CN103882013B - Insect gender modulating construct, modulating method, and application of the construct - Google Patents
Insect gender modulating construct, modulating method, and application of the construct Download PDFInfo
- Publication number
- CN103882013B CN103882013B CN201210559254.8A CN201210559254A CN103882013B CN 103882013 B CN103882013 B CN 103882013B CN 201210559254 A CN201210559254 A CN 201210559254A CN 103882013 B CN103882013 B CN 103882013B
- Authority
- CN
- China
- Prior art keywords
- insect
- construction
- genes
- carrier
- insects
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a transgenic construct with an insect gender modulating function. The construct specifically comprises a promoter, a doublesex gene, and a terminator, and also comprises a tetracycline trans-activated repressor protein coding element (TAV), wherein the element is inserted into the third exon of the doublesex gene. The construct can specifically express tetracycline trans-activated repressor protein in female insects so as to kill the female insects in the early stage of the growth process, while male insects are not affected at the same time; tetracycline can be added into the feed to control the expression of toxic protein, and a transgenic system capable of controlling the insect gender is established thereby.
Description
Technical field
The invention belongs to biotechnology and developmental regulation field, in particular it relates to a kind of insect sex phenotype structure
Thing is built, regulates and controls method and its application.
Background technology
It is a very important technology on sericultural production specially to support hero silkworm, because the Cocoon layer ratio of male silk cocoon and fresh cocoon wire vent
Rate is higher than female silk cocoon, and male mulberry volume taken by the silkworms is few, and conversion rate of silk leaves is high；Additionally, male silkworm constitution is strong, it is easier to raise, and
And male silk fiber number is thin, cleanliness is good, is suitable to put down high-grade raw silk processed.Therefore, countries in the world scientist enters to specially supporting hero silkworm technology
A series of exploration is gone.
Russian scientist Strunnikov V.A. in 1975 etc. have been bred as silkworm with technological means such as radioinductions
Sex linked balanced lethal line.The selfing in sex linked balanced lethal line, its offspring's male and female individuality respectively has half to survive, and makes strain
It is maintained.Can be survived when the offspring that the male worm of balanced lethal system mates with the female adult of conventional strain only has male worm, female adult is complete
Portion is lethal, reaches the purpose for specially supporting hero silkworm.1996, Zhejiang Academy of Agricultural Science was introduced with silkworm property from Russian Academy Of Sciences
Linkage equilibrium lethal system S-8, S-14, and carried out the research for realizing specially supporting hero silkworm using balanced sexlinked lethal technology.Through 10
Year tackling key problem research, creates silkworm sex linked balanced lethal line cell control gene and introduction method（The patent No.：
ZL01132108.3）, backcross improvement silkworm sex linked balanced lethal line method（The patent No.：ZL01126809.3）, it is a kind of miscellaneous
The method for handing over improvement silkworm sex linked balanced lethal line proterties（The patent No.：200810061660.5）Three national inventing patents；
Establish a male silkworm cenospecies mating system for low cost；It has been bred as autumn China × flat 30, Hua Jing × flat 60 grade silkworm sex-kinks
Balanced lethal practicality Silkworm varieties, up to 99.8%, Cocoon layer ratio and silk production rate improve 23%, mixed economy effect to male silkworm rate than conventional variety
Benefit improves 20% or so.
Can make male worm infertility using means such as radiation treatments, then by mass rearing sterile males and carry out field and release
Put, sterile males can not produce offspring with the female adult post-coitum in Natural Population, so as to reach the purpose of control of insect.This elder brother
Worm insect sterile technique（Sterile Insect Technique,SIT）In terms of the insects such as preventing and treating screwfly, citrus fruit fly
Achieve successfully.But the method there is also some problems, as the insect competitiveness after treatment is poor, extensive feeding cost is high
Deng.
By the cultivated silkworm breed variety that balanced sexlinked lethal system is used is obtained by radiomutation, intravarietal dyeing
Body structure and genetic stability have the possibility of variation.Therefore, this area is at present in the urgent need to exploitation new technique makes up
The deficiency of sex linked balanced lethal line system.
The content of the invention
It is an object of the invention to provide a kind of insect sex phenotype construction.
Another object of the present invention is just to provide a kind of insect sex phenotype method and its application.
In the first aspect of the present invention, there is provided a kind of transgenosis construct, described construction has following elements：
Doublesex genes（Double property genes）With tetracycline repressible trans-activator encoder element（TAV）, and tetracycline resistance
Hold back trans-activator encoder element（TAV）It is inserted in doublesex genes（Double property genes）Alternative splicing extron in.
In another preference, the tetracycline repressible trans-activator encoder element（TAV）It is inserted in doublesex
Gene（Double property genes）The 3rd extron in.
In another preference, described construction also includes tetracycline binding sequence（TetO）.
In another preference, described construction also includes the element being selected from the group or its combination：Promoter, termination
Son, poly (A) element, transhipment element, gene target element, riddled basins, enhancer, resistant gene, transposase coding
Gene.
In another preference, described doublesex genes come from lepidopterous insects.
In another preference, described lepidopterous insects include（But it is not limited to）：Silkworm, Ostrinia furnacalis, bollworm,
Or diamondback moth.
In another preference, described doublesex genes come from silkworm.
In another preference, described doublesex genes（Double property genes）With SEQ ID NO：Sequence shown in 1
Row.
In another preference, described tetracycline repressible trans-activator encoder element has SEQ IDNO：Shown in 2
Sequence.
In another preference, described riddled basins are fluorescin encoding gene, preferably gfp (green fluorescences
Protein coding gene or DsRed red fluorescent protein encoding genes.
In another preference, described tetracycline binding sequence（TetO）With SEQ ID NO：Sequence shown in 3.
In another preference, described doublesex genes are operably connected with promoter or terminator.
In the second aspect of the present invention, there is provided a kind of carrier, the carrier contains the transgenosis structure described in first aspect
Build thing.
In another preference, the carrier is selected from：Bacterial plasmid, bacteriophage, yeast plasmid or zooblast carrier,
Shuttle vector.
In another preference, described carrier is transposon vector.
In the third aspect of the present invention, there is provided a kind of host cell, described host cell contains described in first aspect
Construction or second aspect described in carrier, or described host cell chromosome is integrated with the construction described in first aspect
Or the carrier described in second aspect.
In another preference, described host cell also includes the carrier containing encoding transposase gene or its chromosome
On be integrated with transposase gene.
In another preference, described host cell is insect cell.
In another preference, described insect is lepidopterous insects, including（But it is not limited to）：Silkworm, Ostrinia furnacalis,
Bollworm, diamondback moth etc..
In the fourth aspect of the present invention, there is provided the construction described in first aspect, the carrier described in second aspect, or the
The purposes of the host cell described in three aspects, is used for insect sex phenotype, or for preparing the insects other composition of regulation and control.
In the fifth aspect of the present invention, there is provided a kind of method of prepare transgenosis insect, including step：
A () provides insect cell or insect ovum, it is any described that the insect cell or insect ovum contain claim 1-3
Carrier or its chromosomal integration described in construction or claim 4 are had the right any described constructions of requirement 1-3 or right
It is required that the carrier described in 4；
B insect cell described in step (a) or insect ovum are regenerated as insect by (), so as to obtain described transgenosis elder brother
Worm.
In another preference, described insect is lepidopterous insects.
In another preference, the host cell described in step (a) is silkworm primiparity egg cell.
In the sixth aspect of the present invention, there is provided a kind of transgenic insect, described transgenic insect contains the third aspect
Described host cell, or described transgenic insect chromosomal integration has construction or the second aspect institute described in first aspect
The carrier stated, or described transgenic insect is the host cell regeneration as described in the third aspect.
In another preference, described transgenic insect is prepared with the method described in the 5th aspect.
In the seventh aspect of the present invention, there is provided the purposes of the 6th aspect transgenic insect, described transgenosis elder brother
Worm is used for：
(1) silk is produced；
(2) silk cocoon Cocoon layer ratio is improved；
(3) conversion rate of silk leaves is improved；
(4) silk fiber number is improved；
(5) silk cleanliness is improved；
(6) or its combination.
In the eighth aspect of the present invention, there is provided the insects method for distinguishing of one kind regulation, including step：
A () provides insect cell or insect ovum, the insect cell or insect ovum contain the construction described in first aspect
Or the carrier or its chromosomal integration described in second aspect have the load described in construction or second aspect described in first aspect
Body, and described insect cell or insect ovum are regenerated as insect；
B the insect of () under conditions of tetracycline is lacked described in feeding step (a), obtains male insects survival ratio and is more than
95% group of insects.
In another preference, described insect is lepidopterous insects, preferably silkworm.
In another preference, described regulation insect sex is：Improve ratio of the male insects in total insect.
In another preference, step (b) obtains male insects survival ratio and is more than 96%（Preferably greater than 98%）Insect
Group.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Drawings below is used to illustrate specific embodiments of the present invention, rather than limits what is be defined by the claims
The scope of the invention.
Fig. 1：Transgene carrier pBac-IE1DsRed2-tetO-tTA schematic diagrames；DsRed is denoted as the red of molecular labeling
Color fluorescin DsRed；TetOx7 represents tetracycline binding sequence；E1-E5 represents 5 extron knots of doublesex genes
Structure；TAV represents tetracycline repressible trans-activator coded sequence, and it is located in the 3rd extron of doublesex genes
Portion.
Fig. 2：Transgenic bombyx mori positive individuals；Fig. 2A：Silkworm seed under normal visual field；Silkworm under Fig. 2 B red fluorescences visual field
Ovum.
Fig. 3：Dead female individuals and the male of normal development；Left side is dead individuality, and color is deeper；Right side
It is the individuality of normal development, color is shallower.
Specific embodiment
The present inventor turns base there is provided a kind of with regulation and control insect sex function first by in-depth study extensively
Because of construction.Specifically, described construction includes：Promoter, doublesex genes and terminator, also hinder including tetracycline
Hold back trans-activator encoder element（TAV）, and the element is inserted in the 3rd extron of doublesex genes.
Described construction specifically expressing tetracycline repressible trans-activator in female insects individuality, causes female individuals in development
Deaths and male is unaffected；The expression of toxic protein is controlled by adding food tetracycline in feed, so as to build
A kind of insects other transgenic system of controllable is found.The present invention is completed on this basis.
Term
As used herein, term " promoter " or " promoter region (domain) " refer to a kind of accurate and effective initial gene transcription work(
The nucleotide sequence of energy, guiding gene nucleotide sequence is transcribed into mRNA, its upstream (5 ' for being typically found in genes of interest coded sequence
End), usually, promoter or promoter region provide RNA polymerase and the correctly knowledge of other factors necessary to starting transcription
Other site.
Herein, the promoter or promoter region (domain) include the variant of promoter, and promoter variants can pass through
Regulatory region is deleted in insertion, carries out random or rite-directed mutagenesis etc. to obtain.
As used herein, " external source " or " heterologous " refers to the two or more pieces nucleic acid or protein sequence of separate sources
Between relation.For example, if promoter is not usually naturally occurring with the combination of objective gene sequence, promoter for
It is external source for the genes of interest.Particular sequence is " external source " for its cell for being inserted or for organism.
As used herein, " cis-regulating element " refers to the guarantor played regulatory role to the transcription initiation and transcriptional efficiency of gene
Keeping property base sequence.
Promoter of the invention operationally can be connected with foreign gene, and the foreign gene is for promoter
Can be external source (heterologous).
Foreign gene (also referred to as genes of interest) of the present invention preferably doublesex genes, with promoter or termination
Son is operably connected.Described doublesex genes come preferably from lepidopterous insects（Such as silkworm）, and with SEQ ID
NO：Sequence shown in 1.
As used herein, term " lepidopterous insects " has no particular limits, including but not limited to：Silkworm, Asia is beautiful
Rice snout moth's larva, bollworm, diamondback moth.
Doublesex genes（Double property genes）
As used herein, term " doublesex genes " can be with used interchangeably with " double property genes ".Doublesex genes
It is the most downstream gene in insect Sex Determination Mechanism, is played an important role in sex development.
The sequence for encoding silkworm doublesex gene open reading frames is made up of 5 extrons, wherein the 3rd and the 4th is outer aobvious
Son is expressed in female, and is not expressed in male, so as to the alternative splicing due to sex-specific in different sexes is made
The albumen different with coding is ultimately resulted in（See Suzuki etal, Dev Genes Evol.2003）.
In a preference of the invention, the nucleotide sequence such as SEQ ID NO. of doublesex genes:Shown in 1.Should
Understand, described doublesex genes also include SEQ ID NO.:The active fragment of nucleotide sequence shown in 1, variant, or class
Like thing.
Described active fragment, variant, or the like can be SEQ ID NO.:Nucleotide sequence shown in 1 have 50% or
More than (preferably more than 60%, more than 70%, more than 80%, more preferably more than 90%, more preferably more than 95%, most preferably more than 98%, such as
99%) nucleotides with same or similar function of homology.Nucleotide variants can by several (usually 1-60,
Preferably 1-30 is individual, more preferably 1-20, most preferably 1-10) replace, lack or add spreading out obtained by least one nucleotides
Raw sequence, and 5 ' ends and/or 3 ' ends add one or several (usually within 20, within preferably 10,
More preferably within 5) nucleotides.Nucleotide variants include substitution variants, Deletion variants and insert variation.Such as this
Known to field, allelic variant is an alternative forms for polynucleotides, it be probably one or more nucleotides substitution,
Missing is inserted, but will not be from the function of substantially changing the nucleotides.
According to nucleotide sequence as herein described, those skilled in the art easily can be obtained this hair with various known methods
Bright code nucleic acid.These methods are such as, but not limited to：PCR, DNA are artificial synthesized etc., and specific method can be found in J. Pehanorm cloth
Lu Ke,《Molecular Cloning:A Laboratory guide》.As one embodiment of the present invention, can again be entered by subsection synthesis nucleotide sequence
The method of row Overlap extension PCR builds described nucleotide sequence.
Alternative splicing extron
Alternative splicing extron refers to third and fourth extron of doublesex genes（SEQ ID NO:647 of 1-
904）.The extron of doublesex genes has different cut modes in different sexes insect, in male insects,
Third and fourth extron of doublesex genes can be cut away, and female insects can retain.Tetracycline repressible trans-activation egg
White encoder element（TAV）If be inserted into doublesex genes the 3rd or the 4th extron, in male insects, TAV can be with
Doublesex genes the 3rd or the 4th extron are selectively cut off, and TAV is not expressed, in female insects, doublesex
Gene the 3rd or the 4th extron are not sheared, TAV expression.
Tetracycline binding sequence（TetO）With tetracycline repressible trans-activator encoder element（TAV）Regulating and controlling effect,
One of ordinary skill in the art can obtain the technical method and knowledge from conventional route, such as（Hinrichs et al,
Science,1994）.
Transgenosis construct
The invention provides a kind of transgenosis construct, described construction has following elements：Doublesex genes
（Double property genes）With tetracycline repressible trans-activator encoder element（TAV）, and the tetracycline repressible trans-activation egg
White encoder element（TAV）It is inserted in doublesex genes（Double property genes）Alternative splicing extron in.
Preferably, the tetracycline repressible trans-activator encoder element（TAV）It is inserted in doublesex genes（It is double
Property gene）The 3rd extron in.
Described construction can also include tetracycline binding sequence（TetO）；Described tetracycline binding sequence（TetO）
With SEQ ID NO：Sequence shown in 3.
In another preference, described construction also includes the element being selected from the group or its combination：Promoter, termination
Son, poly (A) element, transhipment element, gene target element, riddled basins, enhancer, resistant gene, transposase coding
Gene.
The preferred fluorescin encoding gene of described riddled basins, more preferably gfp (green fluorescent protein encoding genes
Or DsRed red fluorescent protein encoding genes.
In another preference, described tetracycline repressible trans-activator encoder element has SEQ IDNO：Shown in 2
Sequence.
Carrier, host cell
Present invention also offers a kind of carrier, the carrier contains transgenosis construct of the invention.Preferably, the load
Body is selected from：Bacterial plasmid, bacteriophage, yeast plasmid or zooblast carrier, shuttle vector；Described carrier is carried for transposons
Body.Method for Prepare restructuring carrier is well known to those of ordinary skill in the art.As long as it can be answered in host
System and stabilization, any plasmid and carrier all can be adopted.
Those of ordinary skill in the art method can be built containing promoter of the present invention and/or mesh known to
Gene order expression vector.These methods are including recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..
Present invention also offers a kind of host cell, described host cell contains described construction or carrier, or institute
The host cell chromosome stated is integrated with described construction or carrier.In another preference, described host cell is also wrapped
Include and be integrated with transposase gene on the carrier containing encoding transposase gene or its chromosome.
Preferably, described host cell is insect cell.In another preference, described insect is Lepidoptera elder brother
Worm, including（But it is not limited to）：Silkworm, Ostrinia furnacalis, bollworm, diamondback moth etc..
Construction of the invention or carrier, can be used for the appropriate host cell of conversion.Host cell can be that protokaryon is thin
Born of the same parents, such as Escherichia coli, streptomyces, Agrobacterium：Or low eukaryotic, such as yeast cells；Or higher animal cells, such as
Insect cell.Persons skilled in the art are aware that how to select appropriate carrier and host cell.Place is converted with recombinant DNA
Chief cell can be carried out with routine techniques well known to those skilled in the art.When host is prokaryotes (such as Escherichia coli), can
With with CaCl2Method treatment, it is also possible to which electroporation is carried out.When host is eucaryote, following DNA transfection methods are can select：
Calcium phosphate precipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging).Plant also can be used for conversion
The method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as leaf disk method, rataria conversion method, bud infusion method etc..
Using
Present invention also offers a kind of method of prepare transgenosis insect, including step：Conversion of the present invention is provided
Host cell（It is preferred that silkworm primiparity egg cell）；Described host cell is regenerated as insect, so as to obtain described transgenosis
Insect.Described insect is preferably lepidopterous insects.
Present invention also offers a kind of a kind of transgenic insect for preparing in aforementioned manners.And described transgenic insect
Can be used for：Production silk；Improve silk cocoon Cocoon layer ratio；Improve conversion rate of silk leaves；Improve silk fiber number；Improve silk cleanliness；Or
Its combination.
Present invention also offers one kind insects method for distinguishing of regulation, including step：Obtain transgenosis of the present invention
Insect；Described insect is fed under conditions of tetracycline is lacked, male insects survival ratio is obtained and is more than 95%, preferably greater than
96%, more preferably greater than 98% group of insects.In another preference, described regulation insect sex is：Male insects are improved to exist
Ratio in total insect.
In a specific embodiment of present aspect, by transgene carrier of the invention and the auxiliary matter that transposase can be expressed
Proembryo phase silkworm seed is imported by microinjection after grain mixing, protection is extremely hatched under the conditions of 25 DEG C.Newly-hatched silkworm after hatching raise into
After worm, selfing or backcrossing obtain G1 for silkworm seed, in G1 for Late Embryogenesis（6-8 days）Whole body is filtered out under fluorescence microscope
Express the trans genie individual of red fluorescence.
G1 is raised to adult for trans genie individual and is obtained G2 for transgenosis heterozygous individual with normal individual backcrossing.In G2 generations, turn
Genetic heterozygosis are individual to be raised using two ways：1. tetracycline is added in feed, and female and male can normal developments.2.
Without tetracycline in feed, female individuals are in larva Deaths, and male development is unaffected.
Main advantages of the present invention are：
（1）By setting up transgenic insect strain, specifically expressing tetracycline repressible is trans in female individuals swashs the present invention
Living protein, male is unaffected in mesoderm growing early stage death to cause female individuals；
（2）The expression of toxic protein is controlled by adding food tetracycline in feed, is a kind of regulatable transgenosis system
System, is easy to set up pure and mild holding strain；
（3）The present invention develops the lethal transgenic bombyx mori strain of female using transgenic approach first, and female is lethal
Mechanism of action is clear；
（4）The present invention is breached to be wasted time and energy by traditional breeding way seed selection in the past, and the inexplicit bottle of mechanism of action
Neck；
（5）The present invention can not only play an important role in sericultural production, also extend to insect particularly Lepidoptera
The genetic control of insect, for control of insect provides new approach.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) described in condition, or according to the condition proposed by manufacturer.
Embodiment 1
The structure of the special lethal transgenic system of silkworm female
The structure of 1.pBac-IE1DsRed2-tetO-tTA transgene carriers
The pBac-IE1DsRed2-tetO-tTA transgene carriers that the present embodiment is used are to be based on being widely used in insect turn
The Piggybac transposons of gene studies（Piggybac transposons is shown in Fraser et al., Insect Molecular
Biology,1996）Transformed what is completed.
Imported by IE1 promoters in Piggybac transposon vectors first（Kojima et al,
VirusResearch,2008）The color fluorescin DsRed of driving（DsRed gene orders such as SEQ ID NO：Shown in 4）, build
As pBac-IE1DsRed2 transgene carriers.After being successfully transferred to this carrier, transgenic positive individuality can be complete from late embryogenesis
Body expresses red fluorescence, is easy to screening.
Doublesex gene of the clone from silkworm（Doublesex gene orders such as SEQ ID NO：Shown in 1）, and will
The DNA sequence dna of coding tetracycline repressible trans-activator TAV（The DNA sequence dna of TAV such as SEQ IDNO：Shown in 2）Insertion
In 3rd extron of doublesex genes（3rd sequence of extron such as SEQID NO：Shown in 5）, insertion point is
Between 3rd the 44th of extron the-the 45, a fusion is constituted（Fusion gene sequence is SEQ ID NO：
6）.
Due to the special gene selectable splicing of sex（Doublesex genes are expressed in male not need outside the 3rd
Show the intervention of son）, transgenic female individuality is by expressed intact TAV, and male is not expressed then.
Tetracycline binding sequence TetO has been imported in the upstream of TAV and doublesex fusions（The sequence of TetO is such as
SEQ ID NO：Shown in 3）, finally construct purpose transgene carrier pBac-IE1DsRed2-tetO-tTA（Fig. 1）.
Embodiment 2
The foundation of transgenic system
Transgene carrier pBac-IE1DsRed2-tetO-tTA prepared by embodiment 1 with can express Piggybac swivel bases
The plasmid PHA3PIG of enzyme（The plasmid is shown in Tamura et al., Nature Biotechnology, 2000）Noted after mixed in equal amounts
Laid eggs at the beginning of being mapped to silkworm（Give birth to latter 4-8 hours）In.
The purifying of injection plasmid uses the Plasmid Midi kit kits of Qiagen companies, the injecting method ginseng of silkworm seed
Method according to Kanda ＆ Tamura (1991) descriptions carries out microinjection, and the silkworm seed after injection is sealed with nontoxic quick-drying gelatin.Injection
Silkworm seed afterwards is placed in moisturizing box, is hastened the hatching of silkworms under the conditions of 25 DEG C until hatching.
Collect the newly-hatched silkworm mulberry leaf supreme cluster of raising after hatching to cocoon, under the conditions of 25 DEG C of pupa time after protection to change moth, by the present age
（G0）Silkworm moth selfing after, obtain G1 for silkworm seed.G1 for silkworm seed Late Embryogenesis using fluorescence microscope under choose tool
The trans genie individual for having red fluorescence is raised（Fig. 2）.
768 silkworm eggs of inventor's co-injection（G0 generations）, hatched 492, final development to the normal post-coitum of adult
The ovum circle for obtaining has 95（G1 generations）.Detecting 12 ovum circles in the development later stage of these ovum has positive individuals（It is red glimmering
Light）.By further confirmation, the experiment that wherein three strains implement next step is selected, in these three independent lines
The result for arriving has repeatability very high.
Embodiment 3
The identification of female-specific lethal effect
Authentication method：
Female-specific lethal effect can be identified as follows：Spray concentration is 200 μ g/ μ l on mulberry leaf
The tetracycline aqueous solution, feeding transgenic bombyx mori, with this understanding transgenic bombyx mori no matter male and female can normal development；And if
Without tetracycline feeding transgenic bombyx mori on mulberry leaf, then the silkworm for having half or so can juvenile stage death again（Fig. 3）.And it is dead
The larva for dying does not amplify male specific band through PCR detections, can determine whether to be female.
And as control, the non-transgenic silkworm addition 200 μ g/ μ l tetracyclines aqueous solution or no added tetracycline in feeding
Development is had no adverse effects after the aqueous solution.
The all documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, those skilled in the art can
Made various changes or modifications with to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (21)
1. a kind of transgenosis construct, it is characterised in that described construction has following elements：Doublesex genes (double property
Gene) and tetracycline repressible trans-activator encoder element (TAV), and tetracycline repressible trans-activator coding
Element (TAV) is inserted in the alternative splicing extron of doublesex genes (double property genes)；
Wherein, the doublesex genes come from silkworm；
The tetracycline repressible trans-activator encoder element (TAV) is inserted in the of doublesex genes (double property genes)
In three extrons, insertion point is the sequence of the 3rd extron between the 3rd the 44th of extron the-the 45
Such as SEQ ID NO：Shown in 5.
2. construction as claimed in claim 1, it is characterised in that described construction also include the element being selected from the group or its
Combination：Promoter, terminator, poly (A) element, transhipment element, gene target element, riddled basins, enhancer, resistance
Gene, swivel base enzyme coding gene.
3. construction as claimed in claim 1, it is characterised in that described construction also includes tetracycline binding sequence
(TetO)。
4. construction as claimed in claim 1, it is characterised in that described construction has SEQ ID NO：Sequence shown in 6
Row.
5. construction as claimed in claim 2, it is characterised in that described riddled basins are that fluorescin encodes base
Cause.
6. construction as claimed in claim 1, it is characterised in that described doublesex genes (double property genes) have SEQ
ID NO：Sequence shown in 1.
7. construction as claimed in claim 1, it is characterised in that described tetracycline repressible trans-activator encoder element
With SEQ ID NO：Sequence shown in 2.
8. construction as claimed in claim 5, it is characterised in that described riddled basins are gfp green fluorescent proteins
Encoding gene or DsRed red fluorescent protein encoding genes.
9. construction as claimed in claim 1, it is characterised in that described tetracycline binding sequence (TetO) has SEQ ID
NO：Sequence shown in 3.
10. a kind of carrier, it is characterised in that the carrier contains any described transgenosis constructs of claim 1-9.
11. a kind of host cells, it is characterised in that described host cell contains any described constructions of claim 1-9
Or the carrier described in claim 10, or described host cell chromosome is integrated with any described structures of claim 1-9
Carrier described in thing or claim 10.
12. host cells as claimed in claim 11, it is characterised in that described host cell also includes containing coding swivel base
Transposase gene is integrated with the carrier of enzyme gene or its chromosome.
13. host cells as claimed in claim 11, it is characterised in that described host cell is insect cell.
14. host cells as claimed in claim 13, it is characterised in that described insect is lepidopterous insects.
Construction described in 15. claims 1, the carrier described in claim 10, or the host cell described in claim 11
Purposes, it is characterised in that be used for insect sex phenotype, or for preparing the insects other composition of regulation and control.
A kind of 16. methods of prepare transgenosis insect, it is characterised in that including step：
A () provides insect cell or insect ovum, the insect cell or insect ovum contain any described structures of claim 1-9
Carrier or its chromosomal integration described in thing or claim 10 have the right any described constructions of requirement 1-9 or right will
Seek the carrier described in 10；
B insect cell described in step (a) or insect ovum are regenerated as insect by (), so as to obtain described transgenic insect.
The method of 17. prepare transgenosis insects as claimed in claim 16, it is characterised in that described insect is Lepidoptera elder brother
Worm.
The method of 18. prepare transgenosis insects as claimed in claim 16, it is characterised in that the host described in step (a) is thin
Born of the same parents are silkworm primiparity egg cell.
A kind of 19. purposes of transgenic insect, the transgenic insect is silkworm and described transgenic insect containing having the right
It is required that the host cell described in 11, or described transgenic insect chromosomal integration is had the right any described structures of requirement 1-9
Carrier described in thing or claim 10, or described transgenic insect is as the host cell regeneration described in claim 11
Into, it is characterised in that described transgenic insect is used for：
(1) silk is produced；
(2) silk cocoon Cocoon layer ratio is improved；
(3) conversion rate of silk leaves is improved；
(4) silk fiber number is improved；
(5) silk cleanliness is improved；
(6) or its combination.
The purposes of 20. transgenic insects as claimed in claim 19, it is characterised in that described transgenic insect is used for：
Production silk.
21. a kind of insects method for distinguishing of regulation, it is characterised in that including step：
A () provides insect cell or insect ovum, the insect cell or insect ovum contain any described structures of claim 1-9
Construction or claim 10 institute that carrier or its chromosomal integration described in thing or claim 10 are had the right described in requirement 1
The carrier stated, and described insect cell or insect ovum are regenerated as insect；
B the insect of () under conditions of tetracycline is lacked described in feeding step (a), obtains male insects survival ratio and is more than 95%
Group of insects.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210559254.8A CN103882013B (en) | 2012-12-20 | 2012-12-20 | Insect gender modulating construct, modulating method, and application of the construct |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210559254.8A CN103882013B (en) | 2012-12-20 | 2012-12-20 | Insect gender modulating construct, modulating method, and application of the construct |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103882013A CN103882013A (en) | 2014-06-25 |
| CN103882013B true CN103882013B (en) | 2017-05-24 |
Family
ID=50951137
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210559254.8A Active CN103882013B (en) | 2012-12-20 | 2012-12-20 | Insect gender modulating construct, modulating method, and application of the construct |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103882013B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114807144B (en) * | 2022-05-30 | 2023-05-12 | 河南大学 | Asiatic corn borer double gene mutant and construction method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2404382B (en) * | 2003-07-28 | 2008-01-30 | Oxitec Ltd | Pest control |
| CN1561712A (en) * | 2004-04-21 | 2005-01-12 | 浙江省农业科学院 | Method for cultivating ZW type lepidopteraus pest linkage balanced lethal system using molecule genetic label |
| WO2007091099A1 (en) * | 2006-02-10 | 2007-08-16 | Oxitec Limited | Gene expression system using alternative splicing in insects |
| CN102559691B (en) * | 2011-12-31 | 2013-08-28 | 浙江省农业科学院 | Silkworm sex chromosome linked gene PdpI and recessive lethal mutation gene thereof and application of silkworm sex chromosome linked gene PdpI and recessive lethal mutation gene in silkworm sex ratio control |
-
2012
- 2012-12-20 CN CN201210559254.8A patent/CN103882013B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori;Masataka G. Suzuki et al.;《Insect Biochemistry and Molecular Biology》;20011101;第31卷;1201-1211 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103882013A (en) | 2014-06-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1860235B (en) | Expression systems for insect pest control | |
| CN107426986A (en) | A kind of method for cultivating male sterility culex | |
| CN104673815A (en) | Compound type piggyBac recombinant vector as well as preparation method and application of compound type piggyBac recombinant vector | |
| CN111850039B (en) | Silkworm fibroin heavy chain expression system with expressed proteins distributed on silkworm silk glue layer, and preparation method and application thereof | |
| CN107075529A (en) | Gene expression system | |
| CN107619836B (en) | System for reducing activity 20E concentration in spinning period, changing silk pupa nutrition distribution proportion and increasing silkworm cocoon yield, application and method | |
| KR101510437B1 (en) | Transgenic silkworms producing red fluorescent cocoons | |
| KR20150000155A (en) | Transgenic silkworms producing yellow fluorescent cocoons | |
| CN103882013B (en) | Insect gender modulating construct, modulating method, and application of the construct | |
| CN105400817B (en) | Utilize the method for the silkworm simultaneously synthesizing traction of secretion latrodectus mactans silk-fibroin 1 and albumen 2 | |
| CN102851297B (en) | Myzuspersicae hunchback gene cDNA and application thereof | |
| CN106701792A (en) | Artificially synthesized insecticidal gene with high toxicity on lepidoptera pests and application | |
| JP5997772B2 (en) | Mutated sequences of silkworm fibroin heavy chain gene, and methods and applications for inducing mutations | |
| CN107058324B (en) | Rice root specific expression promoter POsRO4 and corresponding rice cultivation method | |
| CN106978416A (en) | A kind of assignment of genes gene mapping integrant expression system and its application | |
| CN102559691B (en) | Silkworm sex chromosome linked gene PdpI and recessive lethal mutation gene thereof and application of silkworm sex chromosome linked gene PdpI and recessive lethal mutation gene in silkworm sex ratio control | |
| CN106148346B (en) | A kind of endosperm isolated does not express promoter SAFES6 and its application | |
| KR101570783B1 (en) | Transgenic silkworms producing antimicrobial peptide | |
| KR101634275B1 (en) | Transgenic silkworms producing blue fluorescent cocoons | |
| CN105463022B (en) | The method for synthesizing secretion latrodectus mactans traction silk-fibroin 2 using domestic natural silk gland bioreactor | |
| CN105063088B (en) | A kind of method of production of ecological safety type fluorescent transgenic ornamental fish | |
| KR101480153B1 (en) | Transgenic silkworms producing cocoons containing melittins antibiotic peptides | |
| US20120054880A1 (en) | Method for enhancing production of pathogen-resistant proteins using bioreactor | |
| CN105969801A (en) | Bombyx mori middle silkgland bioreactor universal plasmid for expressing T4 ligase as well as application and method of universal plasmid | |
| CN108753777A (en) | A kind of promoter and its application with anther tissue specificity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200506 Address after: 200031 building 4, No. 300 Fenglin Road, Xuhui District, Shanghai Patentee after: Center for excellence and innovation in molecular plant science, Chinese Academy of Sciences Address before: 200031 Yueyang Road, Shanghai, No. 319, No. Patentee before: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES |
|
| TR01 | Transfer of patent right |