CN107619836A - A kind of system and application and method for reducing spinning phase activity 20E concentration and changing silk pupa nutrient distribution ratio increase cocoon yield - Google Patents
A kind of system and application and method for reducing spinning phase activity 20E concentration and changing silk pupa nutrient distribution ratio increase cocoon yield Download PDFInfo
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Abstract
The present invention relates to a kind of system and application and method for reducing spinning phase activity 20E concentration and changing silk pupa nutrient distribution ratio increase cocoon yield, foreign protein EGT is expressed in last age silkworm by Gal4/UAS double element systems and extends house silkworms spin silk phase increase silk yield, GAL4 genes are started by BmLP3 and expressed in the system, UAS regulation and control EGT expression, using GAL4/UAS systems using silkworm fat-body as bioreactor, to express the albumen EGT for making 20E inactivate in the silkworm fat-body of the promoter of silkworm LP3 genes phase of being cleverly in that silkworms spin silk, silkworm is prevented to pupate metamorphosis in silkworm hemolymph by the protein secretion to spinning phase, successfully obtain the extension of spinning phase, the increased silkworm material of cocoon yield, thinking is provided to reduce sericulture cost.
Description
Technical field
The invention belongs to genetic engineering field, and in particular to one kind reduces spinning phase activity 20E concentration and changes silk pupa nutrition
The system that allocation proportion increases cocoon yield, further relate to the systematic difference and and application and reduction spinning phase activity 20E concentration
Change the method for silk pupa nutrient distribution ratio increase cocoon yield.
Background technology
Silkworm has important economic value due to its unique spun silk characteristic, so how to make what the silkworm larva phase accumulated
Matter and energy is as much as possible to be distributed towards silk cocoon direction, and silk production is improved on the basis of breed silkworms early stage is not increased in cycle
Amount, is all the topic that related practitioner is keen to the most all the time.But at present by traditional breeding method to Kosé amount cultivated silkworm breed variety
Seed selection has nearly reached the limit.China is sericulture big country of the world, produces silk cocoon few hundred thousand tonnes of per year, and modern molecular biology technique turns into
Further improve family's important means that silkworms spin silk measures.
Silkworm is a kind of complete metamorphosis, and a life cycle will undergo ovum, larva, pupa, moth four in morphosis
With the stage of development entirely different in function.Pupa time is the abnormal stage that silkworm linal-instar larvae is transitioned into adult, wherein last age is young
Worm later stage to prepupal period in this stage is that silkworm carries out nutrition and dispensed materials are the most key once：Linal-instar larvae is told after stopping eating
Silk is cocoond, and the transformation from larva to pupa is completed in cocoon layer, by the silk cocoon of energy and dispensed materials to protection itself and continuation
In the pupa developed backward.It is assigned to the matter and energy in cocoon and in pupa and then shows obvious hereditary capacity, cocoon weight and pupa weight
Between high-positive correlation (coefficient correlation 0.85, generally with Cocoon layer ratio (cocoon weight/(cocoon weight+pupa weight) × 100%) weigh).
Having studied confirms that lepidopterous insects linal-instar larvae-pupa metamorphosis process is in moulting hormone (20-
Hydroxyecdysone, 20E) control under complete.Family silkworms spin silk terminate after prepupa appearance, be in 20E precision controls
Lower completion, the spinning phase 20E concentration before in fact silkworm prepupa occurs is not high, when internal 20E concentration enters very
Spinning terminates after high level, and now silkworm hemolymph middle and high concentration 20E promotes the larva histoorgan drop including sericterium
Solution, while the formation of pupa time histoorgan is induced, promote silkworm to pupate.Husking steroidal uridine diphosphoglucose transferase
UDPG glycosides can be transferred to moulting hormone by (ecdysteroid UDP glucosyltransferase, EGT)
On the hydroxyl of 22nd carbon atom of (20E), inactive 20E compounds -20E-22- β-D- glucopyranosides are formed.
When insect is infected by this viroid, the EGT of this viral secretory can inactivate the 20E in insect bodies, hinder insect just
Often husking and emergence.
Lethal effect is poisoned because EGT has to silkworm in itself, so no matter what mode directly to express EGT eggs using
In vain, conservation work can not be all carried out, is utilized in production.The GAL4/UAS double element systems built using the present invention, make cause
Dead gene is preserved；The system contains two transgenosis systems of UAS and GAL4.In UAS- target genes system, in the absence of transcription
Activator protein, target gene are in silence state；In GAL4 transgenosis systems, transcription activating protein be present, but there is no regulating and controlling sequence
With the target gene being attached thereto, only GAL4 transgenosis systems and UAS transgenosis systems are hybridized, expression target gene could be produced
Filial generation.Therefore, even if target gene is lethal gene, without GAL4 activation, UAS transgenosis systems remain to survive.Utilize GAL4/
UAS double bases crossing system can build period and tissue specificity GAL4 transgenosis systems, so as to form a GAL4 transgenosis system
Storehouse, it is miscellaneous by the GAL4 transgenosis systems and the progress of UAS- target genes transgenosis system that select that there is period and tissue specificity in storehouse
Hand over the specific expressed of can regulation and control target gene.In addition EGT reduces active 20E characteristic, after spinning silkworm also have purify and
Further utilize EGT possibility.
BmLP3 is protein of the last age silkworm later stage to pupa fat-body at initial stage specifically expressing, studies have reported that it has
Tissue and period specific promoter sequence, the tissue of sequence driving red fluorescent protein expression and period and endogenous
LP3 albumen is consistent.And successfully express active foreign protein in five latter stage in age silkworm fat-bodies using it --- phytase.
The content of the invention
In view of this, an object of the present invention is that providing a kind of spinning phase activity 20E concentration that reduces changes silk pupa battalion
The system that nutrient formula example increases cocoon yield, change system and tieed up the 20E of the phase of spinning always using LP3 driving EGT protein expressions
Hold in a relatively low level, the time retardation nutrient distribution of silkworms spin silk by extending house phase has additional nutrients to the time point of pupa
The time of silk cocoon is flowed to, and then improves silkworm cocoon yield；The third purpose is that providing described system is improving silkworm silk cocoon
Application in yield；The third object of the present invention, which is to provide, utilizes described system to prepare reduction spinning phase activity 20E concentration
Change the method for silk pupa nutrient distribution ratio increase cocoon yield.
Family's silkworms spin silk phase moulting hormone content is reduced to extend the spinning phase of silkworm using foreign protein, and then increases silk cocoon
Yield.
For achieving the above object, the present invention provides following technical scheme：
1st, a kind of system for reducing spinning phase activity 20E concentration and changing silk pupa nutrient distribution ratio increase cocoon yield, institute
To state system and include GAL4/UAS double element systems, GAL4 genes start expression by BmLP3 in the system, and UAS regulation and control EGT is expressed,
The nucleotide sequence of the BmLP3 is as shown in SEQ ID NO.1, the nucleotide sequence such as SEQ ID NO.2 institutes of the EGT genes
Show, the nucleotide sequence of the GAL4 genes is as shown in SEQ ID NO.3, the nucleotide sequence such as SEQ ID of the UAS
Shown in NO.4.
Preferably, the GAL4/UAS double element systems are designated as recombinating transgene carrier A respectively respectively on two carriers
Contain the expression cassette for starting GAL4 gene expressions by BmLP3 with restructuring transgene carrier B, the restructuring transgene carrier A；It is described
Restructuring transgene carrier B contains by the expression cassette of UAS regulation and control EGT expression.
Preferably, the restructuring transgene carrier A is prepared by following methods：Using cultivated silkworm breed variety dazao genomes as template,
LP3 promoter sequences are expanded, and are connected at the Sal I/BamH I restriction enzyme sites of pSL1180 plasmids, obtain pSL1180 [Lp3-
SV40] carrier, the GAL4 nucleotide sequences of synthesis are connected into the BamH I/Not I digestions position of pSL1180 [Lp3-SV40] carrier
At point, pSL1180 [Lp3-GAL4-SV40] intermediate carrier is obtained.It will further be carried among pSL1180 [Lp3-GAL4-SV40]
Constitution grain and pBac [3xP3-EGFP] the plasmids single endonuclease digestions of Asc I, the table for starting GAL4 gene expressions containing BmLP3 is separately recovered
Up to frame and carrier framework, connection obtains restructuring transgene carrier A, is named as pBac [3xP3-EGFP, BmLP3-GAL4-SV40].
Preferably, the restructuring transgene carrier B is prepared by following methods：With bombyx mori nuclear polyhydrosis virus BmNPV bases
Because group is template, expands EGT genes and be connected at the EcoR I/Xho I restriction enzyme sites of pSL1180 plasmids, obtain intermediate carrier
PSL1180 [EGT-SV40], synthesize UAS nucleotide sequences and be connected into intermediate carrier pSL1180 [EGT-SV40] I/EcoR of Asc
At I restriction enzyme sites, recombinant vector pSL1180 [UAS-EGT-SV40] is obtained, then by recombinant vector pSL1180 [UAS-EGT-
SV40] and pBac [3xP3-DsRed] plasmid use the single endonuclease digestions of Asc I respectively, be separately recovered containing UAS regulation and control EGT expression expression
Frame and carrier framework, connection obtain restructuring transgene carrier B, are named as pBac [3xP3-DsRed, UAS-EGT-SV40].
2nd, being prepared using described system, which reduces spinning phase activity 20E concentration, changes silk pupa nutrient distribution ratio increase silk cocoon
The method of yield, comprises the following steps：The restructuring transgene carrier A and auxiliary matter of GAL4 gene expressions will be started containing BmLP3
The silkworm seed of termination of diapause is injected after grain mixing, positive transgenic silkworm is screened after hatching, transgenic bombyx mori GAL4 systems are made；
Then the silkworm of termination of diapause is injected after the restructuring transgene carrier B containing UAS regulation and control EGT expression is mixed with helper plasmid
Ovum, positive transgenic silkworm is screened after hatching, transgenic bombyx mori UAS-EGT systems are made；Again by transgenic bombyx mori GAL4 systems
With transgenic bombyx mori UAS-EGT systematic crosses, the silkworm seed simultaneously containing GAL4 and EGT genes is screened, that is, obtains and passes through external source egg
The white transgenic bombyx mori material for reducing house silkworms spin silk phase moulting hormone content silkworms spin silk extending house phase increase cocoon yield.
Further, it is described to start the restructuring transgene carrier of GAL4 gene expressions by pSL1180 [Lp3- containing BmLP3
GAL4-SV40] plasmid and pBac [3xP3-EGFP] the plasmids single endonuclease digestions of Asc I, it is separately recovered and starts GAL4 bases containing BmLP3
Because of the expression cassette and carrier framework of expression, it is made through connecting, pSL1180 [Lp3-GAL4-SV40] plasmid is started by LP3
Subsequence is simultaneously connected at the Sal I/BamH I restriction enzyme sites of pSL1180 plasmids, obtains pSL1180 [Lp3-SV40] carrier, then will
Obtained at the BamH I/Not I restriction enzyme sites of GAL4 genes pSL1180 [Lp3-SV40] carrier.
Further, the restructuring transgene carrier B containing UAS regulation and control EGT expression, pSL1180 is connected into by EGT genes
At the EcoR I/Xho I restriction enzyme sites of plasmid, intermediate carrier pSL1180 [EGT-SV40] is obtained, then connects UAS fragments again
Enter at intermediate carrier pSL1180 [EGT-SV40] Asc I/EcoR I restriction enzyme sites, obtain recombinant vector pSL1180 [UAS-
EGT-SV40], then recombinant vector pSL1180 [UAS-EGT-SV40] and pBac [3xP3-DsRed] plasmid are used into Asc I respectively
Single endonuclease digestion, expression cassette and carrier framework containing UAS regulation and control EGT expression is separately recovered, is most made afterwards through connecting.
Further, silkworm seed of the screening simultaneously containing GAL4 and EGT genes can detect that red and green is glimmering simultaneously for screening
The egg-incubation of light.
Further, the helper plasmid is pHA3PIG plasmids.
The beneficial effects of the present invention are：The invention discloses one kind by GAL4/UAS systems using transgenic technology with
Silkworm fat-body is bioreactor, is cleverly expressed with the promoter of silkworm LP3 genes in the silkworm fat-body of spinning phase
Albumen-the EGT for making 20E inactivate.Activity in hemolymph is reduced in silkworm hemolymph by the protein secretion to spinning phase
20E contents, to extend the spinning phase of silkworm, the increased transgenic bombyx mori material of cocoon yield is successfully obtained, to improve silk cocoon production
Amount reduces sericulture cost and provides thinking.And the present invention is successfully bioreactor using silkworm fat-body, expresses one kind
There is the albumen-EGT of universal toxicity to lepidopterous insects, to be that insecticide improves silkworm macroeconomic valency subsequently by EGT exploitations
Value is laid a good foundation.
Brief description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carried out
Explanation：
Fig. 1 is transgene carrier schematic diagram (A：PBac [3xP3-EGFP, BmLP3-GAL4-SV40] carrier schematic diagram；B：
PBac [3xP3-DsRed, UAS-EGT-SV40] carrier schematic diagram；C：PBac [3xP3-EGFP, BmLP3-GAL4-SV40] is carried
Constitution grain Asc I single endonuclease digestion electrophoresis detections；D：PBac [3xP3-DsRed, UAS-EGT-SV40] vector plasmid Asc I single endonuclease digestions
Electrophoresis detection).
Fig. 2 is positive transgenic screening figure (A1/A2：G0 is for BmLP3-GAL4 transgenic bombyx mori moth fluoroscopic examinations；A3/A4：
G0 is for UAS-EGT transgenic bombyx mori fluoroscopic examinations；B：Silkworm seed fluorescence is examined after the hybridization of BmLP3-GAL4 and UAS-EGT transgenic bombyx moris
Survey).
Fig. 3 is that the correlated traits such as transgenic hybrid strain Cocoon layer ratio, rate of being placed on small straw bundles to spin cocoons investigate (A：Silkworm linal-instar larvae eats Sang Tian
Number；B：Spin duration (spinning phase)；C：Cocoon layer ratio；D：It is placed on small straw bundles to spin cocoons rate).
Fig. 4 is preferred transgenic hybrid product phenotype (A：Cocoon outward appearance；B：Cocoon section；C：15th day table after filial generation cocoons
Type；D：45th day phenotype after filial generation cocoons).
Fig. 5 is transgenic hybrid strain EGT detection of expression figures (A：GAL4/EGT detection of expression in fat-body after being placed on small straw bundles to spin cocoons；B：
EGT Protein Detections in hemolymph after spinning).
Fig. 6 is preliminary assessment figure (As of the transgenic hybrid strain EGT to lepidopterous insects toxicity：Feeding contains EGT silkworm chrysalises
The silkworm death phenotype of powder；B：Feeding contains the silkworm death time statistics of EGT dried silkworm chrysalis meals；C：Feeding contains EGT dried silkworm chrysalis meals
Silkworm mortality statistics).
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
UAS fragments containing I/EcoR of Asc I and the GAL4 pieces containing BamH I/Not I in following examples of the present invention
Section is synthesized by Jin Sirui companies, Basic plasmid pSL1180, pBac [3xP3-EGFP], pBac [3xP3-DsRed] and for turning base
Because the helper plasmid-pHA3PIG of injection is preserved by this laboratory.Duovoltine kind for transgenosis injection is made greatly by southwest
University's domestic silkworm gene resources bank provides.
The acquisition of embodiment 1, silkworm transgene carrier
First, pBac [3xP3-EGFP, BmLP3-GAL4-SV40] vector construction
A.LP3 nucleic acid sequence of promoter obtains
Using cultivated silkworm breed variety dazao genomes as template, with forward primer 5 '-gcgtcgacagtatagtta caacggc-
3 ' (underscore is Sal I restriction enzyme site) (SEQ ID NO.14), and reverse primer 5 '-cgggatccaaagtgtgctatat
Gattc-3 ' (underscore is BamH I restriction enzyme site) (SEQ ID NO.15) expands LP3 promoter sequences, sequence amplification
Condition：94 DEG C of pre-degenerations 5 minutes；94 DEG C are denatured 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 60 seconds, circulate 30 times；72 DEG C of ends prolong
Stretch 10 minutes, 4 DEG C of preservations.Pcr amplification product carries out electrophoresis with 1% Ago-Gel, after EB is dyed, under ultraviolet light
The adhesive tape containing purpose fragment is cut with blade, purpose fragment is reclaimed using glue reclaim kit, through LP3 nucleotide sequences are sequenced such as
Shown in SEQ ID NO.1.
B.pSL1180 [LP3-GAL4-SV40] intermediate carrier is built
Step A is reclaimed into gained purpose fragment and pSL1180 plasmids carry out double digestion with Sal I/BamH I respectively, is reclaimed
Target gene fragment and carrier segments, carrier construction pSL1180 [LP3-SV40] after connection conversion.Further by pSL1180
(GAL4 gene orders are such as [LP3-SV40] plasmid and the GAL4 fragments containing BamH I/Not I restriction enzyme sites of company's synthesis
Shown in SEQ ID NO.3) double digestion is carried out with BamH I/Not I respectively, reclaim target gene fragment and carrier segments, connection
Intermediate carrier pSL1180 [LP3-GAL4-SV40] is built after conversion.
C.pBac [3xP3-EGFP, LP3-GAL4-SV40] transgene carrier is built
By pSL1180 [Lp3-GAL4-SV40] plasmids and pBac [3xP3-EGFP] the plasmids single endonuclease digestions of Asc I, purpose piece
Conversion is attached after section and carrier segments recovery, builds transgene carrier pBac [3xP3-EGFP, BmLP3-GAL4-SV40],
And carry out the single endonuclease digestions of Asc I checking (A and C in such as Fig. 1).
2nd, pBac [3xP3-DsRed, UAS-EGT-SV40] vector construction
A.EGT gene orders obtain
According to total length CDS (coding domain sequence) sequence of EGT genes, using the softwares of primer 5.0 to sequence
Design forward and reverse amplimer, BmEGT-F：5’-ccggaattcatgactattctttgctggct-3’(SEQ ID NO.5)
(underscore is EcoRI restriction enzyme site), BmEGT-R：
(SEQ ID NO.6) (dash area is Myc labels, and underscore is XhoI restriction enzyme site).Enter using BmNPV genomes as template
Performing PCR expands, the condition of sequence amplification：94 DEG C of pre-degenerations 4 minutes；94 DEG C are denatured 40 seconds, and 56 DEG C are annealed 30 seconds, 72 DEG C of extensions 1
Divide 30 seconds, circulate 25 times；72 DEG C extend 10 minutes eventually, 4 DEG C of preservations.Pcr amplification product carries out electricity with 1% Ago-Gel
Swimming, after EB is dyed, cuts the adhesive tape containing purpose fragment with blade under ultraviolet light, and purpose piece is reclaimed using glue reclaim kit
Section, through EGT gene orders are sequenced as shown in SEQ ID NO.2.
B.pSL1180 [UAS-EGT-SV40] intermediate carrier is built
Step A is reclaimed into gained purpose fragment and pSL1180 plasmids Plasmids carry out double digestion with EcoR I/Xho I respectively,
Reclaim target gene fragment and carrier segments, carrier construction pSL1180 [EGT-SV40] after connection conversion.Further will
PSL1180 [EGT-SV40] plasmids and the UAS fragments containing I/EcoR of Asc I of company's synthesis use I/EcoR of Asc I respectively
Double digestion is carried out, reclaims target gene fragment and carrier segments, carrier construction pSL1180 [UAS-EGT- after connection conversion
SV40], and sequence verification is carried out, its sequence is as shown in SEQ ID NO.7, wherein UAS gene orders such as SEQ ID NO.4 institutes
Show.
C.pBac [3xP3-DsRed, UAS-EGT-SV40] transgene carrier is built
By pSL1180 [UAS-EGT-SV40] plasmids and pBac [3xP3-DsRed] the plasmids single endonuclease digestions of Asc I, purpose piece
Conversion is attached after section and carrier segments recovery, builds transgene carrier pBac [3xP3-DsRed, UAS-EGT-SV40], and
Carry out the single endonuclease digestions of Asc I checking (B and D in such as Fig. 1).
The acquisition of embodiment 2, transgenic bombyx mori
With pHA3PIG (Tamura et al.2000) for helper plasmid, respectively with pBac [3xP3-EGFP, BmLP3-
GAL4-SV40] and pBac [3xP3-DsRed, UAS-EGT-SV40] plasmid with 1:Microinjection has released after 1 mixed in molar ratio
The big of diapause makes body early embryo (2~5h after spawning), and the silkworm seed after injection is sealed with nontoxic dehydration, and 25 DEG C are hastened the hatching of silkworms to hatching, are incubated
Larva (G0 generations) conventinal breeding of change, to adult after examined under macroscopical Stereo fluorescence microscope (Olypus MVX10, Japan)
Survey, filter out individual (A in Fig. 2), the production of hybrid seeds that produces the special transgenic positive for exciting fluorescence in eyes, the G1 of acquisition for silkworm seed,
The positive silkworm seed containing fluorescence is selected, continues raising and obtains BmLP3-Gal4 and UAS-EGT transgenosis systems.Two groups of acquisition are turned
Gene line silkworm hybrid, the ovum produced to it carry out fluorescent screening (B in Fig. 2), red and green fluorescence by can detect simultaneously
Egg-incubation, conventinal breeding, spinning phase, Cocoon layer ratio etc. are counted to after being placed on small straw bundles to spin cocoons, detect GAL4 and EGT expression, analysis
The validity of Gal4/UAS systems, EGT validity is analyzed, assess the transgenic bombyx mori " toxicity " containing EGT after spinning.
As a result show, after silkworm seed (B in Fig. 2) hatching of green and red fluorescence is detected simultaneously by after hybridization, conventinal breeding
To five ages (last age), compared with the control, although there are a certain degree of extension (A in Fig. 3), spinning the food mulberry time of hybridization silkworm
Phase (B in Fig. 3), Cocoon layer ratio (C in Fig. 3) are significantly increased.Further investigation discovery EGT expression may be placed on small straw bundles to spin cocoons to silkworm one
It is fixing to ring (D in Fig. 3), select Cocoon layer ratio, spinning phase and rate preferably transgenosis system E4G relatively of being placed on small straw bundles to spin cocoons further to investigate, E4G
Silk cocoon is obvious (A in Fig. 4) bigger than normal, and the silk cocoon of the 7th day is splitted after cocooing, silkworm development situation after observation spinning：It can be seen that
The silkworm of control group has pupated, and experimental group silkworm is still in the prepupa state (B in Fig. 4) after spinning.Experimental group is spun
Silkworm afterwards takes out room temperature from silk cocoon and places 15 days (C in Fig. 4) and 45 days (D in Fig. 4), changes until thirsting and not observing
Pupa phenomenon, illustrate successfully to add cocoon yield and prevent silkworm to pupate.
Embodiment 3, transgenic hybrid strain EGT expressions
Using the positive individuals after hybridization as material, take be placed on small straw bundles to spin cocoons beginning (W0) and after being placed on small straw bundles to spin cocoons 120h (W120) fat-body group
Knit, it is standby to be put into -80 DEG C of refrigerators after instantaneously being cooled down in liquid nitrogen.The drawn materials total serum IgE of extracting, reverse transcription synthesis cDNA, leads to
Crossing RT-PCR detections GAL4, (primer sequence is：SEQ ID NO：8 and SEQ ID NO：9) and EGT (primer sequence is：SEQ ID
NO：10 and SEQ ID NO：11) expression of mRNA level in-site (is used as control, primer sequence using Actin3 in silkworm fat-body
It is classified as：SEQ ID NO：12 and SEQ ID NO：13).Reaction condition is：94 DEG C of pre-degeneration 4min；94 DEG C denaturation 15s, 55 DEG C
Anneal 30s, 72 DEG C of extension 1min, totally 25 circulations；Last 72 DEG C of extensions 10min, PCR products are using 1% Ago-Gel electricity
Swimming detection, as a result as shown in Figure 5.As a result show, LP3 promoters can correctly start GAL4 in the silkworm fat-body after being placed on small straw bundles to spin cocoons
Expression, and EGT gene expression (A in Fig. 5) is detected in the silkworm fat-body after only hybridizing.Illustrate Gal4/UAS systems
Worked in the silkworm united successfully after hybridization.4 DEG C of the silkworm hemolymph of 120h after spinning further is taken, is taken after 12000g centrifugations
Supernatant, determine total protein concentration after these protein samples are mixed with 5x sample loading buffers, 100 DEG C be denatured 10 minutes after be splined on
10%SDS- polyacrylamides glue carries out electrophoresis, and then detecting EGT albumen by Western blot, (EGT antibody is by this experiment
Room prepares and preserved).Make P50 greatly with non-transgenic and non-hybridized transgenosis is made greatly as control, the results showed that in protein level
On, detect and obtain in the same silkworm hemolymphs only after hybridization of EGT, there is no signal (B in Fig. 5) in other samples, say again
It is due to that hybridization makes Gal4/UAS systems work that silkworm after bright spinning, which stops development, and the EGT of fat-body expression is secreted into blood strangury
Caused by Palestine and China play a role.
Embodiment 4, the transgenic hybrid strain containing EGT are to the preliminary assessment of lepidopterous insects toxicity
In addition to extending spinning phase increase cocoon yield, another object of the present invention is to be used as life by the use of silkworm fat-body
Thing reactor expresses useful proteins, and in order to assess EGT utilizability, 120h silkworm is ground by liquid nitrogen flash freezer after cocooing
Cheng Fenhou is freeze-dried, and the silkworm equally processing for making 120h after cocooing greatly with blank and normal non-transgenic is control, with 0.1g
The amount morning and evening feeding of freeze-dried powder/g silkworms has just enter into the silkworm in five ages, and mortality statistics finds the family that feeding contains EGT dried silkworm chrysalis meals
Silkworm the 4th day and the 5th day significantly raised (Fig. 6) after food is added, illustrate that the hybridization silkworm after spinning possesses what subsequent development utilized
Value.Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although by upper
State preferred embodiment the present invention is described in detail, it is to be understood by those skilled in the art that can be in form
Various changes are made to it in upper and details, without departing from claims of the present invention limited range.
Sequence table
<110>Southwest University
<120>It is a kind of reduce spinning phase activity 20E concentration change silk pupa nutrient distribution ratio increase cocoon yield system and
Using and method
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1124
<212> DNA
<213>Silkworm (Bombyx mori)
<400> 1
agtatagtta caacggctgc cccacccttc aaaccgaaac gcattactgc ttcacggcag 60
aaataggcag ggaggtggta tctaaccgtg cgaatccata cgaaaatatt aatattagca 120
aaacaaatat acctttcaca tggcatgttt taaactacca cgaacaatgt gaatttttaa 180
atgtgtccat taaaattaca catttaaatt ataatgttga cggcttgata atttcactca 240
atcaataata aactatatct ttatttcaat gcactttcat ttgacatttg aactatgatg 300
ttgatattgc atctgacgtt ttttaattca aaacaatttc gagtataaaa ggcagagttt 360
caaaggaaac aggcagttcg ttcttgggta acacacaggt gagatacatt ttgtttttaa 420
ctctggagaa tccgtttcgg atacccagtc gtggggggta acagaccggg ttatgtcaga 480
cttcggttcc tccaaggaag agggagaccg aggtcctcct cttcttctaa ttcctgtcga 540
gagtctacgt cttgagatat ctacctacca cacaaaaacg ttttcttcta tttagcgttt 600
gttaaattgt aagagtttga gaaaccaatt ggccgatatt tcgacctctg gcattttttt 660
catcactccg ctgacttttc ttattctttt tattgcttag atgggtgaac gagctcacag 720
cccacctggt gttaagtggt taccggagcc catagacatt tacaacgtaa atgccccacc 780
caccttgaaa tttaaggtct aagatctcaa gtataggtac ttcttgtact ggtctccaaa 840
caccgatcgt atgaattttg tatcagtgga tataaaatta tacactaaga tgtttatgtg 900
tctaagcttt caaagaagtc aaatatataa tatacttttt tatttaacaa ttaatttgtc 960
aagttcgttt ttgctatata ctcacaaaat ctgcgaccgt tttgtctcat atatacatca 1020
aatatacata ttatgttcaa ttctcaatgt gtataattca acttacgttt ttaaaattct 1080
aatccttaac aaataatttt acatattgca ggactcgact cgac 1124
<210> 2
<211> 1518
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
atgactattc tttgctggct tgcactgctg tctacgctta ctgctgtaaa tgcggtcaat 60
atattggccg tgtttcctac gccagcttac agccaccata tagtctacaa agtgtatatt 120
gaagcccttg ccgaaaaatg tcacaacgtt acggtcgtca agcccaaact gtttgcgtat 180
tcgaccaaaa cttattgcgg taatattacg gaagttaatt ccgacatgtc ggtcaagcaa 240
tacaagaaac tagtaacgaa ttcggcaatg tttagaaagc gcggagtggt gtccgataca 300
gacacggtaa ccgccgccaa ctacctgggc ttgattgaaa tgttcaaaga ccagtttgac 360
aatatcaacg tgcgcaatct cattgccaac aaccagacgt ttgatttagt tgtcgtggaa 420
gcgtttgccg attatgcgtt ggtgtttggc cacctgtacg atcctgcgcc cgtaatccaa 480
atcgcgcctg gctacggttt ggcggaaaac tttgacacgg taggcgccgt ggcgcggcac 540
cccgttcacc atcctaacat ttggcgcaac aatttcgacg acacgaaggc gaacttgatg 600
acggaaatgc gtttgtataa agaatttaaa attttggcca acatgtccaa tgcgttgctc 660
aaacagcagt ttggacccga cacaccgaca attgaagaac tgcgcaacaa ggtgcaattg 720
cttttgctga acctacatcc catatttgac aacaaccgac ccgtgtcgcc cagcgtccag 780
tatcttggcg gaggaatcca tcttgtaaaa agtgcgccgt tgaccaaatt aagtccggtc 840
atcgacgcga aaatgaacaa gtcaaaaagc ggagcgattt acgtaagttt tgggtcgagc 900
attgacacca aatcgtttgc aaacgagttt ttttacatgt taatcaatac gcttaaagcg 960
ttggataatt acaccatatt atggaaaatt gacgacgaag tagtaaaaaa cataacgttg 1020
cccgccaacg tgatcacgca aaattggttt aatcaacgcg ccgtgttgcg tcataaaaaa 1080
atggcggcgt ttattacgca aggcggacta caatcgagcg acgaggcctt ggaagccgga 1140
atacccatgg tgtgtctgcc catgatgggc gaccagtttt accatgcgca caaattacag 1200
caactcggcg tagcccgcgc cttggacact gttaccgttt ccagcgatca actattactg 1260
gcgataaacg acgtgttgtt taacgcgtct acatacaaaa aacacatggc cgagttatat 1320
gcgcttatca ataacgataa agcaacgttt ccgcctctag ataaggccat caaatttaca 1380
gaacgcgtaa ttcgatatag acatgacatc agtcgtcgat tgtattcatt aaaaacaaca 1440
gctgccaatg taccgtactc aaattactac atgtataaat ctgtactttc tattgtaatg 1500
aatcatatag cacacttt 1518
<210> 3
<211> 2646
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 3
atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag 60
tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac 120
tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg 180
ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt 240
ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat 300
aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta 360
acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt 420
caaagacagt tgactgtatc gattgactcg gcagctcatc atgataactc cacaattccg 480
ttggatttta tgcccaggga tgctcttcat ggatttgatt ggtctgaaga ggatgacatg 540
tcggatggct tgcccttcct gaaaacggac cccaacaata atgggttctt tggcgacggt 600
tctctcttat gtattcttcg atctattggc tttaaaccgg aaaattacac gaactctaac 660
gttaacaggc tcccgaccat gattacggat agatacacgt tggcttctag atccacaaca 720
tcccgtttac ttcaaagtta tctcaataat tttcacccct actgccctat cgtgcactca 780
ccgacgctaa tgatgttgta taataaccag attgaaatcg cgtcgaagga tcaatggcaa 840
atccttttta actgcatatt agccattgga gcctggtgta tagaggggga atctactgat 900
atagatgttt tttactatca aaatgctaaa tctcatttga cgagcaaggt cttcgagtca 960
ggttccataa ttttggtgac agccctacat cttctgtcgc gatatacaca gtggaggcag 1020
aaaacaaata ctagctataa ttttcacagc ttttccataa gaatggccat atcattgggc 1080
ttgaataggg acctcccctc gtccttcagt gatagcagca ttctggaaca aagacgccga 1140
atttggtggt ctgtctactc ttgggagatc caattgtccc tgctttatgg tcgatccatc 1200
cagctttctc agaatacaat ctccttccct tcttctgtcg acgatgtgca gcgtaccaca 1260
acaggtccca ccatatatca tggcatcatt gaaacagcaa ggctcttaca agttttcaca 1320
aaaatctatg aactagacaa aacagtaact gcagaaaaaa gtcctatatg tgcaaaaaaa 1380
tgcttgatga tttgtaatga gattgaggag gtttcgagac aggcaccaaa gtttttacaa 1440
atggatattt ccaccaccgc tctaaccaat ttgttgaagg aacacccttg gctatccttt 1500
acaagattcg aactgaagtg gaaacagttg tctcttatca tttatgtatt aagagatttt 1560
ttcactaatt ttacccagaa aaagtcacaa ctagaacagg atcaaaatga tcatcaaagt 1620
tatgaagtta aacgatgctc catcatgtta agcgatgcag cacaaagaac tgttatgtct 1680
gtaagtagct atatggacaa tcataatgtc accccatatt ttgcctggaa ttgttcttat 1740
tacttgttca atgcagtcct agtacccata aagactctac tctcaaactc aaaatcgaat 1800
gctgagaata acgagaccgc acaattatta caacaaatta acactgttct gatgctatta 1860
aaaaaactgg ccacttttaa aatccagact tgtgaaaaat acattcaagt actggaagag 1920
gtatgtgcgc cgtttctgtt atcacagtgt gcaatcccat taccgcatat cagttataac 1980
aatagtaatg gtagcgccat taaaaatatt gtcggttctg caactatcgc ccaataccct 2040
actcttccgg aggaaaatgt caacaatatc agtgttaaat atgtttctcc tggctcagta 2100
gggccttcac ctgtgccatt gaaatcagga gcaagtttca gtgatctagt caagctgtta 2160
tctaaccgtc caccctctcg taactctcca gtgacaatac caagaagcac accttcgcat 2220
cgctcagtca cgccttttct agggcaacag caacagctgc aatcattagt gccactgacc 2280
ccgtctgctt tgtttggtgg cgccaatttt aatcaaagtg ggaatattgc tgatagctca 2340
ttgtccttca ctttcactaa cagtagcaac ggtccgaacc tcataacaac tcaaacaaat 2400
tctcaagcgc tttcacaacc aattgcctcc tctaacgttc atgataactt catgaataat 2460
gaaatcacgg ctagtaaaat tgatgatggt aataattcaa aaccactgtc acctggttgg 2520
acggaccaaa ctgcgtataa cgcgtttgga atcactacag ggatgtttaa taccactaca 2580
atggatgatg tatataacta tctattcgat gatgaagata ccccaccaaa cccaaaaaaa 2640
gagtaa 2646
<210> 4
<211> 367
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 4
gtcggagtac tgtcctccga gcggagtact gtcctccgag cggagtactg tcctccgagc 60
ggagtactgt cctccgagcg gagtactgtc ctccgagcgg agactctagc gagcgccgga 120
gtataaatag aggcgcttcg tctacggagc gacaattcaa ttcaaacaag caaagtgaac 180
acgtcgctaa gcgaaagcta agcaaataaa caagcgcagc tgaacaagct aaacaatctg 240
cagtaaagtg caagttaaag tgaatcaatt aaaagtaacc agcaaccaag taaatcaact 300
gcaactactg aaatctgcca agaagtaatt attgaataca agaagagaac tctgaatagg 360
gaattgg 367
<210> 5
<211> 29
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 5
ccggaattca tgactattct ttgctggct 29
<210> 6
<211> 59
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 6
ccgctcgagc tacagatcct cttctgagat gagtttttgt tcaaagtgtg ctatatgat 59
<210> 7
<211> 1938
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 7
ggcgcgccgt cggagtactg tcctccgagc ggagtactgt cctccgagcg gagtactgtc 60
ctccgagcgg agtactgtcc tccgagcgga gtactgtcct ccgagcggag actctagcga 120
gcgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt caaacaagca 180
aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg aacaagctaa 240
acaatctgca gtaaagtgca agttaaagtg aatcaattaa aagtaaccag caaccaagta 300
aatcaactgc aactactgaa atctgccaag aagtaattat tgaatacaag aagagaactc 360
tgaataggga attgggaatt catgactatt ctttgctggc ttgcactgct gtctacgctt 420
actgctgtaa atgcggtcaa tatattggcc gtgtttccta cgccagctta cagccaccat 480
atagtctaca aagtgtatat tgaagccctt gccgaaaaat gtcacaacgt tacggtcgtc 540
aagcccaaac tgtttgcgta ttcgaccaaa acttattgcg gtaatattac ggaagttaat 600
tccgacatgt cggtcaagca atacaagaaa ctagtaacga attcggcaat gtttagaaag 660
cgcggagtgg tgtccgatac agacacggta accgccgcca actacctggg cttgattgaa 720
atgttcaaag accagtttga caatatcaac gtgcgcaatc tcattgccaa caaccagacg 780
tttgatttag ttgtcgtgga agcgtttgcc gattatgcgt tggtgtttgg ccacctgtac 840
gatcctgcgc ccgtaatcca aatcgcgcct ggctacggtt tggcggaaaa ctttgacacg 900
gtaggcgccg tggcgcggca ccccgttcac catcctaaca tttggcgcaa caatttcgac 960
gacacgaagg cgaacttgat gacggaaatg cgtttgtata aagaatttaa aattttggcc 1020
aacatgtcca atgcgttgct caaacagcag tttggacccg acacaccgac aattgaagaa 1080
ctgcgcaaca aggtgcaatt gcttttgctg aacctacatc ccatatttga caacaaccga 1140
cccgtgtcgc ccagcgtcca gtatcttggc ggaggaatcc atcttgtaaa aagtgcgccg 1200
ttgaccaaat taagtccggt catcgacgcg aaaatgaaca agtcaaaaag cggagcgatt 1260
tacgtaagtt ttgggtcgag cattgacacc aaatcgtttg caaacgagtt tttttacatg 1320
ttaatcaata cgcttaaagc gttggataat tacaccatat tatggaaaat tgacgacgaa 1380
gtagtaaaaa acataacgtt gcccgccaac gtgatcacgc aaaattggtt taatcaacgc 1440
gccgtgttgc gtcataaaaa aatggcggcg tttattacgc aaggcggact acaatcgagc 1500
gacgaggcct tggaagccgg aatacccatg gtgtgtctgc ccatgatggg cgaccagttt 1560
taccatgcgc acaaattaca gcaactcggc gtagcccgcg ccttggacac tgttaccgtt 1620
tccagcgatc aactattact ggcgataaac gacgtgttgt ttaacgcgtc tacatacaaa 1680
aaacacatgg ccgagttata tgcgcttatc aataacgata aagcaacgtt tccgcctcta 1740
gataaggcca tcaaatttac agaacgcgta attcgatata gacatgacat cagtcgtcga 1800
ttgtattcat taaaaacaac agctgccaat gtaccgtact caaattacta catgtataaa 1860
tctgtacttt ctattgtaat gaatcatata gcacactttg aacaaaaact catctcagaa 1920
gaggatctgt agctcgag 1938
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 8
gcaatcccat taccgcatat c 21
<210> 9
<211> 22
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 9
aggttcggac cgttgctact gt 22
<210> 10
<211> 18
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 10
ccagcgtcca gtatcttg 18
<210> 11
<211> 19
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 11
cgacgactga tgtcatgtc 19
<210> 12
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 12
aacaccccgt cctgctcact g 21
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 13
gggcgagacg tgtgatttcc t 21
<210> 14
<211> 25
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 14
gcgtcgacag tatagttaca acggc 25
<210> 15
<211> 27
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 15
cgggatccaa agtgtgctat atgattc 27
Claims (10)
1. a kind of system for reducing spinning phase activity 20E concentration and changing silk pupa nutrient distribution ratio increase cocoon yield, its feature
It is：The system includes GAL4/UAS double element systems, and GAL4 genes start expression, UAS regulation and control by BmLP3 in the system
EGT is expressed, and the nucleotide sequence of the BmLP3 is as shown in SEQ ID NO.1, the nucleotide sequence such as SEQ ID of the EGT genes
Shown in NO.2, the nucleotide sequence of the GAL4 genes is as shown in SEQ ID NO.3, the nucleotide sequence such as SEQ of the UAS
Shown in ID NO.4.
2. system according to claim 1, it is characterised in that：The GAL4/UAS double element systems are located at two carriers respectively
On, it is designated as recombinating transgene carrier A respectively and recombinates transgene carrier B, the restructuring transgene carrier A contains is opened by BmLP3
The expression cassette of dynamic GAL4 gene expressions；The restructuring transgene carrier B contains by the expression cassette of UAS regulation and control EGT expression.
3. system according to claim 2, it is characterised in that：The restructuring transgene carrier A is prepared by following methods：With
Cultivated silkworm breed variety dazao genomes are template, expand LP3 promoter sequences, and be connected into the Sal I/BamH I enzymes of pSL1180 plasmids
At enzyme site, pSL1180 [Lp3-SV40] carrier is obtained, the GAL4 nucleotide sequences of synthesis are connected into pSL1180 [Lp3-SV40] carries
At the BamH I/Not I restriction enzyme sites of body, pSL1180 [Lp3-GAL4-SV40] intermediate carrier is obtained, further by pSL1180
[Lp3-GAL4-SV40] intermediate carrier plasmid and pBac [3xP3-EGFP] the plasmids single endonuclease digestions of Asc I, be separately recovered containing
BmLP3 starts the expression cassette and carrier framework of GAL4 gene expressions, and connection obtains restructuring transgene carrier A, is named as pBac
[3xP3-EGFP, BmLP3-GAL4-SV40].
4. system according to claim 2, it is characterised in that：The restructuring transgene carrier B is prepared by following methods：Expand
Increase EGT genes and be connected at the EcoR I/Xho I restriction enzyme sites of pSL1180 plasmids, obtain intermediate carrier pSL1180 [EGT],
Then UAS fragments are connected at intermediate carrier pSL1180 [EGT] Asc I/EcoR I restriction enzyme sites again, obtain recombinant vector
PSL1180 [UAS-EGT], then recombinant vector pSL1180 [UAS-EGT] and pBac [3xP3-DsRed] plasmid are used into Asc respectively
I single endonuclease digestion, expression cassette and carrier framework containing UAS regulation and control EGT expression being separately recovered, connection obtains restructuring transgene carrier B,
It is named as pBac [3xP3-DsRed, UAS-EGT].
5. application of the system in silkworm cocoon yield is improved described in any one of Claims 1 to 4.
6. being prepared using the system described in any one of Claims 1 to 44, which reduces spinning phase activity 20E concentration, changes silk pupa nutrition point
Method with ratio increase cocoon yield, it is characterised in that comprise the following steps：BmLP3 will be contained and start GAL4 gene expressions
Restructuring transgene carrier mixed with helper plasmid after inject the silkworm seed of termination of diapause, positive transgenic man is screened after hatching
Silkworm, transgenic bombyx mori GAL4 systems are made；Then by restructuring transgene carrier and helper plasmid containing UAS regulation and control EGT expression
The silkworm seed of termination of diapause is injected after mixing, positive transgenic silkworm is screened after hatching, transgenic bombyx mori UAS-EGT systems are made
System；Transgenic bombyx mori GAL4 systems and transgenic bombyx mori UAS-EGT systematic crosses, screening are contained into GAL4 and EGT bases simultaneously again
The silkworm seed of cause, that is, obtaining reduces family's silkworms spin silk phase moulting hormone content by foreign protein silkworms spin silk extending house that the phase increases silk cocoon
The transgenic bombyx mori material of yield.
7. according to the method for claim 6, it is characterised in that：The restructuring for starting GAL4 gene expressions containing BmLP3
Transgene carrier is by pSL1180 [Lp3-GAL4-SV40] plasmids and pBac [3xP3-EGFP] the plasmids single endonuclease digestions of Asc I, difference
The expression cassette and carrier framework for starting GAL4 gene expressions containing BmLP3 are reclaimed, is made through connecting, the pSL1180 [Lp3-
GAL4-SV40] plasmid by LP3 promoter sequences and is connected into the Sal I/BamH I restriction enzyme sites of pSL1180 plasmids, obtains
PSL1180 [Lp3-SV40] carrier, then the BamH I/Not I restriction enzyme sites by GAL4 genes pSL1180 [Lp3-SV40] carrier
Place and obtain.
8. according to the method for claim 6, it is characterised in that：The restructuring transgenosis containing UAS regulation and control EGT expression carries
Body is by EGT genes and is connected into the EcoR I/Xho I restriction enzyme sites of pSL1180 plasmids, obtains intermediate carrier pSL1180
[EGT], then UAS fragments are connected at intermediate carrier pSL1180 [EGT] Asc I/EcoR I restriction enzyme sites again, weighed
Group carrier pSL1180 [UAS-EGT], then recombinant vector pSL1180 [UAS-EGT] and pBac [3xP3-DsRed] plasmid are distinguished
With the single endonuclease digestions of Asc I, expression cassette and carrier framework containing UAS regulation and control EGT expression is separately recovered, is most made afterwards through connecting.
9. the method according to claim 7 or 8, it is characterised in that：Screening the silkworm seed simultaneously containing GAL4 and EGT genes is
Screening can detect red and green fluorescence egg-incubation simultaneously.
10. according to the method for claim 6, it is characterised in that：The helper plasmid is pHA3PIG plasmids.
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