CN106973868B - Method for eliminating silkworm diapause egg diapause fertility by extremely early corona and product thereof - Google Patents
Method for eliminating silkworm diapause egg diapause fertility by extremely early corona and product thereof Download PDFInfo
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Abstract
The invention relates to a method for preparing silkworm diapause variety transgenic silkworms by using very early corona, which comprises the following steps: carrying out corona treatment on the diapause silkworm eggs laid for 2-4 h in an electric field with the voltage intensity of 8-12 kV for 30-120 sec, then disinfecting the corona-treated silkworm eggs, then carrying out microinjection, disinfecting again, carrying out incubation and hatching to obtain G0 generation silkworm moths, then carrying out selfing or backcrossing on the obtained G0 generation silkworm moths to obtain G1 generation silkworm eggs, screening transgenic individuals, and finally carrying out breeding and passage on the transgenic individuals to obtain transgenic varieties of the diapause varieties of the silkworms.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for removing diapause of silkworm eggs by very early stage corona, silkworm eggs for removing diapause by very early stage corona, and a method for preparing transgenic silkworms by using the silkworm eggs for removing diapause.
Background
The transgenic technology is an important technology for realizing gene function verification and genetic improvement in modern biological research. In the post-genome era, transgenic technology has become mature day by day and plays a great role in functional genome research, directed genetic breeding, establishment of human disease models, development of bioreactors and the like.
Silkworm is one of the most successful and long-lasting economic insects for human utilization in the world at present, and domestication and breeding of silkworm have about five thousand years history. The silkworm is also a lepidopteran insect model, and the completion of a silkworm genome frame map, a fine map and re-sequencing marks that the research of silkworm genomics has entered the post-genome, namely, the functional genome era. The large-scale study of important gene functions by using highly efficient and stable techniques is an important issue in the functional genome era of silkworms. Meanwhile, the silkworm is an ideal insect factory, and the development of the silk gland bioreactor by using the modern genetic engineering technology has huge market prospect. The silkworm transgenic technology is undoubtedly the most powerful weapon in exploring silkworm gene function, promoting silkworm bioreactor development, expanding silkworm industry chain and promoting the practical technological achievement process.
However, in the reports so far, most transgenic experiments use silkworm diversified varieties as materials, mainly because eggs produced by the diversified silkworm varieties are non-diapause eggs, the eggs can continuously develop and hatch under proper temperature and humidity without any manual treatment, the characteristic greatly facilitates the operation control in the transgenic experiments and further experimental research after the transgenosis, but the diversified silkworm varieties have the characteristic of poor economic characters such as few silks, poor silk quality and the like compared with the fertility-retarding practical silkworm varieties commonly used in production, and the like, summarizing, the utilization of the diversified silkworm varieties as transgenic breeding materials has 2 obvious defects, namely ① diversified economic characters are poor, the additional characters created by the transgenosis cannot reach the standard of direct popularization in terms of yield and quality, the transgenic silkworm varieties only can be used as original silkworm breeding materials, the transgenic silkworm varieties as one relevant character of the silkworms are not easy to be continuously bred and used as transgenic breeding materials for continuous breeding of large-scale silkworm breeding, and the transgenic silkworms cannot be used as a dominant character for continuous breeding of the silkworms in the traditional transgenic breeding method for increasing the fertility of the silkworms in the fertility-increasing the fertility of the silkworms in the breeding period, so that the transgenic silkworms are only capable of obtaining the transgenic silkworms in the traditional transgenic breeding materials, and the transgenic silkworms in the breeding method for increasing the breeding of continuous breeding of the transgenic silkworms in the breeding of the transgenic silkworms, and the breeding of the silkworms, so that the transgenic silkworms, the transgenic silk.
At present, practical silkworm strains produced in the silkworm industry are almost diapause normalized or bivoltine strains. The 2 nd defect of the diversification silkworm variety as a transgenic breeding material can be overcome by utilizing the transgenic research of the common silkworm diapause strain (such as Dazao and the like), and all the defects of the diversification silkworm variety as the transgenic breeding material can be overcome by utilizing the transgenic research of the stock of the practical silkworm diapause strain (such as Dongting/Bibo and the like). Therefore, the transgenic research of the silkworm diapause variety has extremely important theoretical and practical significance. In actual production, although the diapause of the silkworm eggs laid by the diapause strain silkworm moths can be effectively relieved by a conventional instant pickling method at room temperature for about 16-20 hours, because the most effective silkworm embryo transgenic microinjection time is within 3-6 hours after the eggs are laid, small injection holes are left on the silkworm eggs after microinjection operation, and if the silkworm eggs are treated by the instant pickling method after the injection, hydrochloric acid permeates the silkworm eggs to cause death, the genetic transformation research of the silkworm diapause strain cannot be realized at all. The fatal defect brings great limitation to the development of transgenic operation technology of diapause silkworm varieties.
In early studies, chinese patent publication No. CN101195834A explored a method of pickling immediately after the fertilization of silkworm eggs and succeeded in obtaining a corresponding transgenic method; the Chinese patent with publication number CN101195833A discloses that the voltinism of next generation of silkworm diapause variety is changed by using special incubation conditions, and the conventional silkworm transgenic technology is combined, so that the barrier of the silkworm diapause variety transgenic technology is ingeniously broken through, and transgenic silkworms are effectively produced; chinese patent publication No. CN101503704A discloses that a transgenic method for further improving the low-temperature incubation transgenic method is finally established by strictly controlling incubation, feeding, cocoon protection and mating modes of parent silkworms under special environmental conditions to obtain non-diapause offspring silkworms by using the relationship between the environmental conditions of the parent silkworms and the fertility retardation of the offspring silkworms. So far, the literature reports that silkworm diapause varieties are used as materials to create transgenes at home and abroad almost use the early pickling transgenic method or the low-temperature incubation transgenic method. Although the three patents mentioned above have provided corresponding transgenic methods for diapause, respectively, and most transgenes for delayed breeding can be achieved, the methods provided have respective limitations and disadvantages: the early pickling transgenic method (CN101195834A) for silkworm lag breeding requires immediate microinjection after pickling, so that the incubation rate after injection is extremely low due to two continuous stimulations to silkworm eggs, and the effect of early pickling is greatly different among varieties, thus being mainly suitable for the Japanese variety with strong acid-resistant stimulation. Although the hatching rate of the silkworm after injection is comparable to that of a multi-chemical silkworm egg, the low-temperature incubation transgenic method (CN101195833A) for the delayed breeding of silkworms has very large difference between varieties, is mainly suitable for Hua series varieties with sensitive chemical change influenced by temperature, and has poor effect of removing diapause of first-chemical and second-chemical silkworm varieties in the day series. In addition, because the low-temperature incubation period of the parent silkworm eggs is long and the physique of the non-diapause offspring silkworms obtained after the low-temperature incubation is poor, the survival rate of the silkworms after injection incubation is seriously influenced. The improved silkworm slow breeding low-temperature incubation transgenic method (CN101503704A) has certain improvement on the diapause removing effect of a Japanese line bivoltine silkworm variety, but has the same defects as the low-temperature incubation transgenic method (application number: CN101195833A) of silkworm slow breeding. These disadvantages greatly limit the large-scale application of silkworm transgenic technology in functional gene research and bioreactor development.
Therefore, in order to overcome and solve the limitations of the methods provided in the above patents, it is necessary to develop a silkworm slow breeding transgenic method which is simpler, more efficient and suitable for all silkworm varieties. The simple and efficient silkworm diapause seed transgenic method is also an urgent need for establishing a stable and efficient silkworm transgenic technology system, promoting large-scale functional gene research and realizing rapid industrialization of silkworm transgenic achievements.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for removing the diapause of silkworm eggs by very early corona, which comprises performing corona treatment (referred to as very early corona method in this specification) on the diapause eggs of silkworms within a certain time period before the development of silkworm egg embryos and the formation of embryo ribbons, i.e., 2 to 4 hours after the eggs lay, to obtain the silkworm eggs with diapause removed, and then creating transgenic silkworms by referring to the publicly-known and public transgenic method of microinjection of non-diapause silkworm egg embryos, thereby ingeniously breaking through the obstacle of the transgenic technology of the silkworm diapause varieties and simply and efficiently making transgenic silkworms; the second object of the present invention is to provide a diapause-released silkworm egg prepared by the above method; the third purpose of the present invention is to provide a method for preparing transgenic silkworms using eggs for diapause relief.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the method for eliminating the diapause egg fertility of the silkworms by the aid of the corona at the very early stage comprises the following steps: and (3) carrying out corona treatment on the diapause silkworm eggs laid for 2-4 h in an electric field with the voltage intensity of 8-12 kV for 30-120 sec.
In the present invention, the time for the corona treatment is 60 sec.
In the present invention, the electric field strength is 10 kV.
In the invention, a corona instrument with a positive plate and a negative plate at an interval of 15mm, a pole needle length of 10mm and a needle pitch of 8mm is used in the corona treatment.
2. The obtained silkworm egg has diapause relieving effect.
3. The method for preparing the transgenic silkworm by utilizing the eggs laid by the diapause relief comprises the steps of sterilizing, microinjecting, sterilizing again, accelerating incubation and hatching to obtain G0 generation silkworm moths, selfing or backcrossing the obtained G0 generation silkworm moths to obtain G1 generation silkworm eggs, screening transgenic individuals, and finally feeding the transgenic individuals for passage to obtain transgenic varieties of diapause varieties of the silkworms.
In the invention, the microinjection is to inject the transposition carrier plasmid into the disinfected silkworm eggs from the center of the abdominal surface of the silkworm eggs by using a microinjection instrument after the silkworm eggs lay for 3-6 h, and to seal the remained holes for injecting the egg shells by using nontoxic glue.
In the invention, the sterilization is to soak the silkworm eggs subjected to corona treatment in distilled water for 2-3 min, and then place the silkworm eggs in 35-37% formaldehyde steam for sterilization for 5 min.
In the invention, the microinjection method also comprises a disinfection step, wherein the disinfection is carried out in 35-37% formaldehyde steam for 5 min.
In the invention, the incubation is carried out under the conditions that the temperature is 25 ℃ and the relative humidity is more than 85% until incubation.
The corona treatment technology is combined with the silkworm embryo microinjection technology, the fluorescence detection technology and the molecular biology operation technology, so that the bottleneck of the silkworm diapause variety transgenic technology is ingeniously broken through, and a simple, convenient and efficient silkworm diapause variety transgenic technical route method is established. The silkworm diapause variety transgenic method provided by the invention is simple, convenient and effective, and is suitable for all silkworm diapause varieties.
The invention has the beneficial effects that: the invention discloses a method for preparing silkworm diapause variety transgenic silkworms by using very early stage corona, which comprises the steps of obtaining the optimum condition for removing diapause by using very early stage corona, removing diapause needing silkworm egg injection by using very early stage corona, and finally referring to a preparation method of multivoltic strain transgenic silkworm microinjection, thereby effectively breaking through the bottleneck of the silkworm diapause variety transgenic technology. The operation method for eliminating the diapause of the silkworm eggs by the aid of the extremely early corona and the obtaining of the optimal conditions are the first originations of the method, the method for eliminating the diapause of the silkworm eggs by the aid of the extremely early corona is further combined with a silkworm transgenic technology based on embryo microinjection, and transgenic silkworms of diapause varieties are successfully created by the aid of the extremely early corona transgenic method for the first time.
The method has the advantages that the method can directly remove the diapause of the eggs in the current generation for injection through corona treatment, has short breeding period, has strong physical property of the hatched silkworms after injection and high survival rate, can overcome all the defects of transgenic silkworms of diversified lines, has the obvious advantages that ① silkworms diapause varieties, particularly practical varieties adopted in production, have better economic properties and excellent target properties of exogenous or endogenous transgenes, have the yield and quality of target products far exceeding those of transgenic lines of diversified lines when the silkworms are used as bioreactors, can greatly save the yield and quality of transgenic lines of the cultivated silkworms in the production of silkworms and greatly save the transgenic lines of the cultivated silkworms in the production of the silkworms, can be used as a transgenic line of the transgenic silkworms in the long-term production, can be used as a transgenic line of the transgenic breeding of the silkworms in the production of the silkworms, can be used as a transgenic line of the silkworm diapause, can be used as a transgenic line of the silkworm products of the silkworm diapause, can be more favorable for the silkworm, can be used as a transgenic line of the silkworm breeding of the silkworm, can be used for the transgenic line of the silkworm, can be directly used for the silkworm breeding of the silkworm, can be used for the silkworm, can be used as a transgenic line of the silkworm, can be used for the silkworm breeding of the transgenic line of the silkworm breeding of the silkworm, can be used for the silkworm breeding of the silkworm, the silkworm breeding of the silkworm breeding of the silkworm, can be more, can be simply used for the silkworm, the transgenic lines of the silkworm, the silkworm of the silkworm, can be used for the silkworm of the.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a schematic diagram of a transgenic technological process for very early stage corona elimination of diapause of silkworm eggs.
FIG. 2 is a diagram showing the statistical results of silkworm egg hatchability obtained by treating different corona treatment time lengths and different post-egg corona time points of very early corona treatment for removing diapause of silkworm eggs.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1 Explanation of optimum duration of Corona treatment for very early Corona treatment to solve silkworm egg diapause
Collecting the eggs of a diapause silkworm variety 932 (provided by Guangxi silkworm industry technical promotion general station) as a material, and forming a plurality of groups according to about 100 eggs/component after delivery for 2 h; according to the flow of figure 1, the silkworm eggs are placed between a positive plate and a polar needle of a GZ-01 type corona instrument (the specification of the corona instrument is that the specification of a metal shell is 20 multiplied by 12cm, a voltage display can display 0-15kV, a power line is connected with 220V alternating current, the length of the polar needle is 10mm, the needle pitch is 8mm, the specification of the polar plate is 15 multiplied by 8cm, and the interval between the positive polar plate and the negative polar plate is 15mm), and all groups of silkworm eggs are treated in an electric field with the voltage intensity of 10kV according to different time periods of 5sec, 30sec, 1min (namely 60sec) and 2min (namely 120sec), and 3 groups of silkworm eggs are treated under each time period condition; the treated eggs were incubated at 25 ℃ under 85% relative humidity for about 12 days, and the number of eggs finally hatched in each treatment group was counted and the hatching rate was calculated, and the results are shown in table 1.
TABLE 1 statistics of results of treating silkworm eggs for different durations of corona treatment to remove diapause
The results showed that the average hatching rate of the eggs obtained at the time of corona treatment for 5sec was only 83.83%, while the average hatching rates of the eggs obtained at the time of corona treatment for 30sec, 60sec and 120sec all reached 94% or more. Analysis shows that the silkworm egg hatching rate obtained by the corona treatment time of 30-120 sec is obviously higher than that obtained by the corona treatment time of 5sec (figure 2, A), and the corona treatment time of 30-2 min is the optimal corona treatment time for removing silkworm egg diapause.
Example 2 Example Corona treatment Ex very early Corona treatment to remove diapause of silkworm eggs Ex optimal post-egg corona treatment time Point Condition exploration
Taking the silkworm eggs of a diapause silkworm variety 932 (provided by Guangxi silkworm industry technical promotion general station) as materials, respectively collecting the silkworm eggs which are 30min, 60min (namely 1h), 120min (namely 2h), 180min (namely 3h) and 240min (namely 4h) after delivery, and forming a plurality of groups according to about 100 silkworm eggs/component; according to the flow of figure 1, placing silkworm eggs between a positive plate and a polar needle of a GZ-01 type corona instrument, carrying out corona treatment on each group of silkworm eggs for 1min in an electric field with the voltage intensity of 10kV, and collecting 3 groups of silkworm eggs at each time point; the treated eggs were incubated at 25 ℃ under 85% relative humidity for about 12 days, and the number of eggs finally hatched in each treatment group was counted and the hatching rate was calculated, and the results are shown in table 2.
TABLE 2 statistics of results of treating silkworm eggs at different post-egg corona time points to relieve diapause
The results show that the average hatching rate of the silkworm eggs obtained by the corona treatment 30min after egg laying is 87.57%, the average hatching rate of the silkworm eggs obtained by the corona treatment 60min after egg laying is 85.59%, and the average hatching rates of the silkworm eggs obtained by the corona treatment 120min, 180min and 240min after egg laying are all more than 95%. Analysis shows that the silkworm egg hatching rate obtained by corona treatment for 2-4 h after egg laying is obviously higher than that obtained by corona treatment for 30min-1h after egg laying (figure 2, B), and the corona treatment for 2-4 h after egg laying is the optimal post-egg corona treatment time period for eliminating silkworm egg diapause.
Application example 1
Silkworm diapause variety Dazao is used as raw material, silkworm eggs obtained by normal breeding, mating and collecting according to the method shown in figure 1 are subjected to corona treatment for 45sec in an electric field with the voltage intensity of 10kV 2h10min after delivery. Then, 4h after delivery, an Eppendorf microinjector is used to inject 10nL-15nL, a piggyBac vector and an auxiliary vector which have a total concentration of 400ng/uL and are marked by red fluorescent protein with specific promoter of nerve and eye into a general syringe togetherRemoving diapause of 532 silkworm eggs by premature acid dipping, sealing with nontoxic glue, and incubating in high humidity environment at 25 deg.C and 95% relative humidity to obtain 186G 0 generation silkworm with incubation rate of 34.96%. Collecting and feeding the silkworm moth with artificial feed to obtain 123G 0 generation silkworm moths, selfing or backcrossing to obtain 73G 1 generation silkworm eggs, and breeding
Observing and screening by an electric macroscopic fluorescence microscope to obtain 13 positive moth rings, obtaining 69 transgenic silkworms of the silkworm diapause variety Dazao, wherein the transformation efficiency is 17.81 percent.
Application example 2
Silkworm diapause variety Dongting is used as raw material, silkworm eggs obtained by normal breeding, mating and collecting treatment according to the method shown in figure 1 are subjected to corona treatment for 1min in an electric field with the voltage intensity of 12kV 2h30min after delivery. And then, injecting 10nL-15nL of piggyBac vector and auxiliary vector which have total concentration of 400ng/uL and are provided with red fluorescent protein markers started by nerve and eye specific promoters into 572 grainBac eggs which are released from diapause by an early pickling method by using an Eppendorf microinjector 3h after delivery, sealing the carrier by using non-toxic glue, placing the carrier in a high humidity environment with the relative humidity of 95% at 25 ℃ for incubation, and obtaining 154G 0 generation of newly-hatched silkworms with the incubation rate of 26.92%. Collecting and feeding moth-killing by artificial feed, obtaining 96G 0 generation silkworm moth, selfing or backcrossing to obtain 46G 1 generation silkworm egg, and using
4 positive moth rings are obtained by observing and screening through an electric macroscopic fluorescence microscope, 32 silkworm diapause varieties, namely Dongting transgenic silkworms are obtained, and the transformation efficiency is 8.7%.
Application example 3
Silkworm diapause variety 'Bibo' is used as raw material, silkworm eggs obtained by normal breeding, mating and collecting treatment according to the method shown in figure 1 are subjected to corona treatment for 2min in an electric field with the voltage intensity of 11kV 2h50min after delivery. Followed by Eppendorf microinjection at 5h postnatalThe instrument is characterized in that 10nL-15nL of piggyBac vector and auxiliary vector with red fluorescent protein mark and total concentration of 400ng/uL and with nerve and eye specific promoter are injected into 498 silkworm eggs with early pickling, sealed by nontoxic glue and placed in a high humidity environment with 25 ℃ and 95% relative humidity for incubation, 104G 0 generation silkworms are obtained, and the incubation rate is 20.88%. Collecting and feeding moth with artificial feed to obtain 83G 0 generation silkworm moth, selfing or backcrossing to obtain 32 moth rings G1 generation silkworm eggs, and culturing
The electric macroscopic fluorescence microscope is used for observing and screening to obtain 9 positive moth rings, and transgenic silkworms of 32 silkworm diapause varieties 'green wave' are obtained in total, and the transformation efficiency is 28.13%.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (9)
1. The method for eliminating the diapause egg fertility of the silkworms by the aid of the corona at the very early stage is characterized by comprising the following steps of: the method comprises the following steps: and (3) carrying out corona treatment on the diapause silkworm eggs laid for 2-4 h in an electric field with the voltage intensity of 8-12 kV for 30-120 sec.
2. The method of claim 1, wherein: the corona treatment time was 60 sec.
3. The method of claim 1, wherein: the electric field strength is 10 kV.
4. The method according to any one of claims 1 to 3, wherein: in the corona treatment, a corona instrument with a positive plate and a negative plate at an interval of 15mm, a pole needle length of 10mm and a needle pitch of 8mm is used.
5. A method for producing a transgenic silkworm by using the diapause-released silkworm eggs produced by the method according to any one of claims 1 to 4, characterized in that: sterilizing the silkworm eggs subjected to corona treatment, then performing microinjection, then sterilizing again, then hatching to obtain G0 generation silkworm moths, selfing or backcrossing the obtained G0 generation silkworm moths to obtain G1 generation silkworm eggs, screening transgenic individuals, and finally feeding the transgenic individuals for passage to obtain transgenic varieties of diapause varieties of silkworms.
6. The method of claim 5, wherein: the microinjection is to inject the transposition carrier plasmid into the disinfected silkworm eggs from the center of the abdominal surface of the silkworm eggs by using a microinjection instrument after the silkworm eggs lay for 3-6 hours, and to seal the holes left by injecting the egg shells by using nontoxic glue.
7. The method of claim 5, wherein: and the disinfection is to soak the silkworm eggs subjected to corona treatment in distilled water for 2-3 min, and then place the silkworm eggs in 35-37% formaldehyde steam for disinfection for 5 min.
8. The method of claim 5, wherein: the microinjection method also comprises a disinfection step, wherein the disinfection is carried out in 35-37% formaldehyde steam for 5 min.
9. The method of claim 5, wherein: the incubation is carried out under the conditions that the temperature is 25 ℃ and the relative humidity is more than 85 percent until incubation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710240738.9A CN106973868B (en) | 2017-04-13 | 2017-04-13 | Method for eliminating silkworm diapause egg diapause fertility by extremely early corona and product thereof |
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| CN101703035A (en) * | 2009-11-09 | 2010-05-12 | 苏州大学 | Method for preparing silkworm eggs for transgene of eggs of bivoltine silkworm varieties |
| CN106191111A (en) * | 2016-07-19 | 2016-12-07 | 西南大学 | A kind of method blocking transgenic bombyx mori ovum diapause |
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| CN101703035A (en) * | 2009-11-09 | 2010-05-12 | 苏州大学 | Method for preparing silkworm eggs for transgene of eggs of bivoltine silkworm varieties |
| CN106191111A (en) * | 2016-07-19 | 2016-12-07 | 西南大学 | A kind of method blocking transgenic bombyx mori ovum diapause |
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| 《桑蚕卵电晕人工孵化技术研究》;陈家森等;《蚕桑通报》;19950630;第26卷(第2期);16-17 * |
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