CN101418302B - Construction method of cultivated silkworm with controllable upgrowth and upgrowth control method - Google Patents
Construction method of cultivated silkworm with controllable upgrowth and upgrowth control method Download PDFInfo
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- CN101418302B CN101418302B CN2008102431410A CN200810243141A CN101418302B CN 101418302 B CN101418302 B CN 101418302B CN 2008102431410 A CN2008102431410 A CN 2008102431410A CN 200810243141 A CN200810243141 A CN 200810243141A CN 101418302 B CN101418302 B CN 101418302B
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Abstract
The invention discloses a method for constructing a silkworm with controllable growth and a method for controlling growth. A transposon piggy BAC factor is used as a basic vector; a recombining transgenic vector with an egt gene expression box controlled by a silkworm heat shock protein gene promoter is constructed through gene engineering operation; the recombining transgenic vector and an auxiliary plasmid are mixed and are conducted to a first spawn; newly-hatched silkworms after incubation are subjected to routine screening to obtain silkworms with fluorescence protein report genes; the egt gene is detected and is breed to prepare a transgenic silkworm; and the breed transgenic silkworm is subjected to thermal induction at a temperature of between 41 and 43 DEG C for 0.5 to 2 hours so as to control the growth of the silkworm. The method realizes that the rhabdovirus egt gene of the silkworm is utilized to control the growth of the silkworm, increases the flexibility of silkworm breeding, cocoon purchasing and filature process, and has important implementation value.
Description
Technical field
The present invention relates to the preparation method of transgenic bombyx mori in the genetically engineered field, be specifically related to the method that the silkworm transgenosis is improved the production traits, the invention still further relates to a kind of method of controlling the growth of this transgenic bombyx mori.
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.Silkworm is abnormal to be one of fundamental issue of silk industry science with the artificial adjustment of growing, and the metamorphosis of artificially regulating silkworm has great effect with the production structure and the integral production benefit of growing the silk industry.Silkworm is a complete metamorphosis, and pupa time is very short, only is 2 weeks, because moth mouth cocoon is not suitable for filature, must finish the purchase and the drying work of bright cocoon in the production before the nymphosis moth.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 wish by metamorphosis of artificial adjusting silkworm and growth, prolong pupa time, alleviate the operating pressure and the intensity of bright cocoon purchase and oven dry, even wish 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 save 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.In addition, in the sericulture process, people wish that growth by regulating silkworm larva is to reach balance between silkworm and mulberry output, usually, people by use neotonin (juvenilehormone, JH) or moulting hormone (molting hormone, MH) delay or promote the growth of silkworm larva, owing to use the generation of the improper silkworm that often causes not cocooing of period of neotonin, moulting hormone or concentration, therefore, people are devoted to silkworm larva always and grow the novel method of regulating and explore.
The Bombyx complete metamorphosis, it is generally acknowledged insect cast off a skin and metamorphosis is subjected to hormone regulating and controlling, its residing growth and development stage depends on the relative titre of this two parahormone of neotonin and moulting hormone in the hemolymph.Existing many results of study show, by human intervention, change the endocrinosity of insect, can think that the growth of regulating insect is with abnormal.From the outside silkworm is applied JH or juvenile hormone analogue (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 MH also can change silkworm equally, newly-hatched silkworm is raised with the artificial diet that contain MH (100mg/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 be induced 3 hibernating worms usefulness imidazoles chemical substance KK-42 such as Dedos to occur to handle 4 instar larvaes, can induce precocity to pupate; The silkworm in 129-132 hour 5 ages is applied 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 by applying certain chemical substance, does not relate to the growth metamorphosis that changes silkworm by genetic manipulation.
Existing more result of study shows that baculovirus has a significant effect with metamorphosis to the just dormancy of insect.Autographa californica multicapsid nucleopolyhedrosisvirus baculovirus (AcNPV) inoculation silkworm larva in 4 age with to the non-target host of silkworm can cause that larvae development delays to prolong, and gives the silkworm pupa injection of pupating 2 AcNPV, can induce the generation of artificial diapause pupa.The Owain result of study shows, the coded product of baculovirus egt gene is steroidal uridine diphosphoglucose based transferase (the Ecdysone UDP-glucosyl transferase of casting off a skin, EGT), glucosyl group in this enzyme energy 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, David R.O ' Reilly, 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 by molecular biological method, improving the production traits, is the problem that need research and solve.
Summary of the invention
The object of the invention provides a kind of method of growing controlled silkworm that makes up, and prolongs the larval stage of silkworm and the time in pupa time, improves the product silk amount of silkworm, solves bright cocoon and receives baking and the pupa time contradiction between too short.
For achieving the above object, the technical solution used in the present invention is: a kind of construction process of growing controlled silkworm may further comprise the steps:
By the genetically engineered operation piggyBAC transposon is advanced in the silkworm baculovirus egt gene clone under the control of silkworm heat shock protein gene promoter, make up the reorganization transgene carrier, do to mix with helper plasmid, import and just lay eggs, newly-hatched silkworm after the hatching obtains to have the silkworm of fluorescent protein report gene by the routine screening, detect the egt gene, transgenic bombyx mori is made in breeding.
Fluorescent protein report gene described in the above-mentioned technical scheme can be green fluorescence protein gene, also can be the red fluorescent protein gene.
Silkworm heat shock protein gene promoter described in the above-mentioned technical scheme can be hsp20.4, also can be hsp23.3, also can be hsp19.9 etc.
Be to realize that further aim of the present invention, the technical scheme of employing are, the transgenic bombyx mori that breeding is made is through thermal induction, and 41～43 ℃, thermal induction 0.5～2 hour, the growth of may command silkworm;
Optimized technical scheme is:
The transgenic bombyx mori that breeding is made is normally raised the 4th～6 day 5 ages, and 41～43 ℃ of thermal inductions 0.5～2 hour are normally raised then; Perhaps the transgenic bombyx mori that breeding is made is normally raised and is cocoond, and pupates 41～43 ℃ of thermal inductions 0.5～2 hour, normally protection then the 2nd～8 day.
Preferred technical scheme is: the transgenic bombyx mori that obtains is normally raised the 5th day 5 ages, 42 ℃ of thermal inductions 1 hour, after normally raise, compare with the non-transgenic silkworm of same processing, 5 elapsed time in age of transgenic bombyx mori prolong 1～2 day;
Perhaps the transgenic bombyx mori that obtains is normally raised and cocoons, pupated the 4th day, 42 ℃ of thermal inductions 1 hour, after normally protection, compare with the non-transgenic silkworm chrysalis of same processing, the pupa of transgenic bombyx mori prolongs more than 3 days age.
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
1. the present invention is by adopting the transgenic bombyx mori that can obtain silkworm heat shock protein promotor control silkworm baculovirus egt gene based on the carrier of piggyBAC transposon, under normal temps, the egt gene is disabled state, silkworm development is unaffected, and induce down egt genetic expression, MH horizontal down-regulation in the silkworm body in hot activation, thereby the growth of control silkworm has Modulatory character.
2. the present invention has increased the handiness of mulberry silkworm rearing, cocoon purchasing and silk reeling technology owing to adopt the purpose that reaches artificial control silkworm development by control silkworm baculovirus egt expression of gene.
Embodiment
Below in conjunction with embodiment the present invention is further described:
Embodiment one: silkworm heat shock protein gene hsp20.4 promoters driven is expressed the preparation of the transgenic bombyx mori of egt gene
Technical operating procedure is as follows:
1. the preparation of silkworm baculovirus egt gene
Silkworm baculovirus (BmNPV) genome DNA extraction is carried out according to a conventional method.According to announcing BmNPV-T3 pnca gene group complete sequence (GenBank registration number: L33180) design PCR upstream and downstream primer, primer 1:5 '-cg
CtcgagAtgactattctttgctggc-3 ' (underscore is an Xho I restriction enzyme site), primer 2: 5 '-cg
GgtaccgaccGgcacggcaagctggcgc-3 ' (underscore is a Kpn I restriction enzyme site).With the BmNPV genome is template, presses following condition pcr amplification: 95 ℃ of pre-sex change 3min, 95 ℃ of 50sec, 50 ℃ of 50sec, 72 ℃ of 90sec, 35 back 72 ℃ of 10min of circulation.Pcr amplification product is cut the glue test kit with 1% sepharose and is reclaimed, and clones into by TA that the T carrier obtains the pUC-egt plasmid, confirms the sequence of egt gene through order-checking.
2.hsp20.4 promotor preparation
Extract domestic silkworm gene group DNA routinely, according to the hsp20.4 gene order of having announced (GenBank registration number: AF315318) search genome sequence (the GenBank registration number: AADK01000034) design PCR upstream and downstream primer, primer 1:5 '-ta that obtains
C ccg ggC cagaca caa cgcgac gta cg-3 ' (underscore is a Sma I restriction enzyme site), primer 2: 5 '-ta
C tcg agA tta ctc ccg aaaatc gct tgg-3 ' (underscore is an Xho I restriction enzyme site).With the domestic silkworm gene group is template, presses following condition pcr amplification: 95 ℃ of pre-sex change 3min, 95 ℃ of 50sec, 50 ℃ of 50sec, 72 ℃ of 60sec, 35 back 72 ℃ of 10min of circulation.Pcr amplification product is cut the glue test kit with 1% sepharose and is reclaimed, and clones into by TA that the T carrier obtains the pUC-hsp20.4 plasmid, confirms the hsp20.4 promoter sequence through order-checking.
3. make up pBluescriptIISK (+) recombinant plasmid that has the egt gene
The pUC-egt plasmid reclaims the endonuclease bamhi of 1.74kb with Kpn I and Xho I double digestion, conventional method, is connected with pBluescript II SK (+) plasmid (Stratagene company product) with Kpn I and Xho I double digestion.The T4 dna ligase is a GIBCO BRL company product, and condition of contact is 16 ℃, 12 hours.Connect product transformed into escherichia coli TG1 bacterial strain, cultivate screening by blue hickie and obtain recombinant conversion, short run is extracted plasmid DNA (referring to " molecular cloning ", 1989, Science Press), cuts evaluation, the fragment of a treaty 1.74kb is arranged with Kpn I and Xho I enzyme, the egt gene of proof silkworm baculovirus has been cloned into pBluescript II SK (+) plasmid, and the recombinant plasmid that is obtained is referred to as pSK-egt.
4.hsp20.4 the vector construction of the egt gene of promotor control silkworm baculovirus
The pUC-hsp20.4 plasmid reclaims the fragment of 0.66kb with Sma I and Xho I double digestion, and is connected with the pSK-egt plasmid of Xho I double digestion with Sma I.The T4 dna ligase is a GIBCO BRL company product, and condition of contact is 16 ℃, 12 hours.Connect product transformed into escherichia coli TG1 bacterial strain, cultivate screening by blue hickie and obtain recombinant conversion, short run is extracted plasmid DNA, fragment with Sma I and the visible 0.66kb of Xho I double digestion, proof hsp20.4 promotor is cloned the upstream of the egt gene of the baculovirus of being on request, and the recombinant plasmid of carrying that obtains is referred to as psk-hsp20.4-egt.
5. make up the recombinant vectors that has silkworm baculovirus ie-1 gene promoter control neomycin resistance gene neo
DNA with silkworm baculovirus is a template, with the Auele Specific Primer of ie-1 gene promoter to (5 '-ttc
Gaa ttcGat ttg cag ttc ggg ac-3 ', 5 '-gcg
Gat atcAgt cgt ttg gtt gtt ca-3) (underscore is respectively EcoR I and EcoR V restriction enzyme site) carries out pcr amplification, behind PCR product EcoR I, the EcoR V double digestion, be cloned in pBluescript II SK (+) carrier, obtain to have the carrier pSK-IE of silkworm baculovirus ie-1 gene promoter.
With pcDNA3.1 carrier (Invitrogen company product) is template, with Neo1 (5 '-ct
G ata tcAtga ttg aac aag atg g-3 ') and Neo3 (5 '-ag
C tcg aga att cTa gct aga ggt cga c-3 ') (underscore is respectively EcoR V and Xho I, EcoR I restriction enzyme site) is primer, pcr amplification goes out the encoding sequence of neo gene and the polyA signal sequence in downstream (about 1.1kb) thereof, after Xho I, EcoR V enzyme are cut digestion, the clone advances through in the same pSK-IE carrier of handling, and obtains the pSK-IE-Neo carrier.
6. make up the reorganization transgene carrier of the egt gene, silkworm baculovirus ie-1 gene promoter control neomycin resistance gene neo and the fluorescent protein report gene that have hsp20.4 promotor control silkworm baculovirus
The pSK-IE-Neo carrier is cut with EcoR I enzyme, recovery has the fragment ie-neo of ie-1 promotor control neomycin resistance gene, the clone advances pigA3GFP plasmid that same enzyme cuts (referring to Cary, L.C.et al.Transposon mutagenesis of baculoviruses:analysis of Trichoplusia nitransposon IFP2 insertions within the FP-locus of nuclear polyhedrosisviruses.Virology, 1989,172:156-69), the heavy grain called after pigA3GFP-Neo that obtains.
Psk-hsp20.4-egt plasmid Sma I and Kpn I double digestion, reclaim the fragment hsp20.4-egt of hsp20.4 promotor control egt gene, the clone advances between the Sma I and Kpn I site of pigA3GFP-Neo plasmid, obtains transgene carrier pigA3GFP-Neo-hsp20.4-egt.
7. to obtain to have the transgenic bombyx mori cultivated silkworm breed variety of fluorescence report gene GFP and neomycin resistance be the bright moon kind to semen transformation, and normal raising is to changing moth.
With pigA3GFP-Neo-hsp20.4-egt and helper plasmid (referring to Tamura Toshiki.et al.Germline transformation of the silkworm Bombyx mori L.using a piggy Bactransposon-derived vector.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/, normal mating, lay eggs the newly-hatched silkworm (G after the hatching
0) add continuously the food 10000 μ g/mL G418 to 4 sleep age, the combined with fluorescent inverted microscope detects, filter out anti-G418, have the silkworm of fluorescent protein report gene, keep and have the silkworm (transgenic bombyx mori) that obvious fluorescence shows, carry out egt gene test and conventional silkworm breeding.
8. the egt gene PCR of transgenic bombyx mori detects
The take a morsel hemolymph (about 20 μ L hemolymphs/every) of fluorescence silkworm of acupuncture, add 500 μ L TE damping fluids (pH8.0), add 500 μ L supersaturation phenol (pH8.0) effect 10min, the centrifugal 10min of 12000r/min gets supernatant, use the supersaturation phenol extraction more once, add chloroform: primary isoamyl alcohol (24:1) effect 10min, the centrifugal 10min of 12000r/min gets and resets and add 2 times of volumes and do not have water-cooled ethanol, mix, put-20 ℃, use the centrifugal 10min of 12000r/min after 2 hours again, abandon supernatant, wash precipitation with 70% ethanol, the centrifugal 1min of 12000r/min abandons supernatant, seasoning, precipitation obtains the total DNA of domestic silkworm gene group with the dissolving of 10 μ L TE damping fluids.
To extract the total DNA of silkworm hemolymph, with egt gene-specific primer 1:5 '-c as template
Gc tcg AgA tga cta ttc ttt gct ggc-3 ' (underscore is an Xho I restriction enzyme site) and primer 2: 5 '-c
Gg gta CcG acc ggc acg gca agc tgg cgc-3 ' (underscore is a Kpn I restriction enzyme site) establishes the negative contrast of normal silkworm, the positive contrast of pigA3GFP-Neo-hsp20.4-egt plasmid DNA simultaneously.PCR detects demonstration, at G
0, G
1, G
2, G
3The equivalent egt gene specific fragment that all can amplify about 1.74kb, negative control do not have this fragment and produce, and show that the egt gene has entered the domestic silkworm gene group, and genetic stability.
Embodiment two: the larvae development control of transgenic bombyx mori
The transgenic bombyx mori that embodiment one is obtained is normally raised the 5th day 5 ages, 42 ℃ of thermal inductions 1 hour, after normally raise, compare with the non-transgenic silkworm of same processing, 5 elapsed time in age of transgenic bombyx mori prolong 1～2 day.
Embodiment three: grow control the pupa time of transgenic bombyx mori
The transgenic bombyx mori that embodiment one is obtained is normally raised and is cocoond, pupated the 4th day, 42 ℃ of thermal inductions 1 hour, after normally protection, compare with the non-transgenic silkworm chrysalis of same processing, the pupa of transgenic bombyx mori prolongs more than 3 days age.
Embodiment four: grow control the pupa time of transgenic bombyx mori
The transgenic bombyx mori that embodiment one is obtained is normally raised and is cocoond, pupated the 8th day, 42 ℃ of thermal inductions 0.5 hour, after normally protection, compare with the non-transgenic silkworm chrysalis of same processing, the pupa of transgenic bombyx mori prolongs more than 3 days age.
Claims (4)
1. construction process of growing controlled silkworm is characterized in that may further comprise the steps:
By the genetically engineered operation piggyBAC transposon is advanced in the silkworm baculovirus egt gene clone under the control of silkworm heat shock protein gene promoter, structure has the reorganization transgene carrier of egt gene, silkworm baculovirus ie-1 gene promoter control neomycin resistance gene neo and the fluorescent protein report gene of hsp20.4 promotor control silkworm baculovirus, do to mix with helper plasmid, import and just lay eggs, newly-hatched silkworm after the hatching obtains to have the silkworm of fluorescent protein report gene by the routine screening, detect the egt gene, transgenic bombyx mori is made in breeding; Described silkworm heat shock protein gene promoter is hsp20.4.
2. method of controlling silkworm development is characterized in that may further comprise the steps:
Adopt transgenic bombyx mori that claim 1 breeding makes through thermal induction, 41～43 ℃, thermal induction 0.5～2 hour.
3. a kind of method of controlling silkworm development according to claim 2 is characterized in that:
The transgenic bombyx mori that adopts claim 1 breeding to make is normally raised the 4th～6 day 5 ages, and 41～43 ℃ of thermal inductions 0.5～2 hour are normally raised then.
4. a kind of method of controlling silkworm development according to claim 2 is characterized in that:
The transgenic bombyx mori that adopts claim 1 breeding to make is normally raised and is cocoond, and pupates 41～43 ℃ of thermal inductions 0.5～2 hour, normally protection then the 2nd～8 day.
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| CN102250932B (en) * | 2011-06-27 | 2012-10-31 | 苏州大学 | Method for controlling domestic silkworm pupal stage growth |
| CN102367450A (en) * | 2011-07-11 | 2012-03-07 | 西北农林科技大学 | PiggyBac transposon mediated transgenic vector for inducing cell immortalization, its construction method and its application |
| CN104293918B (en) * | 2014-09-15 | 2016-08-17 | 浙江大学 | Utilize the method that RT-PCR method carries out the most strong Fruit variety of 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 |
| CN108477082B (en) * | 2018-03-09 | 2019-12-17 | 北京中农富通园艺有限公司 | Method for instantly relieving diapause by heat shock of silkworms and delaying incubation by refrigeration |
| CN109929853B (en) * | 2019-03-13 | 2020-10-02 | 中国科学院微生物研究所 | Application of thermophilic bacteria source heat shock protein gene |
| CN114216870B (en) * | 2021-10-29 | 2023-10-03 | 辽宁省蚕业科学研究所 | Screening method of tussah breeding material and application thereof |
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