CN102250932B - Method for controlling domestic silkworm pupal stage growth - Google Patents
Method for controlling domestic silkworm pupal stage growth Download PDFInfo
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Abstract
The invention belongs to the field of genetic engineering, and specifically relates to a method of domestic silkworm transgene for controlling domestic silkworm pupal stage growth through UAS/GAL4 dual system. According to the method, piggyBAC transposon vector pigA3GFP is adopted as a base vector; through genetic engineering operation, GAL4 gene expressed transgenic domestic silkworm PDP-GAL4 system controlled by domestic silkworm cocoon protease gene promoter (PDP) is established; through genetic engineering operation, domestic silkworm baculovirus egt gene or charybdotoxin BmKIT3R gene expressed transgenic domestic silkworm UAS-egt or UAS-BmKIT3R system controlled by UAS promoter is established; the PDP-GAL4 system and the UAS-egt or UAS-BmKIT3R system are hybridized, such that larval stage growth of an F1 generation is normal, and pupal stage of the F1 generation is prolonged for 7 to 10 days. According to the invention, with an UAS/GAL4 dual system, the F1 generation silkworm pupal stage is prolonged by using domestic silkworm baculovirus egt gene or BmKIT3R gene. Flexibilities of silkworm cocoon purchasing and filature technology are improved. Therefore, the invention has important application value.
Description
Technical field
The invention belongs to the genetically engineered field, be specifically related to the method for a kind of transgenic bombyx mori through the growth in pupa time of UAS/GAL4 double element system control silkworm.
Background technology
Domestic silkworm silk protein is efficiently synthetic, grow with abnormal, immune and resistance and sex determination be silkworm most important four big proterties.The abnormal artificial adjustment with growth of silkworm is the important content that silk is produced, and the metamorphosis of artificially regulating silkworm has great effect with the production structure and the production benefit of growing the silk industry.Silkworm is a complete metamorphosis, and pupa time is very short, is merely for 2 weeks, because moth mouth cocoon is not suitable for filature, must before the nymphosis moth, accomplish the purchase and the drying work of bright cocoon in the production.Since the receipts of bright cocoon baking link can not be in time and the high temperature of cocoon drying process to the destruction of silk-protein, cause the quality of raw material cocoon seriously to descend.People hope through metamorphosis of artificial adjusting silkworm and growth, prolong pupa time, alleviate the WP and the intensity of bright cocoon purchase and oven dry, even hope to grow termination pupa time, realize fresh cocoon reeling.Like this, not only can only solve bright cocoon receive baking with pupa time the contradiction between too short, make raising raw silk grade become possibility, but also can practice thrift the required energy of cocoon drying greatly.At present, conventional method also is difficult to realize having pupa time the prolongation of long period, therefore, is necessary the novel method of fundamentally exploring prolongation process in pupa time even ending pupa development.The research of the parties concerned is received aspects such as drying by the fire present situation, the major reform of realization raw silk complete processing at the optimization silk existing silk cocoon of the industrial structure, reform already and is significant.
The Bombyx complete metamorphosis, it is generally acknowledged insect cast off a skin and metamorphosis receives hormone regulating and controlling, its residing growth and development stage depends on the relative titre of neotonin and this two parahormone of moulting hormone in the hemolymph.Existing many results of study show, through human intervention, change the endocrinosity of insect, can think that the growth of regulating insect is with abnormal.From the outside silkworm is applied neotonin (JH) or JHA (JHA) can promote or suppress to cast off a skin, handle time end larva in age (4 age) that has just casted off a skin, can increase by 1 time and sleep and cast off a skin, induce 6 instar larvaes with JHA; Apply the number of times of casting off a skin that moulting hormone (MH) also can change silkworm equally, newly-hatched silkworm is raised with the artificial diet that contain MH (100 mg/kg), can obviously increase the number of times of casting off a skin, and indivedual silkworms can cast off a skin 12 times; Silkworm is applied precocene can induce 3 hibernating worms to occur; Usefulness imidazoles chemical substance KK-42 such as Dedos handle 4 instar larvaes, can induce precocity to pupate; The silkworm in 129-132 hour 5 ages is applied fenoxycarb (fenoxycarb) can be induced and produce permanent pupa (referring to Dedos SG, J Insect Physiol, 2002,48 (9): 857-865).Aforesaid method is all intervened silkworm development metamorphosis through applying certain chemical substance, does not relate to the growth metamorphosis that changes silkworm through genetic manipulation.
Existing more result of study shows that baculovirus has a significant effect with metamorphosis to the just dormancy of insect.Use Autographa californica multicapsid nucleopolyhedrosisvirus baculovirus (AcNPV) inoculation silkworm larva in 4 age, can cause that larvae development delays, give the silkworm pupa injection of pupating 2 AcNPV, can induce the generation of artificial diapause pupa the non-target host of silkworm.The Owain result of study shows, baculovirus
EgtThe coded product of gene for the steroidal uridine diphosphoglucose based transferase of casting off a skin (Ecdysone UDP-glucosyl transferase, EGT), this enzyme glucone in can intravital uridine diphosphoglucose of catalysis insect (UDP-glucosyl) is transferred on the MH; Form moulting hormone 22-O-β-D-Glucopyranose, make host insect excretory MH inactivation, thereby stop the metamorphosis of larva; Prolong getting the food time of larva; Impel the pupa premature ripening (referring to Owain PE, O ' Reilly DR, Biochem J; 1998,330:1265-1270).
Infer that from the mechanism of hormone regulating and controlling insectean metamorphosis the MH level of regulating silkworm can be controlled the growth of silkworm, still; How, the MH level of silkworm is controlled, to delay the growth of larva or pupa through molecular biological method; Improving the production traits, is the problem that need research and solve.
Publication number is that China's invention Shen Qing Publication specification sheets of CN 101418302 discloses a kind of construction process and growth control method of growing controlled silkworm, and this method is with silkworm HSP promotor control silkworm baculovirus
EgtGene makes up transgenic bombyx mori, through to transgenic bombyx mori with 41 ~ 43 ℃ of thermal inductions prolongation in 0.5 ~ 2 hour silkworm development, but the temperature and time during the strict control inductive of this method careless slightlyly will cause detrimentally affect.
Scorpion venom (Scorpion venom) is the poison that is stored in scorpion anal spine poison capsule, is the weapon that the scorpion predation is resist the enemy, and also can be used to treat diseases such as tumour, thrombus.From scorpion venom, separate the anti-insect specific neurotoxin that obtains, act on insect because most ability is single-minded, and have characteristics harmless to Mammals or that toxicity is very little, thereby cause the concern of Chinese scholars.Anti-insect scorpion venom have two big types, and one type is the long-chain toxin, is made up of 60～70 amino acid, acts on Na specifically
+Passage contains 3～4 pairs of disulfide linkage; Another kind of is short chain toxin, specific inhibition K
+Passage.(BmK) there are 3 toxoid gene, i.e. α in Chinese Scorpion Buthus martensii Karsch at least from Scorpio
-Neurotoxin gene, spasm type insect toxins gene and the type insect toxins gene that collapses from physical exhaustion.BmKIT
3 RBe a kind of of Scorpio inhibition type insect toxins gene, the scorpion toxin of this genetic expression has the single-minded optionally neural paralysis of insect lethal effect, the reorganization BmKIT of injection trace
3 RCan cause the silkworm neural paralysis, cause silkworm to collapse from physical exhaustion, and this silkworm that collapses from physical exhaustion is not perishable.Therefore,
BmKIT 3 RCan be used as and end the candidate gene that silkworm chrysalis is grown, through transgenic technology, will
BmKIT 3 RGene imports silkworm, at the abduction delivering in pupa time, might cause silkworm chrysalis self neural paralysis, ends silkworm chrysalis and grows, and prolongs pupa time.
But, how to guarantee
BmKIT 3 RGene is specific expressed in the pupa time of silkworm, is still an open question, does not see the research report of related fields.
Summary of the invention
Goal of the invention of the present invention provides a kind of method of growing in silkworm pupa time of controlling, and prolongs the silkworm time in pupa time, solve bright cocoon receive baking with pupa time the contradiction between too short, increase the handiness of production.
For reaching the foregoing invention purpose, basic ideas of the present invention are: through the GAL4/UAS double element system, at the F1 of silkworm for the specifically expressing in pupa time
EgtGene causes pupal cell MH horizontal down-regulation, or the F1 that makes silkworm is for the specifically expressing in pupa time
BmKIT 3 RGene causes the silkworm chrysalis paralysis developmental arrest of poisoning, thereby automatically prolongs pupa time, and the growth of the larval stage of silkworm is not influenced.
For reaching the foregoing invention purpose, the technical scheme that the present invention adopts is: a kind of method of controlling silkworm growth in pupa time, it is characterized in that, and may further comprise the steps:
(1) operates tame silk cocoon proteinase gene promotor through genetically engineered
PDPUnder the control
GAL4 gene clones are advanced
PiggyBACTransposon carrier pigA3GFP makes up reorganization transgene carrier A, mixes with helper plasmid, imports and just lays eggs, and the newly-hatched silkworm after the hatching is screened the silkworm that obtains to have fluorescent protein report gene through routine, detects
GFPWith
GAL4 genes, breeding are processed transgenic bombyx mori PDP-GAL4 system; Will through the genetically engineered operation
UASSilkworm baculovirus under the promotor control
EgtGene or scorpion toxin
BmKIT 3 RGene clone is advanced
PiggyBACTransposon carrier pigA3GFP; Make up reorganization transgene carrier B; Mix with helper plasmid, import and just lay eggs, the newly-hatched silkworm after the hatching obtains to have the silkworm of fluorescent protein report gene through the routine screening; Detect foreign gene, transgenic bombyx mori UAS-egt or transgenic bombyx mori UAS-BmKIT are processed in breeding
3 RSystem;
(2) the transgenic bombyx mori PDP-GAL4 system and transgenic bombyx mori UAS-egt system or the transgenic bombyx mori UAS-BmKIT that breeding are processed
3 RSystematic cross, the F1 that obtains silkworm are for larva, and the F1 of gained silkworm grows normal for larval stage, prolong 7-10 days pupa time.
In the technique scheme, said reorganization transgene carrier A is pigA3GFP-ie-neo-PDP-Gal4-PA; Said reorganization transgene carrier B is piggyA3GFP-ie-neo-UAS-IT3R-polyA or pigA3GFP-ie-neo-USA-egt.
Because the technique scheme utilization, the present invention compared with prior art has advantage:
1. the present invention is through the GAL4/UAS double element system, at F1 for the specifically expressing in pupa time
EgtGene cause pupal cell MH horizontal down-regulation, or F1 is for the specifically expressing in pupa time
BmKIT 3 RGene causes the silkworm chrysalis paralysis developmental arrest of poisoning, thereby automatically prolongs pupa time, and the growth of the larval stage of silkworm is not influenced.
2. the present invention is through specific expressed for pupa time at F1
EgtGene or
BmKIT 3 RGene, pupa time, significant prolongation increased the handiness of cocoon purchasing and silk reeling technology.
Description of drawings
Fig. 1 is the qualification result figure of piggyA3GFP-ie-neo-UAS-IT3R-polyA carrier in embodiment one step 2;
Fig. 2 is the qualification result figure of pigA3GFP-ie-neo-PDP-Gal4-PA carrier in embodiment one step 3;
Fig. 3 is the fluorescence silkworm larva in embodiment one step 4;
Fig. 4 is UAS-BmKIT in embodiment one step 4
3 RThe qualification result of system;
Fig. 5 is the fluorescence silkworm chrysalis in embodiment one step 5;
In Fig. 6 embodiment one step 5 in the PDP-GAL4 system
GFPIdentify; M:DNA maker; Swimming lane 1-5: different transgenic bombyx moris individualities
GFPThe PCR product;
Fig. 7 is the fluorescence silkworm chrysalis in embodiment two steps 4;
Fig. 8 is in the UAS-egt system in embodiment two steps 4
EgtQualification result figure.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Embodiment one: tame silk cocoon proteinase gene promotor (
PDP) control
GAL4,
UASControl
BmKIT 3 RThe structure of transgenic double element system
1.
BmKIT3 RThe synthetic of gene
According to disclosed scorpion toxin
BmKITThe sequence of 3 genes with reference to the preferences of silkworm to codon, has been synthesized and has been comprised initiator codon ATG and terminator codon TGA is 189 nt's in interior length
BmKIT3
RGene, concrete nucleotide sequence and corresponding amino acid sequence are respectively shown in SEQ ID No.1 and SEQ ID No.2.The synthetic sequence clone is advanced pUCm-T (Shanghai Shenergy Biocolor BioScience & Technology Company's product), obtain recombinant plasmid pUCm-IT3R, order-checking confirms with the sequence of design in full accord.
2. the structure of effect transgene carrier piggyA3GFP-ie-neo-UAS-IT3R-polyA
(1) pSK-UAS plasmid construction: with the pUAST DNA (referring to Brand and Perimon, Development, 1993,118:401-415) be template, with UAS-1 (SEQ ID No.3:ct
G aat tcG cat gcc tgc agg tcg, underscore does
EcoR I site) and UAS-2 (SEQ ID No.4:tt
G ata tcC aat tcc cta ttc aga g, underscore does
EcoR V site) be primer, pcr amplification goes out
UASFragment (about 350 bp).
EcoThe R I/
EcoR V double digestion rear clone advances pBluescript II SK (+) plasmid (Stratagene Company products)
EcoThe R I/
EcoThe pSK-UAS plasmid is obtained in R V site.
(2) structure of pSK-IT3R-polyA plasmid: with IT3R-1 (SEQ ID No.5:ca
C tcg agA tgg acg gct ata ttc gc, underscore does
XhoThe I site) and IT3R-2 (SEQ ID No.6:ca
G ggc ccT caa ccg cat gta ttg c, underscore does
ApaThe I site) being primer, is masterplate with pUCm-IT3R, and PCR obtains
BmKIT3 RGene fragment,
XhoI with
ApaThe I enzyme is cut the identical restriction enzyme site of rear clone to pSK, obtains the pSK-IT3R plasmid; With YCFib-PA-1 (SEQ ID No.7:gg
G ata tcA aat tgt gtt tgc gtt agg, underscore does
EcoR V site) and YCFib-PA-2 (SEQ ID No.8:gc
G gat ccg gta ccC act gtc caa tcc acc gtc, underscore does
BamThe H I,
KpnThe I site) be primer, amplification obtains bombyx mori silk fibroin light chain gene poly (A) tailing signal sequence from the domestic silkworm gene group,
EcoThe R V with
KpnThe I enzyme is cut rear clone to pSK-IT3R's
ApaI with
KpnThe I site obtains the pSK-IT3R-polyA plasmid.
(3) structure of pSK-UAS-IT3R-polyA plasmid: from the pSK-IT3R-polyA plasmid, use
XhoI with
KpnI cuts out the IT3R-polyA fragment, and the clone advances the pSK-UAS plasmid that same enzyme is cut, and obtains pSK-UAS-IT3R-polyA.
(4) structure of piggyA3GFP-UAS-IT3R-polyA: the pSK-UAS-IT3R-polyA plasmid is used
EcoThe R I/
KpnAfter the I double digestion cut out the UAS-IT3R-polyA fragment, the clone advanced the piggyA3GFP carrier (referring to Cary LC et al., Virology, 1989,172:156 – 69) that same enzyme cuts and obtains the piggyA3GFP-UAS-IT3R-polyA plasmid.
(5) structure of piggyA3GFP-ie-neo-UAS-IT3R-polyA: will use
EcoIE-neo fragment (the about 1.7 kb) clone that the R I cuts out from pSK-IE-Neo (referring to Zhou Wenlin, etc. silkworm industry science, 2007,33 (1): 30 – 35) advances the piggyA3GFP-UAS-IT3R-polyA plasmid
EcoFinal transgene carrier piggyA3GFP-ie-neo-UAS-IT3R-polyA is obtained in R I site.
The piggyA3GFP-ie-neo-UAS-IT3R-polyA carrier is used respectively
EcoThe R I with
EcoThe R V,
EcoThe R I with
XhoI,
XhoI with
KpnThe I double digestion identifies that figure is as shown in Figure 1 as a result, wherein, and M: λ/
HinD Ш standard molecular weight; Swimming lane 2: use
EcoThe R I with
EcoR V enzyme is cut; Swimming lane 3:
EcoThe R I with
XhoThe I double digestion; Swimming lane 4:
XhoI with
KpnThe I enzyme is cut; Fig. 1 can know: band number, molecular weight size that enzyme is cut generation are consistent with theoretical value.
3. activate the structure of transgene carrier pigA3GFP-ie-neo-PDP-Gal4-PA
(1) structure of pSK-Fib-L-polyA plasmid: with TPFib-L-3 (SEQ ID No.9:gg
C tcg agC aaa ttg tgt ttg cgt tag g, underscore does
XhoThe I site), TPFib-L-4 (SEQ ID No.10:gc
G gta ccC act gtc caa tcc acc gtc, underscore does
KpnThe I site) being primer, is template with the domestic silkworm gene group, and pcr amplification goes out the fragment of about 290 bp, through warp
XhoI with
KpnBehind the I double digestion, the carrier that the clone advances the pBluescript II SK (+) that same enzyme cuts obtains the pSK-Fib-L-polyA plasmid.
(2) structure of pSK-Gal4-PA: design primer GAL4-1 (SEQ ID No.11:ag
G ata tcA tga agc tac tgt ctt cta tcg, underscore does
EcoR V restriction enzyme site) and GAL4-2 (SEQ ID No.12:gc
A agc ttG cac agt tga agt gaa ctt gcg g, underscore does
HinD III restriction enzyme site), with pBGT1 (with reference to Lukacsovich T
Et al., Arch Insect Biochem Physiol, 2008,69 (4): be template 168-175), pcr amplification goes out total length
GAL4 genes; With
EcoThe R V with
HinD III double digestion PCR product and clone advance the pSK-Fib-L-polyA carrier
EcoThe R V with
HinThe pSK-Gal4-PA plasmid is obtained in the site of d III.
(3) structure of pSK-PDP-Gal4-PA plasmid: with domestic silkworm gene group DNA is template, with PDP1 (SEQ ID No.13:tt
G gat ccg aat tcT aca tcc ata acc ctg g, underscore does
BamThe H I with
EcoR I restriction enzyme site) and PDP2 (SEQ ID No.14:tt
G ata tcT tca gtt tcg atc cgg cg, underscore does
EcoR V restriction enzyme site) for primer PCR amplify cocoon proteinase gene promotor (
PDP), warp
BamThe H I/
EcoR V double digestion rear clone advances pSK-Gal4-PA's
BamThe H I/
EcoThe pSK-PDP-Gal4-PA plasmid is obtained in R V site.
(4) structure of pigA3GFP-PDP-Gal4-PA plasmid: use
EcoThe R I with
KpnI cuts out the PDP-Gal4-PA fragment from the pSK-PDP-Gal4-PA plasmid, and the clone advances piggyA3GFP's
EcoThe R I with
KpnThe pigA3GFP-PDP-Gal4-PA plasmid is obtained in the I site.
(5) structure of pigA3GFP-ie-neo-PDP-Gal4-PA: will use
EcoThe IE-neo fragment cloning that the R I cuts out from pSK-IE-Neo (referring to Zhou Wenlin, etc. silkworm industry science, 2007,33 (1): 30 – 35) advances pigA3GFP-PDP-Gal4-PA's
EcoFinal carrier pigA3GFP-ie-neo-PDP-Gal4-PA is obtained in R I site.
Fig. 2 is the qualification result figure of pigA3GFP-ie-neo-PDP-Gal4-PA carrier in the step 3, wherein, and M: λ/
HinD Ш standard molecular weight; Swimming lane 2 is used with swimming lane 3:pigA3GFP-ie-neo-PDP-Gal4-PA carrier
EcoThe R I with
HinD Ш double digestion; Swimming lane 4:pigA3GFP-ie-neo-PDP-Gal4-PA carrier is used
EcoThe R I with
BglThe II enzyme is cut; Band number, molecular weight size that enzyme is cut generation are consistent with theoretical value.
4.UAS-BmKIT
3 RThe structure of system and evaluation
(1) UAS-BmKIT
3 RThe structure of system: piggyA3GFP-ie-neo-UAS-IT3R-polyA and helper plasmid are (referring to Tamura T
Et al., Nature Biotechnology, 2000,18:81-84) press 1:1 and mix (melting concn 2 μ g/ μ L), only inject virgin moth mating capsule with the kapillary glass needle with 1.5 ~ 2.0 μ L/, the newly-hatched silkworm (G after the hatching is laid eggs in normal mating
0) add continuously the food 10 000 μ g/mL G418 to 4 sleep age; The combined with fluorescent inverted microscope detects, and filters out anti-G418, has the silkworm of fluorescent protein report gene, keeps to have the silkworm (transgenic bombyx mori that obvious fluorescence shows; Referring to Fig. 3), carry out Molecular Detection and conventional silkworm breeding.
(2) UAS-BmKIT
3 RThe evaluation of system: extract the transgenic bombyx mori genomic dna, with DEGFP-1 (SEQ ID No.15:tg
G aat tcA tgg tga gca agg gcg agg, underscore shows
BamH I site) and DEGFP-2 (SEQ ID No.16:tt
G gat ccT tac ttg tac agc tcg tcc atg, underscore shows respectively
EcoR I site) for primer carries out pcr amplification, but specific amplification goes out
GFPSpecific band; With IT3R-1 (SEQ ID No.5) and IT3R-2 (SEQ ID No.6) is that primer also can amplify from the transgenic bombyx mori genome
BmKIT 3 RGene.
Qualification result such as Fig. 4, Fig. 4 are UAS-BmKIT in the step 4
3 RThe qualification result of system, wherein, M:DNA maker; Swimming lane 1:
BmKIT 3 RThe PCR product; Swimming lane 2:
GFPThe PCR product; The result shows really be transgenic bombyx mori.
5. the structure of PDP-GAL4 system and evaluation
(1) structure of PDP-GAL4 system: pigA3GFP-ie-neo-PDP-Gal4-PA and helper plasmid are pressed 1:1 mixing (melting concn 2 μ g/ μ L); Only inject virgin moth mating capsule with the kapillary glass needle with 1.5 ~ 2.0 μ L/; Newly-hatched silkworm (the G after the hatching is laid eggs in normal mating
0) add continuously the food 10 000 μ g/mL G418 to 4 sleep age; The combined with fluorescent inverted microscope detects, and filters out anti-G418, has the silkworm of fluorescent protein report gene, keeps to have the silkworm (transgenic bombyx mori that obvious fluorescence shows; Referring to Fig. 5), carry out Molecular Detection and conventional silkworm breeding.
(2) evaluation of PDP-GAL4 system: extracting the transgenic bombyx mori genomic dna, be that primer carries out pcr amplification with DEGFP-1 (SEQ ID No.15) and DEGFP-2 (SEQ ID No.16), but specific amplification goes out the specific band of GFP; With GAL4-1 (SEQ ID No.11) and GAL4-2 (SEQ ID No.12) is that primer also can amplify the GAL4 gene from the transgenic bombyx mori genome.Qualification result such as Fig. 6, the GFP qualification result in Fig. 6 step 5 in the PDP-GAL4 system, wherein, M:DNA maker; Swimming lane 1-5: different transgenic bombyx moris individualities
GFPThe PCR product; The result shows really be transgenic bombyx mori.
6. PDP-GAL4 system and UAS-BmKIT
3 RThe systematic cross offspring grows pupa time: PDP-GAL4 system and UAS-BmKIT
3 RSystem's phase mutual cross, first familiar generation is normally raised, and larval stage, grows and normal, pupa time to prolong 7-10 days.
Embodiment two: tame silk cocoon proteinase gene promotor (PDP) control GAL4, UAS control
EgtThe structure of transgenic double element system
Technical operating procedure is following:
1. activate the structure of transgene carrier pigA3GFP-ie-neo-PDP-Gal4-PA: with the step 3 of embodiment one.
2.PDP-GAL4 the structure of system and evaluation: with the step 5 of embodiment one.
3. the structure of effect transgene carrier pigA3GFP-ie-neo-UAS-EGT
(1) structure of pSK-UAS-egt carrier: silkworm baculovirus (BmNPV) genome DNA extraction is undertaken by ordinary method.According to announcing BmNPV-T3 pnca gene group complete sequence (GenBank registration number: L33180) design PCR upstream and downstream primer Egt-1 (SEQ ID No.17:cg
C tcg agA tga cta ttc ttt gct ggc, underscore shows
XhoThe I site) and Egt-2b (SEQ ID No.18:cg
G gta ccT tgg ttg cta ctg gca cat aag cgc, underscore shows
KpnThe I site); With the BmNPV genome is template, carries out pcr amplification, reclaims 1.7 kb's
EgtGene fragment.
KpnI/
XhoBehind the I double digestion, clone the pSK-UAS-IT3R-polyA's of embodiment one step 2
KpnI/XhoI obtains pSK-UAS-egt in the site.
(2) structure of pigA3GFP-ie-neo carrier: use
EcoThe R I cuts out the IE-neo fragment from pSK-IE-Neo (referring to Zhou Wenlin, etc. silkworm industry science, 2,007 33 (1): 30 – 35), the clone advances the piggyA3GFP carrier
EcoThe pigA3GFP-ie-neo carrier is obtained in R I site.
(3) structure of pigA3GFP-ie-neo-UAS-egt:
BamHI/
KpnI double digestion pSK-UAS-egt carrier returns the UAS-egt fragment, and the clone advances pigA3GFP-ie-neo's
BglII with
KpnI obtains pigA3GFP-ie-neo-USA-egt in the site.
4. the structure of UAS-egt system and evaluation
(1) structure of UAS-egt system: pigA3GFP-ie-neo-USA-egt and helper plasmid are pressed 1:1 mixing (melting concn 2 μ g/ μ L); Only inject virgin moth mating capsule with the kapillary glass needle with 1.5 ~ 2.0 μ L/; Newly-hatched silkworm (the G after the hatching is laid eggs in normal mating
0) add continuously the food 10 000 μ g/mL G418 to 4 sleep age; The combined with fluorescent inverted microscope detects, and filters out anti-G418, has the silkworm of fluorescent protein report gene, keeps to have the silkworm (transgenic bombyx mori that obvious fluorescence shows; Referring to Fig. 7), carry out Molecular Detection and conventional silkworm breeding.
(2) evaluation of UAS-egt system: extracting the transgenic bombyx mori genomic dna, be that primer carries out pcr amplification with DEGFP-1 (SEQ ID No.15) and DEGFP-2 (SEQ ID No.16), but specific amplification goes out
GFPSpecific band; With Egt-1 (SEQ ID No.17) and Egt-2b (SEQ ID No.18) is that primer also can amplify from the transgenic bombyx mori genome
EgtGene, qualification result is as shown in Figure 8, and Fig. 8 is in the UAS-egt system in embodiment two steps 4
EgtIdentify, wherein, M:DNA maker; Swimming lane 1-3: different transgenic bombyx moris individualities
EgtThe PCR product.The result shows really be transgenic bombyx mori.
5.PDP-GAL4 system grows with UAS-egt systematic cross offspring pupa time: PDP-GAL4 system and the mutual cross mutually of UAS-egt system, first familiar generation is normally raised, and larval stage, grows and normal, pupa time to prolong 8-10 days.
SEQUENCE LISTING
< 110>University Of Suzhou
< 120>a kind of method of controlling silkworm growth in pupa time
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 189
<212> DNA
< 213>synthetic
<400> 1
atggacggct atattcgcgg cagcaacggc tgcaaagtat cctgcttatg gggcaatgaa 60
ggctgcaata aagaatgcag agcctacggc gcctcttatg gatattgctg gacctgggga 120
cttgcctgct ggtgcgaagg ccttcctgac gacaagacat ggaaatctga aagcaataca 180
tgcggttga 189
<210> 2
<211> 62
<212> PRT
< 213>synthetic
<400> 2
Met Asp Gly Tyr Ile Arg Gly Ser Asn Gly Cys Lys Val Ser Cys Leu
1 5 10 15
Trp Gly Asn Glu Gly Cys Asn Lys Glu Cys Arg Ala Tyr Gly Ala Ser
20 25 30
Tyr Gly Tyr Cys Trp Thr Trp Gly Leu Ala Cys Trp Cys Glu Gly Leu
35 40 45
Pro Asp Asp Lys Thr Trp Lys Ser Glu Ser Asn Thr Cys Gly
50 55 60
<210> 3
<211> 24
<212> DNA
< 213>synthetic
<400> 3
ctgaattcgc atgcctgcag gtcg 24
<210> 4
<211> 25
<212> DNA
< 213>synthetic
<400> 4
ttgatatcca attccctatt cagag 25
<210> 5
<211> 26
<212> DNA
< 213>synthetic
<400> 5
cactcgagat ggacggctat attcgc 26
<210> 6
<211> 25
<212> DNA
< 213>synthetic
<400> 6
cagggccctc aaccgcatgt attgc 25
<210> 7
<211> 27
<212> DNA
< 213>synthetic
<400> 7
gggatatcaa attgtgtttg cgttagg 27
<210> 8
<211> 33
<212> DNA
< 213>synthetic
<400> 8
gcggatccgg tacccactgt ccaatccacc gtc 33
<210> 9
<211> 28
<212> DNA
< 213>synthetic
<400> 9
ggctcgagca aattgtgttt gcgttagg 28
<210> 10
<211> 27
<212> DNA
< 213>synthetic
<400> 10
gcggtaccca ctgtccaatc caccgtc 27
<210> 11
<211> 30
<212> DNA
< 213>synthetic
<400> 11
aggatatcat gaagctactg tcttctatcg 30
<210> 12
<211> 31
<212> DNA
< 213>synthetic
<400> 12
gcaagcttgc acagttgaag tgaacttgcg g 31
<210> 13
<211> 31
<212> DNA
< 213>synthetic
<400> 13
ttggatccga attctacatc cataaccctg g 31
<210> 14
<211> 26
<212> DNA
< 213>synthetic
<400> 14
ttgatatctt cagtttcgat ccggcg 26
<210> 15
<211> 27
<212> DNA
< 213>synthetic
<400> 15
tggaattcat ggtgagcaag ggcgagg 27
<210> 16
<211> 30
<212> DNA
< 213>synthetic
<400> 16
ttggatcctt acttgtacag ctcgtccatg 30
<210> 17
<211> 27
<212> DNA
< 213>synthetic
<400> 17
cgctcgagat gactattctt tgctggc 27
<210> 18
<211> 33
<212> DNA
< 213>synthetic
<400> 18
cgggtacctt ggttgctact ggcacataag cgc 33
Claims (3)
1. a method of controlling silkworm growth in pupa time is characterized in that, may further comprise the steps:
(1) operates tame silk cocoon proteinase gene promotor through genetically engineered
PDPGAL4 gene clone under the control is advanced
PiggyBACTransposon carrier pigA3GFP makes up reorganization transgene carrier A, mixes with helper plasmid, imports and just lays eggs, and the newly-hatched silkworm after the hatching is screened the silkworm that obtains to have fluorescent protein report gene through routine, detects
GFPWith
GAL4 genes, breeding are processed transgenic bombyx mori PDP-GAL4 system; Will through the genetically engineered operation
UASSilkworm baculovirus under the promotor control
EgtGene or scorpion toxin
BmKIT 3 RGene clone is advanced
PiggyBACTransposon carrier pigA3GFP; Make up reorganization transgene carrier B; Mix with helper plasmid, import and just lay eggs, the newly-hatched silkworm after the hatching obtains to have the silkworm of fluorescent protein report gene through the routine screening; Detect foreign gene, transgenic bombyx mori UAS-egt or transgenic bombyx mori UAS-BmKIT are processed in breeding
3 RSystem;
Wherein, said family silk cocoon proteinase gene promotor
PDPBeing to be template with domestic silkworm gene group DNA, is that the primer PCR amplification obtains with PDP1 and PDP2; Said PDP1 is SEQ ID No.13:tt
G gat ccg aat tcT aca tcc ata acc ctg g, underscore does
BamThe H I with
EcoR I restriction enzyme site, said PDP2 are SEQ ID No.14:tt
G ata tcT tca gtt tcg atc cgg cg, underscore does
EcoR V restriction enzyme site;
(2) the transgenic bombyx mori PDP-GAL4 system and transgenic bombyx mori UAS-egt system or the transgenic bombyx mori UAS-BmKIT that breeding are processed
3 RSystematic cross, the F1 that obtains silkworm are for larva, and the F1 of gained silkworm grows normal for larval stage, prolong 7-10 days pupa time.
2. according to the method for the said control of claim 1 silkworm growth in pupa time, it is characterized in that said reorganization transgene carrier A is pigA3GFP-ie-neo-PDP-Gal4-PA.
3. according to the method for the said control of claim 1 silkworm growth in pupa time, it is characterized in that said reorganization transgene carrier B is piggyA3GFP-ie-neo-UAS-IT
3 R-polyA or pigA3GFP-ie-neo-UAS-egt.
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| CN102250932B true CN102250932B (en) | 2012-10-31 |
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| CN103088066B (en) | 2013-02-07 | 2014-01-15 | 中国科学院水生生物研究所 | Method for controlling fish reproduction |
| CN103243121B (en) * | 2013-05-17 | 2014-07-02 | 苏州大学 | Method for improving egg laying amount of original strain of mulberry silkworm |
| CN107619836B (en) * | 2017-09-30 | 2020-10-09 | 西南大学 | System for reducing activity 20E concentration in spinning period, changing silk pupa nutrition distribution proportion and increasing silkworm cocoon yield, application and method |
| BR102020005666A2 (en) * | 2020-03-20 | 2021-09-28 | Ctc - Centro De Tecnologia Canavieira S.A. | POLYNUCLEOTIDE, PAIRS OF INITIATORS, VEGETABLE MATERIAL DETECTION METHODS, GENETIC CONSTRUCTION, KIT FOR DETECTION OF THE PRESENCE IN A VEGETABLE MATERIAL SAMPLE, EVENT CTC93209-4, INSECT RESISTANT PLANT, COMODITE PRODUCT- A PLANT TO PRODUCE DE-SUGAR RESISTANT TO INSECT AND USE OF A PLANT, PLANT CELL, PART OF PLANT OR SEED |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101418302A (en) * | 2008-12-09 | 2009-04-29 | 苏州大学 | Construction method of cultivated silkworm with controllable upgrowth and upgrowth control method |
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|---|---|---|---|---|
| CN101418302A (en) * | 2008-12-09 | 2009-04-29 | 苏州大学 | Construction method of cultivated silkworm with controllable upgrowth and upgrowth control method |
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| Targeted gene expression using the GAL4/UAS system in the silkworm bombyx mori;Imamura M，等;《Genetics》;20031101;第165卷(第3期);第1329-1340页 * |
| 基于piggyBac转座子转hGM-CSF基因家蚕的研究;曹广力;《蚕业科学》;20060930;第32卷(第03期);第324-328页 * |
| 杆状病毒egt基因对昆虫激素表达的调控影响;王厚伟，等;《蚕业科学》;20001130;第26卷(第增刊期);第102-106页 * |
| 转BmKIT3~R基因对家蚕发育与生存率的影响;王晓娟;《中国优秀硕士学位论文全文数据库》;20110131;第34,40,90,100,108页 * |
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