CN110272916A - A kind of technical method increasing female silkworm moth oviposition quantity - Google Patents
A kind of technical method increasing female silkworm moth oviposition quantity Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/04—Silkworms
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
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- A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
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Abstract
The invention discloses a kind of technical methods of increase female silkworm moth oviposition quantity.Total serum IgE is extracted from silkworm pupa fat-body, reverse transcription obtains the 1st chain cDNA, and it is expanded using it as template RT-PCR, clone obtains BmAKHR cDNA and as template PCR amplifications, DNA fragmentation connection carrier is obtained into pFLAG-BmAKHR after digestion, PCR amplification obtains reaction template again, and is further processed to obtain BmAKHR siRNA；By BmAKHR siRNA liquid filter membrane degerming, it is injected into silkworm abdomen, mulberry leaf, which are raised to maturation, cocoons, and pupates and normally mates after changing moth, and the silkworm seed quantity given birth to increases.The technology of the present invention method increases the quantity of female silkworm moth oviposition, it is not increase mulberry volume taken by the silkworms, it is realized by regulation silkworm energetic supersession, this has positive effect for material energy metabolism mechanism, the production of hybrid seeds quantity for further increasing silkworm and the physiological function etc. of studying silkworm.
Description
Technical field
The present invention relates to a kind of technical methods of increase female silkworm moth oviposition quantity.
Background technique
Silkworm (Bombyx mori) is the maximum economic resources insect of the indoor number of animals raised on the current earth originating from China.
It breeds silkworms as special advantage industries and in China has more than 5000 history, China is still maximum sericulture state in the world at present.Silkworm
Various nutriments are absorbed by eating mulberry leaf in larval phase, and supply in each period silkworm body each group by metabolic physiology process
Organ is knitted, to be grown, be developed, bred, wherein nutriment supply ovary is carried out silkworm seed manufacture.Silkworm scale is raised
Nutrient is silk cocoon production and silkworm egg produces two seed types, and the main purpose of silkworm egg production is to obtain high-quality, high yield silkworm seed, it is
The basis of silk cocoon production.For this purpose, taking, various methods increase the quantity of female Bombycis mori oviposition and the important technology of silkworm egg production is arranged
One of apply, it is constantly subjected to the extensive attention of people.
The metabolic physiologies such as material energy metabolism of Silkworm, Bombyx mori process is to participate in by neuropeptide.Silkworm neuropeptide by
Neurosecretory cell secretion, it is active very high although content is very low, it is the important regulating and controlling factor of silkworm vital movement.Since silkworm
After first neuropeptide (bombyxin/insulin related peptide) is accredited, more and more neuropeptides are separated from Silkworm, Bombyx mori to be identified
Out, separation identifies more than 30 kinds of neuropeptides and some isomers from Silkworm, Bombyx mori so far, and predicts there is 190 in Silkworm, Bombyx mori
Multiple neuropeptides.Silkworm adipokinetic hormone (BmAKH) is a kind of important neuropeptide of silkworm, and major function is regulation Silkworm, Bombyx mori
Homeostasis energy and distribution.The effect of silkworm adipokinetic hormone is mediated by specific silkworm adipokinetic hormone receptor (BmAKHR)
's.But the related receptor-mediated silkworm adipokinetic hormone effect of silkworm adipokinetic hormone, so that the energy distribution of silkworm body is adjusted, to increase house
Female moth egg quantity of silkworm etc. lacks research.
Summary of the invention
In order to solve the problems, such as background technique, the purpose of the present invention is to provide a kind of ovipositions of increase female silkworm moth
The technical method of quantity is cloned using RT-PCR, PCR amplification, agarose gel electrophoresis recycling, with mammalian expression vector
PFlag-CMV-3' connection, and T7 reaction buffer, ATP liquid, CTP liquid, GTP liquid, UTP solution, T7 enzyme hybrid reaction, then give house
The method of 5 instar larvae abdomen injection of silkworm increases female silkworm moth oviposition quantity by regulating and controlling silkworm material energy metabolism.
In order to achieve the above object, The technical solution adopted by the invention is as follows:
1) it dissects and collects silkworm chrysalis fat-body, therefrom extract total serum IgE, then reverse transcription obtains the 1st chain cDNA；
2) it using the 1st chain cDNA as template, is expanded by reverse transcriptase polymerase chain reaction (RT-PCR), clone obtains
BmAKHR cDNA (silkworm adipokinetic hormone receptor cdna)；
3) using BmAKHR cDNA as template, BmAKHR DNA fragmentation is obtained by PCR amplification, agarose gel electrophoresis returns
It receives target DNA fragment to connect after HindIII/BamHI digestion with mammalian expression vector pFlag-CMV-3', obtain
pFLAG-BmAKHR；
4) using pFLAG-BmAKHR as template, pFLAG-BmAKHR DNA fragmentation, Ago-Gel are obtained by PCR amplification
Electrophoresis recycles the target DNA fragment that the size purified is 781bp, as reaction template；
5) reaction template is mixed into progress with 10 × T7 reaction buffer, ATP liquid, CTP liquid, GTP liquid, UTP solution, T7 enzyme
Then reaction carries out repeatedly processing and obtains BmAKHR siRNA；
6) five age 2-3 giant silkworm abdomen injection 15-25 μ g BmAKHR siRNA are given, continues raising to maturation with mulberry leaf and ties
Cocoon, and normally mate after protecting its pupating moth, the silkworm seed quantity given birth to increases.
The step 1) specifically: the fat-body for dissecting and collecting the 3rd day silkworm chrysalis of pupating is extracted using Trizol kit
Total serum IgE, then reverse transcription is carried out to total serum IgE with the 1st chain cDNA synthetic agent of reverse transcription, obtain the 1st chain cDNA, reverse transcription condition
It is: 30 DEG C of denaturation 10min, 50 DEG C of reactions 60min, 95 DEG C of inactivation 5min.
The step 2) specifically: using the 1st chain cDNA as template, respectively with the forward primer 5 '-such as SEQ ID NO.1
The 5 '-TTAAACCATACCGTTCGTTACG- of reverse primer of ATGGATATAGACGAGAAAGTGTC-3 ' and such as SEQ ID NO.2
3 ' carry out reverse transcriptase polymerase chain reaction (RT-PCR) amplification, and clone obtains BmAKHR cDNA；Its RT-PCR amplification condition
Be: 94 DEG C of initial denaturations 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s carry out 30 circulations.
The step 3) obtains BmAKHR DNA fragmentation specifically in the following ways are as follows: using BmAKHR cDNA as template, point
It Yong not be such as the forward primer 5 '-ATGGATATAGACGAGAAAGTGTCCG-3 ' of SEQ ID NO.3 and anti-such as SEQ ID NO.4
PCR amplification is carried out to primer 5 '-TTAAACCATACCGTTCGTTACGTGG-3 ', obtains BmAKHR DNA fragmentation；PCR amplification item
Part is: 94 DEG C of initial denaturations 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s, carries out 30 circulations, adds rTaq enzyme
Continue to extend 10min.
The step 4) obtains pFLAG-BmAKHR DNA fragmentation specifically in the following ways are as follows:
Using pFLAG-BmAKHR as template, respectively with the 5 '-CCCAAGCTTGATG of specific primer such as SEQ ID NO.5
5 '-the CGCGGATCCGCTTAAACCATACC of specific primer of GATATAGACGAGAAAGTGTC-3 ' and such as SEQ ID NO.6
GTTCGTTACGTGG-3 ' carry out PCR amplification, obtains pFLAG-BmAKHR DNA fragmentation；PCR amplification condition is: 94 DEG C of initial denaturations
2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s carry out 30 circulations.
The step 5) specifically:
5.1) 1-2 μ g reaction template, 2 μ 10 × T7 of L reaction buffers, 2 μ L ATP liquid, 2 μ L are in vitro sequentially added
CTP liquid, 2 μ L GTP liquid, 2 μ L UTP solution and 2 μ L T7 enzymatic mixtures；
5.2) double distilled water of nuclease free is supplemented to 20 μ L, is sufficiently mixed, is placed in 37 DEG C of reaction 16h, is cooled to often
Temperature；
5.3) 1 μ L TURBO DNA enzymatic is added in the mixed liquor that step 5.2) obtains, after mixing, is incubated at 37 DEG C
15min；
5.4) double distilled water of 30 μ L nuclease frees, the 7.5M lithium chloride of 30 μ L and 50mM EDTA (ethylenediamine tetraacetic are added
Acetic acid), terminate reaction；
5.5) 15000rpm is centrifuged 15min at 4 DEG C, removes supernatant, and the ethyl alcohol that 1mL mass percent is 70% is added
Solution washing；
5.6) 15000rpm is centrifuged 15min at 4 DEG C, alcoholic supernatant is removed, by double distillations of precipitating nuclease free
Water dissolution, obtains BmAKHR siRNA.
The step 6) specifically: to the BmAKHR siRNA of five age 2-3 giant silkworm abdomen injection 15-25 μ g, use
Mulberry leaf continue to be placed in raising to maturation in 24-25 DEG C of temperature, 70-80% humidity, bright night in daytime dark environment and cocoon, and in 23-24
DEG C temperature, 70-75% humidity, light normally mate after uniformly protecting its pupating moth under partially dark environmental condition, the silkworm given birth to
Ovum quantity increases.
The BmAKHR siRNA (silkworm adipokinetic hormone receptor siRNA) that the present invention is obtained with specially treated carries out 5 age silkworms note
It penetrates, is then further processed the increase for obtaining female silkworm moth oviposition quantity.
The invention has the advantages that:
The present invention increases the technical method of female silkworm moth oviposition quantity, is not increase mulberry volume taken by the silkworms, by regulating and controlling silkworm
Energetic supersession is come what is realized, this is for the substance and energetic supersession mechanism of research silkworm, the production of hybrid seeds quantity for further increasing silkworm
With physiological function etc., there is positive effect.
The present invention is confirmed by many experiments in the case where not increasing mulberry volume taken by the silkworms, after technical method of the invention
The silkworm seed quantity that female Bombycis mori is given birth to is compared with the silkworm seed quantity increase about 18.1% that check plot female Bombycis mori is given birth to.
Specific embodiment
Below with reference to embodiment, the invention will be further described.
Embodiment 1:
The fat-body for dissecting and collecting the 3rd day silkworm chrysalis of pupating extracts total serum IgE using Trizol kit, then uses reverse transcription
1st chain cDNA synthetic agent carries out reverse transcription to total serum IgE, obtains the 1st chain cDNA；Its reverse transcription condition is: 30 DEG C of denaturation
10min, 50 DEG C of reactions 60min, 95 DEG C of inactivation 5min.
Using the 1st chain cDNA as template, draw respectively with forward primer 5 '-ATGGATATAGACGAGAAAGTGTC-3 ' and reversely
Object 5 '-TTAAACCATACCGTTCGTTACG-3 ' carries out reverse transcriptase polymerase chain reaction (RT-PCR) amplification, and clone obtains
BmAKHR cDNA；Its RT-PCR amplification condition is: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C are prolonged
80s is stretched, 30 circulations are carried out.
Using BmAKHR cDNA as template, respectively with forward primer 5 '-ATGGATATAGACGAGAAAGTGTCCG-3 ' and instead
PCR amplification is carried out to primer 5 '-TTAAACCATACCGTTCGTTACGTGG-3 ', obtains BmAKHR DNA fragmentation；Its PCR amplification
Condition is: 94 DEG C of initial denaturations 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s, carries out 30 circulations, adds rTaq
Enzyme continues to extend 10min.Agarose gel electrophoresis recycles target DNA fragment, dynamic with lactation after HindIII/BamHI digestion
The pFlag-CMV-3' connection of object expression vector obtains pFLAG-BmAKHR.
Using pFLAG-BmAKHR as template, two 5 '-CCCAAGCTTGATGGATATAGACGAGA of specific primer are used
AAGTGTC-3 ' and 5 '-CGCGGATCCGCTTAAACCATACCGTTCGTTACGTGG-3 ' carry out PCR amplification, obtain pFLAG-
BmAKHR DNA fragmentation；Its PCR amplification condition is: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C are prolonged
80s is stretched, 30 circulations are carried out.Agarose gel electrophoresis recycles the target DNA fragment that the size purified is 781bp, as
Reaction template.
2 μ g reaction templates, 2 μ 10 × T7 of L reaction buffers, 2 μ L ATP liquid, 2 μ L CTP are sequentially added in a test tube
Liquid, 2 μ L GTP liquid, 2 μ L UTP solution and 2 μ L T7 enzymatic mixtures (above-mentioned raw materials derive from U.S. Promega company)；Then
The double distilled water of nuclease free is supplemented to 20 μ L, is sufficiently mixed, is placed in 37 DEG C of reaction 16h, is cooled to room temperature；Then above-mentioned
1 μ L TURBO DNA enzymatic (above-mentioned raw materials derive from U.S. Promega company) is added in mixed liquor, after mixing, is incubated at 37 DEG C
15min；It is added followed by the double distilled water of 30 μ L nuclease frees, the 7.5M lithium chloride and 50mM EDTA of 30 μ L, terminates reaction；
Then 15000rpm is centrifuged 15min at 4 DEG C, removes supernatant, and 70% ethanol washing of 1mL is added；Then at 4 DEG C
15000rpm is centrifuged 15min, removes alcoholic supernatant, and the double distilled water of precipitating nuclease free is dissolved, BmAKHR is obtained
siRNA。
20 obtained μ g BmAKHR dsRNA are injected into the 2nd giant silkworm abdomen of five ages, are continued to be placed in 25 DEG C of temperature with mulberry leaf
Raising to maturation is cocoond in degree, 75% humidity, bright night in daytime dark environment, and uniformly inclined in 23.5 DEG C of temperature, 75% humidity, light
It normally mates after protecting its pupating moth under dark environmental condition, the silkworm seed quantity that female Bombycis mori is given birth to is given birth to compared with check plot female Bombycis mori
Silkworm seed quantity increase by 18.2%.
Embodiment 2:
The fat-body for dissecting and collecting the 3rd day silkworm chrysalis of pupating extracts total serum IgE using Trizol kit, then uses reverse transcription
1st chain cDNA synthetic agent carries out reverse transcription to total serum IgE, obtains the 1st chain cDNA；Its reverse transcription condition is: 30 DEG C of denaturation
10min, 50 DEG C of reactions 60min, 95 DEG C of inactivation 5min.
Using the 1st chain cDNA as template, draw respectively with forward primer 5 '-ATGGATATAGACGAGAAAGTGTC-3 ' and reversely
Object 5 '-TTAAACCATACCGTTCGTTACG-3 ' carries out reverse transcriptase polymerase chain reaction (RT-PCR) amplification, and clone obtains
BmAKHR cDNA；Its RT-PCR amplification condition is: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C are prolonged
80s is stretched, 30 circulations are carried out.
Using BmAKHR cDNA as template, respectively with forward primer 5 '-ATGGATATAGACGAGAAAGTGTCCG-3 ' and instead
PCR amplification is carried out to primer 5 '-TTAAACCATACCGTTCGTTACGTGG-3 ', obtains BmAKHR DNA fragmentation；Its PCR amplification
Condition is: 94 DEG C of initial denaturations 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s, carries out 30 circulations, adds rTaq
Enzyme continues to extend 10min.Agarose gel electrophoresis recycles target DNA fragment, dynamic with lactation after HindIII/BamHI digestion
The pFlag-CMV-3' connection of object expression vector obtains pFLAG-BmAKHR.
Using pFLAG-BmAKHR as template, two 5 '-CCCAAGCTTGATGGATATAGACGAGA of specific primer are used
AAGTGTC-3 ' and 5 '-CGCGGATCCGCTTAAACCATACCGTTCGTTACGTGG-3 ' carry out PCR amplification, obtain pFLAG-
BmAKHR DNA fragmentation；Its PCR amplification condition is: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C are prolonged
80s is stretched, 30 circulations are carried out.Agarose gel electrophoresis recycles the target DNA fragment that the size purified is 781bp, as
Reaction template.
1 μ g reaction template, 2 μ 10 × T7 of L reaction buffers, 2 μ L ATP liquid, 2 μ L CTP are sequentially added in a test tube
Liquid, 2 μ L GTP liquid, 2 μ L UTP solution and 2 μ L T7 enzymatic mixtures (above-mentioned raw materials derive from U.S. Promega company)；Then
The double distilled water of nuclease free is supplemented to 20 μ L, is sufficiently mixed, is placed in 37 DEG C of reaction 16h, is cooled to room temperature；Then above-mentioned
1 μ L TURBO DNA enzymatic (above-mentioned raw materials derive from U.S. Promega company) is added in mixed liquor, after mixing, is incubated at 37 DEG C
15min；It is added followed by the double distilled water of 30 μ L nuclease frees, the 7.5M lithium chloride and 50mM EDTA of 30 μ L, terminates reaction；
Then 15000rpm is centrifuged 15min at 4 DEG C, removes supernatant, and 70% ethanol washing of 1mL is added；Then at 4 DEG C
15000rpm is centrifuged 15min, removes alcoholic supernatant, and the double distilled water of precipitating nuclease free is dissolved, BmAKHR is obtained
siRNA。
15 obtained μ g BmAKHR dsRNA are injected into the 3rd giant silkworm abdomen of five ages, are continued to be placed in 24.5 DEG C of temperature with mulberry leaf
Raising to maturation is cocoond in degree, 80% humidity, bright night in daytime dark environment, and uniformly partially dark in 23 DEG C of temperature, 73% humidity, light
Environmental condition under protect its pupating moth after normally mate, what the silkworm seed quantity that female Bombycis mori is given birth to was given birth to compared with check plot female Bombycis mori
Silkworm seed quantity increases by 17.6%.
Embodiment 3:
The fat-body for dissecting and collecting the 3rd day silkworm chrysalis of pupating extracts total serum IgE using Trizol kit, then uses reverse transcription
1st chain cDNA synthetic agent carries out reverse transcription to total serum IgE, obtains the 1st chain cDNA；Its reverse transcription condition is: 30 DEG C of denaturation
10min, 50 DEG C of reactions 60min, 95 DEG C of inactivation 5min.
Using the 1st chain cDNA as template, draw respectively with forward primer 5 '-ATGGATATAGACGAGAAAGTGTC-3 ' and reversely
Object 5 '-TTAAACCATACCGTTCGTTACG-3 ' carries out reverse transcriptase polymerase chain reaction (RT-PCR) amplification, and clone obtains
BmAKHR cDNA；Its RT-PCR amplification condition is: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C are prolonged
80s is stretched, 30 circulations are carried out.
Using BmAKHR cDNA as template, respectively with forward primer 5 '-ATGGATATAGACGAGAAAGTGTCCG-3 ' and instead
PCR amplification is carried out to primer 5 '-TTAAACCATACCGTTCGTTACGTGG-3 ', obtains BmAKHR DNA fragmentation；Its PCR amplification
Condition is: 94 DEG C of initial denaturations 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s, carries out 30 circulations, adds rTaq
Enzyme continues to extend 10min.Agarose gel electrophoresis recycles target DNA fragment, dynamic with lactation after HindIII/BamHI digestion
The pFlag-CMV-3' connection of object expression vector obtains pFLAG-BmAKHR.
Using pFLAG-BmAKHR as template, two 5 '-CCCAAGCTTGATGGATATAGACGAGA of specific primer are used
AAGTGTC-3 ' and 5 '-CGCGGATCCGCTTAAACCATACCGTTCGTTACGTGG-3 ' carry out PCR amplification, obtain pFLAG-
BmAKHR DNA fragmentation；Its PCR amplification condition is: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C are prolonged
80s is stretched, 30 circulations are carried out.Agarose gel electrophoresis recycles the target DNA fragment that the size purified is 781bp, as
Reaction template.
1.5 μ g reaction templates, 2 μ 10 × T7 of L reaction buffers, 2 μ L ATP liquid, 2 μ L are sequentially added in a test tube
CTP liquid, 2 μ L GTP liquid, 2 μ L UTP solution and 2 μ L T7 enzymatic mixtures (above-mentioned raw materials derive from U.S. Promega company)；It connects
Supplement nuclease free double distilled water to 20 μ L, be sufficiently mixed, be placed in 37 DEG C of reaction 16h, be cooled to room temperature；Then upper
It states and 1 μ L TURBO DNA enzymatic (above-mentioned raw materials derive from U.S. Promega company) is added in mixed liquor, after mixing, incubated at 37 DEG C
Educate 15min；It is added followed by the double distilled water of 30 μ L nuclease frees, the 7.5M lithium chloride and 50mM EDTA of 30 μ L, is terminated anti-
It answers；Then 15000rpm is centrifuged 15min at 4 DEG C, removes supernatant, and 70% ethanol washing of 1mL is added；Then at 4 DEG C
15000rpm is centrifuged 15min, removes alcoholic supernatant, and the double distilled water of precipitating nuclease free is dissolved, BmAKHR is obtained
siRNA。
25 obtained μ g BmAKHR dsRNA are injected into the 2nd giant silkworm abdomen of five ages, are continued to be placed in 24 DEG C of temperature with mulberry leaf
Raising to maturation is cocoond in degree, 70% humidity, bright night in daytime dark environment, and uniformly partially dark in 24 DEG C of temperature, 70% humidity, light
Environmental condition under protect its pupating moth after normally mate, what the silkworm seed quantity that female Bombycis mori is given birth to was given birth to compared with check plot female Bombycis mori
Silkworm seed quantity increases by 18.6%.
As seen from the above-described embodiment, the present invention regulates and controls silkworm energy by way of silkworm adipokinetic hormone receptors signal transduction
The distribution of amount, improves the quantity of female moth egg, obvious technical effects, this for study silkworm substance and energetic supersession mechanism,
The production of hybrid seeds quantity of silkworm and the technological innovation etc. of physiological function and traditional sericulture industry are further increased, there is positive effect.
This application involves the sequence tables arrived:
SEQ ID NO.1: primer 1
Source: artificial synthesized
5’-ATGGATATAGACGAGAAAGTGTC-3’
SEQ ID NO.2: primer 2
Source: artificial synthesized
5’-TTAAACCATACCGTTCGTTACG-3’
SEQ ID NO.3: primer 3
Source: artificial synthesized
5’-ATGGATATAGACGAGAAAGTGTCCG-3’
SEQ ID NO.4: primer 4
Source: artificial synthesized
5’-TTAAACCATACCGTTCGTTACGTGG-3’
SEQ ID NO.5: primer 5
Source: artificial synthesized
5′-CCCAAGCTTGATGGATATAGACGAGAAAGTGTC-3′
SEQ ID NO.6: primer 6
Source: artificial synthesized
5′-CGCGGATCCGCTTAAACCATACCGTTCGTTACGTGG-3′。
Sequence table
<110>Zhejiang University
<120>a kind of technical method for increasing female silkworm moth oviposition quantity
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atggatatag acgagaaagt gtc 23
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttaaaccata ccgttcgtta cg 22
<210> 3
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atggatatag acgagaaagt gtccg 25
<210> 4
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ttaaaccata ccgttcgtta cgtgg 25
<210> 5
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cccaagcttg atggatatag acgagaaagt gtc 33
<210> 6
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cgcggatccg cttaaaccat accgttcgtt acgtgg 36
Claims (7)
1. a kind of technical method for increasing female silkworm moth oviposition quantity, it is characterised in that:
1) it dissects and collects silkworm chrysalis fat-body, therefrom extract total serum IgE, then reverse transcription obtains the 1st chain cDNA；
2) it using the 1st chain cDNA as template, is expanded by reverse transcriptase polymerase chain reaction (RT-PCR), clone obtains BmAKHR
CDNA (silkworm adipokinetic hormone receptor cdna)；
3) using BmAKHR cDNA as template, BmAKHR DNA fragmentation is obtained by PCR amplification, agarose gel electrophoresis recycles mesh
DNA fragmentation connect after HindIII/BamHI digestion with mammalian expression vector pFlag-CMV-3', obtain pFLAG-
BmAKHR；
4) using pFLAG-BmAKHR as template, pFLAG-BmAKHR DNA fragmentation, agarose gel electrophoresis are obtained by PCR amplification
The target DNA fragment that the size purified is 781bp is recycled, as reaction template；
5) it is anti-that reaction template is mixed to progress with 10 × T7 reaction buffer, ATP liquid, CTP liquid, GTP liquid, UTP solution, T7 enzyme
It answers, then carries out repeatedly processing and obtain BmAKHR siRNA；
6) five age 2-3 giant silkworm abdomen injection 15-25 μ g BmAKHR siRNA are given, continues raising to maturation with mulberry leaf and cocoons, and
It normally mates after protecting its pupating moth, the silkworm seed quantity given birth to increases.
2. a kind of technical method for increasing female silkworm moth oviposition quantity according to claim 1, it is characterised in that: the step
It is rapid 1) specifically: the fat-body for dissecting and collecting the 3rd day silkworm chrysalis of pupating, using Trizol kit extract total serum IgE, then with reverse
It records the 1st chain cDNA synthetic agent and reverse transcription is carried out to total serum IgE, obtain the 1st chain cDNA, reverse transcription condition is: 30 DEG C of denaturation
10min, 50 DEG C of reactions 60min, 95 DEG C of inactivation 5min.
3. a kind of technical method for increasing female silkworm moth oviposition quantity according to claim 1, it is characterised in that: the step
It is rapid 2) specifically: using the 1st chain cDNA as template, respectively with as SEQ ID NO.1 forward primer 5 '-
The 5 '-TTAAACCATACCGTTCGTTACG- of reverse primer of ATGGATATAGACGAGAAAGTGTC-3 ' and such as SEQ ID NO.2
3 ' carry out reverse transcriptase polymerase chain reaction (RT-PCR) amplification, and clone obtains BmAKHR cDNA；Its RT-PCR amplification condition
Be: 94 DEG C of initial denaturations 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s carry out 30 circulations.
4. a kind of technical method for increasing female silkworm moth oviposition quantity according to claim 1, it is characterised in that: the step
It is rapid 3) to obtain BmAKHR DNA fragmentation specifically in the following ways are as follows: using BmAKHR cDNA as template, respectively with such as SEQ ID
The reverse primer 5 '-of the forward primer 5 '-ATGGATATAGACGAGAAAGTGTCCG-3 ' of NO.3 and such as SEQ ID NO.4
TTAAACCATACCGTTCGTTACGTGG-3 ' carries out PCR amplification, obtains BmAKHR DNA fragmentation；PCR amplification condition is: 94 DEG C
Initial denaturation 2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s carry out 30 circulations, rTaq enzyme are added to continue to extend
10min。
5. a kind of technical method for increasing female silkworm moth oviposition quantity according to claim 1, it is characterised in that: the step
It is rapid 4) to obtain pFLAG-BmAKHR DNA fragmentation specifically in the following ways are as follows:
Using pFLAG-BmAKHR as template, respectively with the 5 '-CCCAAGCTTGATGGATA of specific primer such as SEQ ID NO.5
5 '-the CGCGGATCCGCTTAAACCATACCGTTC of specific primer of TAGACGAGAAAGTGTC-3 ' and such as SEQ ID NO.6
GTTACGTGG-3 ' carry out PCR amplification, obtains pFLAG-BmAKHR DNA fragmentation；PCR amplification condition is: 94 DEG C of initial denaturations
2min, 94 DEG C of denaturation 15s, 50 DEG C of annealing 30s, 68 DEG C of extension 80s carry out 30 circulations.
6. a kind of technical method for increasing female silkworm moth oviposition quantity according to claim 1, it is characterised in that: the step
It is rapid 5) specifically:
5.1) 1-2 μ g reaction template, 2 μ 10 × T7 of L reaction buffers, 2 μ L ATP liquid, 2 μ L CTP are in vitro sequentially added
Liquid, 2 μ L GTP liquid, 2 μ L UTP solution and 2 μ L T7 enzymatic mixtures；
5.2) double distilled water of nuclease free is supplemented to 20 μ L, is sufficiently mixed, is placed in 37 DEG C of reaction 16h, is cooled to room temperature；
5.3) 1 μ L TURBO DNA enzymatic is added in the mixed liquor that step 5.2) obtains, after mixing, is incubated for 15min at 37 DEG C；
5.4) double distilled water of 30 μ L nuclease frees, the 7.5M lithium chloride of 30 μ L and 50mM EDTA (ethylenediamine tetrem are added
Acid), terminate reaction；
5.5) 15000rpm is centrifuged 15min at 4 DEG C, removes supernatant, and the ethanol solution that 1mL mass percent is 70% is added
Washing；
5.6) 15000rpm is centrifuged 15min at 4 DEG C, removes alcoholic supernatant, and the double distilled water of precipitating nuclease free is molten
Solution, obtains BmAKHR siRNA.
7. a kind of technical method for increasing female silkworm moth oviposition quantity according to claim 1, it is characterised in that: the step
It is rapid 6) specifically: to the BmAKHR siRNA of five age 2-3 giant silkworm abdomen injection 15-25 μ g, continued to be placed in 24- with mulberry leaf
Raising to maturation is cocoond in 25 DEG C of temperature, 70-80% humidity, bright night in daytime dark environment, and wet in 23-24 DEG C of temperature, 70-75%
Degree, light normally mate after uniformly protecting its pupating moth under partially dark environmental condition, and the silkworm seed quantity given birth to increases.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349547A (en) * | 2015-12-02 | 2016-02-24 | 浙江大学 | Method for increasing weight of Bombyx mori pupa bodies |
| CN108048464A (en) * | 2017-12-19 | 2018-05-18 | 浙江大学 | It is a kind of that univoltine kind silkworm is allowed to produce the siRNA of non-Diapausing egg, preparation method and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349547A (en) * | 2015-12-02 | 2016-02-24 | 浙江大学 | Method for increasing weight of Bombyx mori pupa bodies |
| CN108048464A (en) * | 2017-12-19 | 2018-05-18 | 浙江大学 | It is a kind of that univoltine kind silkworm is allowed to produce the siRNA of non-Diapausing egg, preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| MATTHIAS W. LORENZ等: "Adipokinetic hormone inhibits the formation of energy stores and egg production in the cricket Gryllus bimaculatus", 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY PART B》 * |
| YING SHI等: "Identification and Functional Characterization of Two Orphan G-protein-coupled Receptors for Adipokinetic Hormones from Silkworm Bombyx mori", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 黄海山: "家蚕脂动激素受体信号转导机制研究", 《中国博士学位论文全文数据库基础科学辑》 * |
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