CN105349548A - Method for increasing weight of Bombyx mori larvae - Google Patents
Method for increasing weight of Bombyx mori larvae Download PDFInfo
- Publication number
- CN105349548A CN105349548A CN201510874406.7A CN201510874406A CN105349548A CN 105349548 A CN105349548 A CN 105349548A CN 201510874406 A CN201510874406 A CN 201510874406A CN 105349548 A CN105349548 A CN 105349548A
- Authority
- CN
- China
- Prior art keywords
- template
- reaction
- bmcrzr
- liquid
- pcr amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for increasing weight of Bombyx mori larvae. The method includes the following steps: extracting total RNA from fifth instar Bombyx mori silk glands; performing reverse transcription to obtain first-chain cDNA, and performing Rt-PCR amplification by taking the first-chain cDNA as a template; obtaining BmCrzR cDNA through cloning; performing PCR amplification by taking BmCrzR cDNA as a template; after enzyme digestion, connecting DNA segments with a carrier to obtain pFLAG-BmCrzR; performing PCR amplification by taking pFLAG-BmCrzR as a template to obtain a reaction template; mixing the reaction template with T7 reaction buffer, ATP liquid, CTP liquid, GTP liquid, UTP liquid and T7 enzyme for reaction, and obtaining BmCrzR dsRNA after further treatment; finally, injecting BmCrzR dsRNA into abdomens of larvae of fourth instar Bombyx mori; feeding with folium mori until weight of fifth instar Bombyx mori is increased. The method for increasing weight of Bombyx mori larvae is realized through controlling Bombyx mori metabolism rather than increasing eating quantity of folium mori, so that the method has positive functions on studying the substance and energy metabolism mechanism of Bombyx mori and further improving the economic character and physiological function of Bombyx mori and improving the technical innovation of the traditional silkworm breeding industry.
Description
Technical field
The present invention relates to a kind of method improving silkworm weight, especially relate to a kind of method improving silkworm larva body weight.
Background technology
Silkworm (Bombyxmori) is the economic resources insect that on the current earth, indoor feeding amount is maximum, it absorbs various nutritive substance from mulberry leaf (feed), and undertaken by each histoorgan in metabolic physiology process supply silkworm body growing, grow, breeding etc.Silkworm is raised on a large scale and is divided into silk cocoon production and silkworm egg to produce two types, and the main purpose that silk cocoon is produced is the silk obtaining high-quality, high yield, and it and silkworm larva Growth and metabolism physiological process regulate and control closely related.
Neuropeptide plays a significant role in the important physiological process such as growth and material energy metabolism of insect regulates and controls.In silkworm, have nearly 40 kinds of neuropeptides, Corazonin is one of them, and its physiological function, and is equaled to be separated in silkworm first for 2000 by Hua in 1989 first identifieds by Ventra.The effect of silkworm Corazonin is mediated by silkworm Corazonin acceptor (BmCrzR), but relevant BmCrzR mediates Corazonin effect, regulates and controls silkworm larva growth and material energy metabolism etc., all lacks research.
Summary of the invention
In order to solve Problems existing in background technology, the object of the present invention is to provide a kind of method improving silkworm larva body weight, adopt Rt-PCR clone, pcr amplification, agarose gel electrophoresis reclaims, and is connected with mammalian expression vector pFlag-CMV-3', with T7 reaction buffer, ATP liquid, CTP liquid, GTP liquid, UTP solution, T7 enzyme hybrid reaction, give the method for silkworm larva abdomen injection again, by regulation and control silkworm material energy metabolism, improve silkworm larva body weight.
In order to achieve the above object, the technical solution used in the present invention is as follows:
1) dissect and collect the domestic natural silk gland in 3-4 days five age, therefrom extracting total serum IgE, then reverse transcription obtains the 1st chain cDNA;
2) with the 1st chain cDNA for template, by reverse transcriptase polymerase chain reaction (RT-PCR) amplification, obtain silkworm Corazonin acceptor (BmCrzR) cDNA;
3) with BmCrzRcDNA be template again, obtain BmCrzRDNA fragment by pcr amplification, agarose gel electrophoresis reclaims target DNA fragment, after HindIII/BamHI enzyme is cut, be connected with mammalian expression vector pFlag-CMV-3', obtain pFLAG-BmCrzR;
4) take pFLAG-BmCrzR as template, obtain pFLAG-BmCrzRDNA fragment by pcr amplification, it is the target DNA fragment of 893bp that agarose gel electrophoresis reclaims the size obtaining purifying, as reaction template;
5) reaction template is mixed with 10 × T7 reaction buffer, ATP liquid, CTP liquid, GTP liquid, UTP solution, T7 enzyme react, then repeatedly process and obtain BmCrzRdsRNA;
6) to the silkworm abdomen injection 10-20 μ gBmCrzRdsRNA in 2-3 days four age, with mulberry leaf continue raise, obtain weight increase five age glutonous stage silkworm larva body.
Described step 1) be specially: dissect and collect the domestic natural silk gland in 3-4 days five age, using Trizol test kit to extract total serum IgE, then with reverse transcription the 1st chain cDNA synthetic agent, reverse transcription is carried out to total serum IgE; Its reverse transcription condition is: 65 DEG C of sex change 5min, and 42 DEG C of reactions 60min, 70 DEG C of deactivation 15min, obtain the 1st chain cDNA.
Described step 2) be specially: with the 1st chain cDNA for template, use forward primer 5 '-AAGCTTGCCACCATGGACAACGAAGGCAACAGTAC-3 ' (primer 1 as SEQIDNO.1) and reverse primer 5 '-GGATCCCGTAACAAACTAATGTTCTGTCCATTCG-3 ' (primer 2 as SEQIDNO.2) to carry out reverse transcriptase polymerase chain reaction (RT-PCR) amplification respectively, clone obtains silkworm Corazonin acceptor (BmCrzR) cDNA.
Described reverse transcriptase polymerase chain reaction (RT-PCR) amplification condition is: 94 DEG C of denaturation 30s, and 58 DEG C of annealing 30s, 72 DEG C extend 2min, carry out 30 circulations.
Described step 3) obtain BmCrzRDNA fragment and be in the following ways specifically: take BmCrzRcDNA as template, forward primer 5 '-AAGCTTATGGACAACGAAGGCAACAGTAC-3 ' (primer 3 as SEQIDNO.3) and reverse primer 5 '-GGATCCTTATAACAAACTAATGTTCTGTCCAT-3 ' (primer 4 as SEQIDNO.4) is used to carry out pcr amplification respectively, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 2min, 30 circulations, add rTaq enzyme to continue to extend 15min, obtain DNA fragmentation.
Described step 4) obtain pFLAG-BmCrzRDNA fragment and be in the following ways specifically: take pFLAG-BmCrzR as template, two Auele Specific Primer 5 '-AAGCTTATGAACTCAGAAACAATAAACGACAC-3 ' (primer 5 as SEQIDNO.5) and 5 '-GGATCCTTAACAGTTCGTGTTGAAGTATGTTTC-3 ' (primer 6 as SEQIDNO.6) are used to carry out pcr amplification respectively, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, obtain DNA fragmentation.
Described step 5) be specially:
5.1) 1-2 μ g reaction template, 2 μ L10 × T7 reaction buffers, 2 μ LATP liquid, 2 μ LCTP liquid, 2 μ LGTP liquid, 2 μ LUTP solution and 2 μ LT7 enzyme mixtures are in vitro added successively one;
5.2) supplement bi-distilled water to the 20 μ L of nuclease free, fully mix, be positioned over 37 DEG C of reaction 16h, be cooled to normal temperature;
5.3) in above-mentioned mixed solution, add 1 μ LTURBODNA enzyme, after mixing, at 37 DEG C, hatch 15min, to digest unnecessary reaction template;
5.4) bi-distilled water of 30 μ L nuclease free, 30 μ L7.5M lithium chloride and 50mMEDTA are added again, termination reaction;
5.5) the centrifugal 15min of 15000rpm at 4 DEG C, removing supernatant liquor, adds 1mL70% washing with alcohol;
5.6) the centrifugal 15min of 15000rpm at 4 DEG C, removes 70% alcoholic supernatant, is dissolved by the bi-distilled water of precipitation nuclease free, obtains BmCrzRdsRNA.
Confirmed when not increasing mulberry volume taken by the silkworms by great many of experiments, to adopt after the inventive method five age glutonous stage silkworm larva body weight compared with the increase about 25% of check plot.
The beneficial effect that the present invention has is:
The present invention improves the method for silkworm larva body weight, do not increase mulberry volume taken by the silkworms, realized by the metabolism of regulation and control silkworm, this material for research silkworm and machine-processed, the further technical renovation etc. improving silkworm economic characters and physiological function and traditional sericulture industry of energy metabolism, have active effect.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1:
Total serum IgE is extracted from the domestic natural silk gland in the 3rd day five ages, then reverse transcription obtains the 1st chain cDNA: dissect and collect the domestic natural silk gland in the 3rd day five ages, use Trizol test kit to extract total serum IgE, then with reverse transcription the 1st chain cDNA synthetic agent, reverse transcription is carried out to total serum IgE; Its reverse transcription condition is: 65 DEG C of sex change 5min, and 42 DEG C of reactions 60min, 70 DEG C of deactivation 15min, obtain the 1st chain cDNA.
With the 1st chain cDNA for template, use forward primer 5 '-AAGCTTGCCACCATGGACAACGAAGGCAACAGTAC-3 ' and reverse primer 5 '-GGATCCCGTAACAAACTAATGTTCTGTCCATTCG-3 ' respectively, carry out reverse transcriptase polymerase chain reaction (RT-PCR) amplification, clone obtains silkworm Corazonin acceptor (BmCrzR) cDNA.RT-PCR amplification condition is: 94 DEG C of denaturation 30s, and 58 DEG C of annealing 30s, 72 DEG C extend 2min, carry out 30 circulations.
Take BmCrzRcDNA as template, use forward primer 5 '-AAGCTTATGGACAACGAAGGCAACAGTAC-3 ' and reverse primer 5 '-GGATCCTTATAACAAACTAATGTTCTGTCCAT-3 ' respectively, carry out pcr amplification, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 2min, 30 circulations, add rTaq enzyme to continue to extend 15min, obtain DNA fragmentation.Agarose gel electrophoresis reclaims target DNA fragment, after HindIII/BamHI enzyme is cut, is connected with mammalian expression vector pFlag-CMV-3', obtains pFLAG-BmCrzR.
Take pFLAG-BmCrzR as template, two Auele Specific Primer 5 '-AAGCTTATGAACTCAGAAACAATAAACGACAC-3 ' (primer 1) and 5 '-GGATCCTTAACAGTTCGTGTTGAAGTATGTTTC-3 ' (primer 2) is used to carry out pcr amplification, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, obtain DNA fragmentation.It is the target DNA fragment of 893bp that agarose gel electrophoresis reclaims the size obtaining purifying, as reaction template.
1 μ g reaction template, 2 μ L10 × T7 reaction buffers, 2 μ LATP liquid, 2 μ LCTP liquid, 2 μ LGTP liquid, 2 μ LUTP solution and 2 μ LT7 enzyme mixtures (above-mentioned raw materials derives from TaKaRa company) are in vitro added successively one, then bi-distilled water to the 20 μ L of nuclease free is supplemented, abundant mixing, be positioned over 37 DEG C of reaction 16h, be cooled to normal temperature; Then in above-mentioned mixed solution, add 1 μ LTURBODNA enzyme (above-mentioned raw materials derives from TaKaRa company), after mixing, at 37 DEG C, hatch 15min; Then the bi-distilled water of 30 μ L nuclease free, 30 μ L7.5M lithium chloride and 50mMEDTA are added again, termination reaction; Then the centrifugal 15min of 15000rpm at 4 DEG C, removing supernatant liquor, adds 1mL70% washing with alcohol; Then the centrifugal 15min of 15000rpm at 4 DEG C, removes 70% alcoholic supernatant, is dissolved by the bi-distilled water of precipitation nuclease free, obtains BmCrzRdsRNA.
The 10 μ gBmCrzRdsRNA obtained are injected into silkworm larva belly in the 2nd day four ages, and mulberry leaf are raised, five glutonous stage body weight of silkworm larva in age comparatively check plot body weight of silkworm larva increases by 23.8%.
Embodiment 2:
Total serum IgE is extracted from the domestic natural silk gland in the 4th day five ages, then reverse transcription obtains the 1st chain cDNA: dissect and collect the domestic natural silk gland in the 4th day five ages, use Trizol test kit to extract total serum IgE, then with reverse transcription the 1st chain cDNA synthetic agent, reverse transcription is carried out to total serum IgE; Its reverse transcription condition is: 65 DEG C of sex change 5min, and 42 DEG C of reactions 60min, 70 DEG C of deactivation 15min, obtain the 1st chain cDNA.
With the 1st chain cDNA for template, use forward primer 5 '-AAGCTTGCCACCATGGACAACGAAGGCAACAGTAC-3 ' and reverse primer 5 '-GGATCCCGTAACAAACTAATGTTCTGTCCATTCG-3 ' respectively, carry out reverse transcriptase polymerase chain reaction (RT-PCR) amplification, clone obtains silkworm Corazonin acceptor (BmCrzR) cDNA.RT-PCR amplification condition is: 94 DEG C of denaturation 30s, and 58 DEG C of annealing 30s, 72 DEG C extend 2min, carry out 30 circulations.
Take BmCrzRcDNA as template, use forward primer 5 '-AAGCTTATGGACAACGAAGGCAACAGTAC-3 ' and reverse primer 5 '-GGATCCTTATAACAAACTAATGTTCTGTCCAT-3 ' respectively, carry out pcr amplification, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 2min, 30 circulations, add rTaq enzyme to continue to extend 15min, obtain DNA fragmentation.Agarose gel electrophoresis reclaims target DNA fragment, after HindIII/BamHI enzyme is cut, is connected with mammalian expression vector pFlag-CMV-3', obtains pFLAG-BmCrzR.
Take pFLAG-BmCrzR as template, two Auele Specific Primer 5 '-AAGCTTATGAACTCAGAAACAATAAACGACAC-3 ' (primer 1) and 5 '-GGATCCTTAACAGTTCGTGTTGAAGTATGTTTC-3 ' (primer 2) is used to carry out pcr amplification, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, obtain DNA fragmentation.It is the target DNA fragment of 893bp that agarose gel electrophoresis reclaims the size obtaining purifying, as reaction template.
2 μ g reaction template, 2 μ L10 × T7 reaction buffers, 2 μ LATP liquid, 2 μ LCTP liquid, 2 μ LGTP liquid, 2 μ LUTP solution and 2 μ LT7 enzyme mixtures (above-mentioned raw materials derives from TaKaRa company) are in vitro added successively one, then bi-distilled water to the 20 μ L of nuclease free is supplemented, abundant mixing, be positioned over 37 DEG C of reaction 16h, be cooled to normal temperature; Then in above-mentioned mixed solution, add 1 μ LTURBODNA enzyme (above-mentioned raw materials derives from TaKaRa company), after mixing, at 37 DEG C, hatch 15min; Then the bi-distilled water of 30 μ L nuclease free, 30 μ L7.5M lithium chloride and 50mMEDTA are added again, termination reaction; Then the centrifugal 15min of 15000rpm at 4 DEG C, removing supernatant liquor, adds 1mL70% washing with alcohol; Then the centrifugal 15min of 15000rpm at 4 DEG C, removes 70% alcoholic supernatant, is dissolved by the bi-distilled water of precipitation nuclease free, obtains BmCrzRdsRNA.
The 15 μ gBmCrzRdsRNA obtained are injected into silkworm larva belly in the 3rd day four ages, and mulberry leaf are raised, five glutonous stage body weight of silkworm larva in age comparatively check plot body weight of silkworm larva increases by 24.9%.
Embodiment 3:
Total serum IgE is extracted from the domestic natural silk gland in the 3rd day five ages, then reverse transcription obtains the 1st chain cDNA: dissect and collect the domestic natural silk gland in the 3rd day five ages, use Trizol test kit to extract total serum IgE, then with reverse transcription the 1st chain cDNA synthetic agent, reverse transcription is carried out to total serum IgE; Its reverse transcription condition is: 65 DEG C of sex change 5min, and 42 DEG C of reactions 60min, 70 DEG C of deactivation 15min, obtain the 1st chain cDNA.
With the 1st chain cDNA for template, use forward primer 5 '-AAGCTTGCCACCATGGACAACGAAGGCAACAGTAC-3 ' and reverse primer 5 '-GGATCCCGTAACAAACTAATGTTCTGTCCATTCG-3 ' respectively, carry out reverse transcriptase polymerase chain reaction (RT-PCR) amplification, clone obtains silkworm Corazonin acceptor (BmCrzR) cDNA.RT-PCR amplification condition is: 94 DEG C of denaturation 30s, and 58 DEG C of annealing 30s, 72 DEG C extend 2min, carry out 30 circulations.
Take BmCrzRcDNA as template, use forward primer 5 '-AAGCTTATGGACAACGAAGGCAACAGTAC-3 ' and reverse primer 5 '-GGATCCTTATAACAAACTAATGTTCTGTCCAT-3 ' respectively, carry out pcr amplification, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 2min, 30 circulations, add rTaq enzyme to continue to extend 15min, obtain DNA fragmentation.Agarose gel electrophoresis reclaims target DNA fragment, after HindIII/BamHI enzyme is cut, is connected with mammalian expression vector pFlag-CMV-3', obtains pFLAG-BmCrzR.
Take pFLAG-BmCrzR as template, two Auele Specific Primer 5 '-AAGCTTATGAACTCAGAAACAATAAACGACAC-3 ' (primer 1) and 5 '-GGATCCTTAACAGTTCGTGTTGAAGTATGTTTC-3 ' (primer 2) is used to carry out pcr amplification, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, obtain DNA fragmentation.It is the target DNA fragment of 893bp that agarose gel electrophoresis reclaims the size obtaining purifying, as reaction template.
2 μ g reaction template, 2 μ L10 × T7 reaction buffers, 2 μ LATP liquid, 2 μ LCTP liquid, 2 μ LGTP liquid, 2 μ LUTP solution and 2 μ LT7 enzyme mixtures (above-mentioned raw materials derives from TaKaRa company) are in vitro added successively one, then bi-distilled water to the 20 μ L of nuclease free is supplemented, abundant mixing, be positioned over 37 DEG C of reaction 16h, be cooled to normal temperature; Then in above-mentioned mixed solution, add 1 μ LTURBODNA enzyme (above-mentioned raw materials derives from TaKaRa company), after mixing, at 37 DEG C, hatch 15min; Then the bi-distilled water of 30 μ L nuclease free, 30 μ L7.5M lithium chloride and 50mMEDTA are added again, termination reaction; Then the centrifugal 15min of 15000rpm at 4 DEG C, removing supernatant liquor, adds 1mL70% washing with alcohol; Then the centrifugal 15min of 15000rpm at 4 DEG C, removes 70% alcoholic supernatant, is dissolved by the bi-distilled water of precipitation nuclease free, obtains BmCrzRdsRNA.
The 20 μ gBmCrzRdsRNA obtained are injected into silkworm larva belly in the 3rd day four ages, and mulberry leaf are raised, five glutonous stage body weight of silkworm larva in age comparatively check plot body weight of silkworm larva increases by 25.7%.
As seen from the above-described embodiment, the present invention improves silkworm larva body weight, obvious technical effects by regulation and control silkworm metabolic way, and this material for research silkworm is machine-processed with energy metabolism, improve silkworm physiological function etc. further, has active effect.
Claims (7)
1. improve a method for silkworm larva body weight, it is characterized in that:
1) dissect and collect domestic natural silk gland in 3-4 days five age, therefrom extracting total serum IgE, then reverse transcription obtains the 1st chain cDNA;
2) with the 1st chain cDNA for template, by reverse transcriptase polymerase chain reaction (RT-PCR) amplification, clone obtains silkworm Corazonin acceptor (BmCrzR) cDNA;
3) with BmCrzRcDNA be template again, obtain BmCrzRDNA fragment by pcr amplification, agarose gel electrophoresis reclaims target DNA fragment, after HindIII/BamHI enzyme is cut, be connected with mammalian expression vector pFlag-CMV-3', obtain pFLAG-BmCrzR;
4) take pFLAG-BmCrzR as template, obtain pFLAG-BmCrzRDNA fragment by pcr amplification, it is the target DNA fragment of 893bp that agarose gel electrophoresis reclaims the size obtaining purifying, as reaction template;
5) reaction template is mixed with 10 × T7 reaction buffer, ATP liquid, CTP liquid, GTP liquid, UTP solution, T7 enzyme react, then repeatedly process and obtain BmCrzRdsRNA;
6) to the silkworm abdomen injection 10-20 μ gBmCrzRdsRNA in 2-3 days four age, with mulberry leaf continue raise, obtain weight increase five age glutonous stage silkworm larva body.
2. a kind of method improving silkworm larva body weight according to claim 1, is characterized in that:
Described step 1) be specially: dissect and collect the domestic natural silk gland in 3-4 days five age, using Trizol test kit to extract total serum IgE, then with reverse transcription the 1st chain cDNA synthetic agent, reverse transcription is carried out to total serum IgE; Its reverse transcription condition is: 65 DEG C of sex change 5min, and 42 DEG C of reactions 60min, 70 DEG C of deactivation 15min, obtain the 1st chain cDNA.
3. a kind of method improving silkworm larva body weight according to claim 1, is characterized in that:
Described step 2) be specially: with the 1st chain cDNA for template, use forward primer 5 '-AAGCTTGCCACCATGGACAACGAAGGCAACAGTAC-3 ' and reverse primer 5 '-GGATCCCGTAACAAACTAATGTTCTGTCCATTCG-3 ' to carry out reverse transcriptase polymerase chain reaction (RT-PCR) amplification respectively, clone obtains silkworm Corazonin acceptor (BmCrzR) cDNA.
4. a kind of method improving silkworm larva body weight according to claim 3, is characterized in that:
Described reverse transcriptase polymerase chain reaction (RT-PCR) amplification condition is: 94 DEG C of denaturation 30s, and 58 DEG C of annealing 30s, 72 DEG C extend 2min, carry out 30 circulations.
5. a kind of method improving silkworm larva body weight according to claim 1, is characterized in that:
Described step 3) obtain BmCrzRDNA fragment and be in the following ways specifically: take BmCrzRcDNA as template, forward primer 5 '-AAGCTTATGGACAACGAAGGCAACAGTAC-3 ' and reverse primer 5 '-GGATCCTTATAACAAACTAATGTTCTGTCCAT-3 ' is used to carry out pcr amplification respectively, its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 2min, 30 circulations, add rTaq enzyme and continue to extend 15min, obtain DNA fragmentation.
6. a kind of method improving silkworm larva body weight according to claim 1, is characterized in that:
Described step 4) obtain pFLAG-BmCrzRDNA fragment and be in the following ways specifically: take pFLAG-BmCrzR as template, pcr amplification is carried out respectively with two Auele Specific Primer 5 '-AAGCTTATGAACTCAGAAACAATAAACGACAC-3 ' and 5 '-GGATCCTTAACAGTTCGTGTTGAAGTATGTTTC-3 ', its pcr amplification condition is: 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, obtain DNA fragmentation.
7. a kind of method improving silkworm larva body weight according to claim 1, is characterized in that:
Described step 5) be specially:
5.1) 1-2 μ g reaction template, 2 μ L10 × T7 reaction buffers, 2 μ LATP liquid, 2 μ LCTP liquid, 2 μ LGTP liquid, 2 μ LUTP solution and 2 μ LT7 enzyme mixtures are in vitro added successively one;
5.2) supplement bi-distilled water to the 20 μ L of nuclease free, fully mix, be positioned over 37 DEG C of reaction 16h, be cooled to normal temperature;
5.3) in above-mentioned mixed solution, add 1 μ LTURBODNA enzyme, after mixing, at 37 DEG C, hatch 15min;
5.4) bi-distilled water of 30 μ L nuclease free, 30 μ L7.5M lithium chloride and 50mMEDTA are added again, termination reaction;
5.5) the centrifugal 15min of 15000rpm at 4 DEG C, removing supernatant liquor, adds 1mL70% washing with alcohol;
5.6) the centrifugal 15min of 15000rpm at 4 DEG C, removes 70% alcoholic supernatant, is dissolved by the bi-distilled water of precipitation nuclease free, obtains BmCrzRdsRNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510874406.7A CN105349548A (en) | 2015-12-02 | 2015-12-02 | Method for increasing weight of Bombyx mori larvae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510874406.7A CN105349548A (en) | 2015-12-02 | 2015-12-02 | Method for increasing weight of Bombyx mori larvae |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105349548A true CN105349548A (en) | 2016-02-24 |
Family
ID=55325610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510874406.7A Pending CN105349548A (en) | 2015-12-02 | 2015-12-02 | Method for increasing weight of Bombyx mori larvae |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105349548A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110372776A (en) * | 2019-06-25 | 2019-10-25 | 浙江大学 | A method of improving silk cocoon cocoon shell weight |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1703625A (en) * | 2002-08-06 | 2005-11-30 | 法赫马西亚及普强有限公司 | Drosophila g protein coupled receptors, nucleic acids, and methods related to the same |
| US20060019347A1 (en) * | 2004-07-21 | 2006-01-26 | Ambrx, Inc. | Biosynthetic polypeptides utilizing non-naturally encoded amino acids |
-
2015
- 2015-12-02 CN CN201510874406.7A patent/CN105349548A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1703625A (en) * | 2002-08-06 | 2005-11-30 | 法赫马西亚及普强有限公司 | Drosophila g protein coupled receptors, nucleic acids, and methods related to the same |
| US20060019347A1 (en) * | 2004-07-21 | 2006-01-26 | Ambrx, Inc. | Biosynthetic polypeptides utilizing non-naturally encoded amino acids |
Non-Patent Citations (2)
| Title |
|---|
| 杨静文 等: "昆虫神经肽Corazonin 及其受体的研究进展", 《蚕业科学》 * |
| 杨静文: "家蚕corazonin受体信号转导机制及生理功能的研究", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110372776A (en) * | 2019-06-25 | 2019-10-25 | 浙江大学 | A method of improving silk cocoon cocoon shell weight |
| CN110372776B (en) * | 2019-06-25 | 2020-12-11 | 浙江大学 | Method for increasing cocoon layer quantity of silkworm cocoons |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104542497B (en) | Fly larvae two step cultural method under room temperature and cryogenic conditions | |
| CN103525920B (en) | A kind of molecule marking method for seed selection Altai Sheep meat production and application thereof | |
| CN105349547A (en) | Method for increasing weight of Bombyx mori pupa bodies | |
| CN104099330A (en) | Sheep lambing number trait related molecular marker and application thereof | |
| CN107549059B (en) | Hybrid breeding method for female sturgeon and male paddlefish | |
| Zhang et al. | Molecular cloning and expression analysis of extra sex combs gene during development in Macrobrachium nipponense | |
| CN107619836B (en) | System for reducing activity 20E concentration in spinning period, changing silk pupa nutrition distribution proportion and increasing silkworm cocoon yield, application and method | |
| CN103430883A (en) | Method for efficiently breeding South American white shrimps | |
| CN105349548A (en) | Method for increasing weight of Bombyx mori larvae | |
| CN105875463A (en) | Pond high-yield culture method for Tai-lake white fish | |
| CN108048464B (en) | siRNA for enabling first-rate silkworm to produce non-diapause eggs, preparation method and application | |
| CN103999808A (en) | Bait feeding method for farmed hippocampus erectus | |
| CN105420255A (en) | Toxoptera citricida chitin synthase gene and dsRNA thereof | |
| CN101347458B (en) | Preparation method of sheep X/Y sperm separation freezing sperm and use thereof | |
| CN105230548B (en) | A kind of Tilapia mossambica high-yield cultivation method | |
| CN101933471B (en) | Method for delaying sex reversal of finless eels | |
| CN101503704A (en) | Transgenic method for cultivated silkworm diapause variety | |
| CN103004649A (en) | Ecology regulation and control method for delaying sex reversal time of eels | |
| CN106973868B (en) | Method for eliminating silkworm diapause egg diapause fertility by extremely early corona and product thereof | |
| CN110272916B (en) | Technical method for increasing egg laying number of female silkworm moths | |
| CN105622254A (en) | Preparation method and use method of bed soil for dry-raising of rice seedlings | |
| CN102228132A (en) | Air-drying method for improving quality of alfalfa | |
| CN103636563B (en) | A kind of preparation method being beneficial to leech and passing the winter | |
| CN107034176A (en) | A kind of nutrient solution and its cultural method for improving in vitro culture porcine oocytes developmental potentiality | |
| CN104067961A (en) | Directional breeding method of female parents of finless eel |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160224 |