer (Diatraea saccharalis), Mexico rice borer (Eoreuma loftini), tobacco powder
Snout moth's larva (Ephestia elutella), galleria mellonella waxmoth (Galleria mellonella), rice cut leaf open country snout moth's larva (Herpetogramma
Licarsisalis), sunflower phycitid (Homoeosoma electellum), South America maize seedling phycitid (Elasmopalpus
Lignosellus), lesser wax-moth (Achroia grisella), loxostege sticticalis (Loxostege sticticalis), beans open country snout moth's larva
(Maruca testulalis), Indian meal moth (Plodia interpunctella), yellow rice borer (Scirpophaga
Incertulas), greenhouse snout moth's larva (Udea rubigalis)；And the pest of Tortricidae (Tortricidae): blackhead Acleris spp
(Acleris variana), fruit tree Huang roll up moth (Archips argyrospila), European leaf roller (Archips rosana),
Tangerine Argyrotaenia spp (Argyrotaenia citrana), rose Choristoneura spp (Choristoneura rosaceana)；And other:
Adoxophyes moth (Adoxophyes orana), striped sunflower moth (Cochylis hospes), hazel steinernema (Cydia
Latiferreana), carpocapsa pononella (Cydia.pomonella), variegated leaf roller (Platynota flavedana), Holland
China pink steinernema (Platynota stultana), European grape flower wing olethreutid (Lobesia botrana), apple Bai little Juan
Moth (Spilonota ocellana), grape fruit moth (Endopiza viteana), ligustrum fine tortricidae (Eupoecilia
Ambiguella), Brazilian apple skin worm (Bonagota salubricola), oriental fruit months (Grapholita
) and sunflower bud moth (Suleima helianthana) etc. molesta.
Selected other agronomy pests of Lepidoptera include but is not limited to: fall cankerworm (Alsophila pometaria), cotton
Lyonetid (Bucculatrix thurberiella), clover Huang butterfly (Colias eurytheme), English walnut Huang butterfly (Datana
Integerrima), white looper (Ennomos subsignaria), bodhi looper (Erannis tiliaria), pornography and drug moth
(Euproctis chrysorrhoea), black quasi- sandfly moth (Harrisina americana), fall webworms (Hyphantria
Cunea), tomato pinworm moth (Keiferia lycopersicella), Polyura narcaea (Lambdina fiscellaria
Fiscellaria), western hemlock looper (Lambdina fiscellaria lugubrosa), leucoma candida (Leucoma
Salicis), gypsymoth (Lymantria dispar), tomato hawkmoth (Manduca quinquemaculata), maduca sexta
(Manduca sexta), winter looper (Operophtera brumata), spring looper (Paleacrita vernata), big swallowtail butterfly
(Papilio cresphontes), California strain worm (Phryganidia californica), phyllocnistis citrella stainton
(Phyllocnistis citrella), spot curtain leaf miner (Phyllonorycter blancardella), large white butterfly
(Pieris brassicae), pieris rapae (Pieris rapae), dark arteries and veins cabbage butterfly (Pieris napi), diamondback moth
(Plutella xylostella), pink bollworm (Pectinophora gossypiella), omnivorous looper (Sabulodes
Aegrotata), gelechiid (Sitotroga cerealella), Song Yizhou moth (Thaumetopoea pityocampa), bag clothing
Moth (Tineola bisselliella), Liriomyza brponiae (Tuta absoluta) and apple ermine moth (Yponomeuta
padella)。
Embodiment
The cry1CgerZM coded sequence and its signature analysis that embodiment 1 synthesizes
It is close according to monocotyledon to encode the genecry1C a5 of bacillus thuringiensis insecticidal crystal proteins as template
Numeral Preference (monocotyledon codon usage bias is as shown in Figure 1), transformation obtain genecry1C gerZM sequence, such as
Shown in sequence SEQ ID NO:1.Reforming composite cry1CgerZM gene has a characteristic that codon preference index (CAI)
Up to 86%；G+C content is up to 60.13%；Eliminate 11 restriction enzyme action sites and 13 cis-acting elements；Eliminate weight
Complex sequences.
Include: its 3 ' end, 128 amino acid coding of removal to the specific steps that cry1Ca5 is transformed, retains 5 '
Hold 630 amino acid sequences；Its coding nucleotide sequence is pushed away using amino acid sequence is counter, according to monocotyledon codon preference
Property it is preferable to use monocot genes codon high frequency use sequence, obtain that sequence tentatively is transformed；To in preliminary transformation sequence
Common restriction enzyme action site, rich in the site of AT and other be likely to form the sequence of unstable secondary structure
The codon replacement of conserved amino acid sequences is carried out to avoid above-mentioned site, final acquisition transformation sequence is generated.
2 vector construction of embodiment
To improve expression of the gene transcripts in plant cell, design vector expression cassette, carrier P35s-
The region cry1CgerZm-Tnos is as shown in Fig. 2, the expression cassette comprising cry1CgerZM gene further includes following element: 35S starting
Son, PolyA sequential structure, omega enhancer and T-nos terminator.It is artificial synthesized according to sequence shown in SEQ ID NO:1
Cry1CgerZm gene, while from PCR amplification bar gene on pCambia3300 carrier, at the same for its both ends add BamHI and
The site SacI.It is building up in same genetic transformation carrier pZHZH25025.
The specific building process of carrier pZHZH25025 are as follows: bar genetic fragment BamHI and the SacI enzyme for obtaining PCR
Cut, by PU130 carrier (carrier is the derivative vector of pCambia3300, and building process is: pCambia3300 through XhoI and
HindIII digestion processing replaces the part P35s-bar to obtain PU130 with maize ubiquitin promoter Pubi) use BglII and SacI enzyme
It cuts, skeleton carrier Pubi-bar-T35spolyA is obtained after connection；PCR amplification 35S promoter simultaneously adds at its both ends respectively
HindIII and BamHI restriction enzyme site, PCR amplification no terminator simultaneously add EcoRI and PmeI restriction enzyme site at its both ends；Manually
Cry1CgerZm genetic fragment is synthesized, 5 ' end addition enhancer element omega enhancers are another to add restriction enzyme site 5 '-
HindIII-BamHI and EcoRI-PmeI-3 ', the cloning vector pUC57simple that the gene cloning to Synesis Company is provided
Upper formation intermediate vector pUC57-cry1CgerZm；Respectively with HindIII and BamHI cutting P35S segment and intermediate vector
PUC57-cry1CgerZm obtains intermediate vector P35S-cry1CgerZm after connection；It is cut respectively using EcoRI and PmeI again
Nos and intermediate vector P35S-cry1CgerZm, obtains intermediate vector P35S-cry1CgerZm-Tnos after connection.It uses
HindIII and PmeI double digestion skeleton carrier Pubi-bar-T35spolyA and intermediate vector P35S-cry1CgerZm-
Tnos.It connects treated skeleton segment and Insert Fragment P35S-cry1CgerZm-Tnos, building obtains
PZHZH25025 carrier.
The acquisition of 3 transgenic corns of embodiment
According to method described in the patent application that applicant's application publication number in a review is CN104745622A, will recombinate
PZHZH25025 carrier imported into Chinese subset by agrobcterium-mediated transformation and rolls into a ball the jade that Co., Ltd provides
In rice self-mating system auspicious 249, transgenic insect-resistant corn is obtained.
Specifically, conversion carrier pZHZH25025 is transferred to Agrobacterium (Agrobacterium by electric shocking method
Tumefaciens) in EHA105.By 10 days after the fresh pollination self stripped of corn inbred line auspicious 249 ratarias, (size was
It 1.0-2.0mm) is put into the 2ml plastic centrifuge tube containing suspension.After sucking suspension, Agrobacterium bacterium solution is added, infects
5min is poured into and is co-cultured on base, and the Agrobacterium bacterium solution of excess surface is sucked with pipettor, is co-cultured 3 days in 23 DEG C of dark.Altogether
After culture, rataria is transferred in rest culture medium, in 28 DEG C after dark culturing 6 days, is put to double propylamine phosphines containing debita spissitudo
On screening and culturing medium, 2 periods of screening and culturing, each period is 2 weeks.Resistant calli is transferred in differential medium I,
25 DEG C, 5000lx, illumination cultivation 1 week.Callus is transferred in differential medium II again, illumination cultivation 2 weeks；It will differentiate
Seedling be transferred on root media, 25 DEG C, 5000lx, illumination cultivation is to taking root；Seedling is transferred in small basin and is grown, simultaneously
Sampling is used for Molecular Detection.
The Molecular Detection of 4 transgenic corns of embodiment
T0 includes that positive, copy number and skeleton detect for the Molecular Detection of transformation seedlings.Using Tiangeng kit Tiagen
DP320 is stripped corn gene group DNA.Real-time PCR reactions system is as shown in the table, the forward direction (F) of reference gene IVR and
Reverse primer (R) is as follows；The forward and reverse primer of target gene cry1CgerZm is as follows；Skeleton segment ORI's
Forward and reverse primer is as follows.
Corn internal reference IVR (single copy) primer sequence:
F-ACTAGGCATCCAAGGCGAACG
R-AGTGCGAGAAGAACGAGTGTCC
Target gene cry1CgerZm primer sequence:
F-CGACATTTCCCTCTCCCTGGTTC
R-AGGTTAGCGATAGCAGCGTTCC
Skeleton segment ORI primer sequence:
F-AACAAGACGAACTCCAATTCACTG
R-TGTTGATTGTAACGATGACAGAGC
1 real-time PCR reactions system of table
PCR reaction is carried out on ABI 7900, response procedures are as follows: 95 DEG C initial denaturation 10 minutes；30 or less circulations: 95 DEG C
Denaturation 10 seconds, 60 DEG C of renaturation and extension 55 seconds, detection example schematic diagram is as shown in Figure 3.
After the completion of real-time PCR reactions, the average Ct (amplification cycles of the reference gene and target gene that are generated according to instrument
Number) value and prediction equation RQ=2-ΔCt, calculate the RQ value of corresponding sample.Reference gene IVR is corn single copy gene.According to RQ
Value, can calculate the Relative copy number of target gene.Concrete foundation are as follows: single copy RQ value is 0.5 or so, and double copy RQ values are
1.0 left and right.
Testing result is as shown in table 2, selects material of the RQ value within the scope of 0.1-1.0, i.e. foreign gene low-copy is inserted into
The material of Maize genome carries out subsequent experimental.
2 T0 of table is for the real-time PCR testing result of transgenic corns
The identification of 5 transgenic corns insect resistace of embodiment
Using two leaf identification methods identify transgenic corns insect resistace in vitro: when corn grows to 6-7 leaf, clip
Two leaf end of blade part, is put into the culture dish of diameter 9cm, and filter paper is drenched with 1300 μ L distilled water to keep high humidity in ware bottom.Often
Connect Ostrinia furnacalis (Ostrinia furnacalis) newly hatched larvae 10 in ware, and with the preferable medical adhesive fabric width of gas permeability
Mouthful.Every plant of 3 repetitions, are compared with the self-mating system of non-transgenosis.28 DEG C of temperature, humidity 75%, illumination (L): dark (D)=
It is cultivated under the conditions of 14:10.The dead worm head number of every ware is checked after 4 days, and calculates larval mortality and corrected mortality.
Corrected mortality (%)=(the processing death rate-control death rate)/(1- compares the death rate)
Take the T0 of two control cry1C genes containing different editions for anti insect gene low-copy (1-2 copy) insertion
Transgenic corns material as the corn for turning cry1CgerZm gene of check plant and the application to do insect resistance identification comparison real
It tests.Both versions cry1C gene is respectively: cry1C* (CN 1483823A) and cry1C (US 6043415).It is carried to eliminate
Volume elements part tests three conversion carriers used for the influence factor of gene expression, except anti insect gene is different, other yuan of carrier
Part is consistent, and the step of converting and growth conditions of check plant are also identical as conversion plant.The preparation of check plant is referring to embodiment 2
The step of with 3.
Laboratory biological measurement result is as shown in table 3, the cry1CgerZm base of monocotyledon codon preference optimization
The insecticidal effect of cause is best, and averagely the correction death rate is 63%, for examination 10 T0 for there is 1 up to 100% in transformation event
The correction death rate, and the equal correction death rate without up to 100% of the transformation event of two genes of cry1C* and cry1Cger, and it is average
The correction death rate is below the transformation event of cry1CgerZm gene, and respectively 32% and 41%.Variance analysis shows, 3 versions
The correction death rate of this cry1C gene conversion events corn borer reaches significant difference (p=0.02, < 0.05).
Insect resistance identification of the T0 of 3 different editions cry1C genetic transformation carrier of table for transformation event
Although above having made detailed description to the application with a general description of the specific embodiments,
On the basis of the application, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, without departing from the application spirit on the basis of these modifications or improvements, belong to this application claims range.
Claims (20)
1. isolated nucleic acid molecules, which is characterized in that nucleic acid molecules nucleotide sequence shown in SEQ ID NO:1 forms.
2. expression cassette, which is characterized in that include the nucleic acid molecules described in claim 1.
3. the expression cassette as described in claim 2, which is characterized in that wherein the nucleic acid molecules and 35S promoter and no are whole
Only son is operably connected.
4. expression vector, which is characterized in that include expression cassette described in claim 2 or 3.
5. the expression vector as described in claim 4, which is characterized in that the expression vector also include PolyA sequence and
Omega enhancer.
6. host cell, which is characterized in that include expression vector described in claim 4 or 5.
7. the host cell as described in claim 6, which is characterized in that the host cell is plant cell or prokaryotes
Cell.
8. the host cell as described in claim 7, which is characterized in that the host cell is that Escherichia coli or Agrobacterium are thin
Born of the same parents.
9. the method for generating genetically modified plants, which is characterized in that the expression vector described in claim 4 or 5 or right
It is required that transformation of host cells plant described in any one of 6 to 8, obtains genetically modified plants.
10. the method as described in claim 9, which is characterized in that the plant is monocotyledon.
11. the method as described in claim 10, which is characterized in that the plant is selected from: corn, rice, wheat, oat, big
Wheat, highland barley, grain, sorghum and sugarcane.
12. the method for generating transgenic seed, which is characterized in that generated from any one of claim 9 to 11 the method
Genetically modified plants generate transgenic seed.
13. the method for controlling lepidopteran pest population, including make lepidopteran pest population feed by claim 9 to
The genetically modified plants that any one of 11 the methods obtain.
14. the method as described in claim 13, which is characterized in that the lepidoptera pest is European corn borer (Pyrausta
) or Ostrinia furnacalis (Ostrinia furnacalis) nubilalis.
15. the method for killing lepidoptera pest, including passing through claim 9 to the lepidoptera pest feeding insecticidal effective dose
The genetically modified plants obtained to any one of 11 the methods.
16. the method as described in claim 15, which is characterized in that the lepidoptera pest is European corn borer (Pyrausta
) or Ostrinia furnacalis (Ostrinia furnacalis) nubilalis.
17. mitigate lepidoptera pest to the method for the injury of plant, including the genome by expression vector stable integration into plant,
It is characterized in that, the expression vector includes the nucleic acid molecules for encoding anti-lepidoptera pest gene, the nucleic acid molecules are by SEQ
The composition of nucleotide sequence shown in ID NO:1.
18. the method as described in claim 17, which is characterized in that the plant is monocotyledon.
19. the method as described in claim 18, which is characterized in that the plant is selected from: corn, rice, wheat, oat, big
Wheat, grain, sorghum and sugarcane.
20. the method as described in any one of claim 17 to 19, which is characterized in that the lepidoptera pest is that Europe is beautiful
Rice snout moth's larva (Pyrausta nubilalis) or Ostrinia furnacalis (Ostrinia
Furnacalis).
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| Transgenic Potato Lines Expressing cry1Ba1 or cry1Ca5 Genes are Resistant to Potato Tuber Moth;S. Meiyalaghan, et al.;《Potato Research》;20061231(第49期);第203-216页 * |
| 转 Bt 基因抗虫作物培育现状及Bt蛋白的改造和聚合策略的利用;李晨，刘博林;《生物工程学报》;20150125;第31卷(第1期);第53-64页 * |
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