Transgenic silkworm purefaction method based on heat shock induced parthenogenesis

Abstract

The invention relates to the technical field of transgenic bombyx mori pure linearization, and discloses a transgenic bombyx mori pure linearization method based on heat shock induced parthenogenesis, which comprises the following steps: S1, bombyx mori breeding and silkworm egg collection; s2, performing parthenogenesis induction treatment and subculture; s3, performing parthenogenesis condition investigation and data analysis. According to the heat shock induced parthenogenesis-based transgenic bombyx mori pure lineation method, animals with specific gene modification, such as animals capable of producing heterologous gene proteins, are bred by using a parthenogenesis method, pure lineation can be faster than natural mating breeding, and transgenosis bombyx mori is subcultured by using a parthenogenesis induction technology, so that the transgenic bombyx mori can be bred more quickly. According to the method, the process of hybridization breeding is avoided, the time of pure line is saved, the breeding cost is reduced, and a foundation is laid for improving the transgenosis efficiency, the genetic stability and the product safety.

Description

Technical Field

[0001] The present invention relates to the technical field of transgenic silkworm inbred lines, and specifically to a method for inbreeding transgenic silkworms based on heat shock-induced parthenogenesis. Background Technique

[0002] Silkworm breeding has a very long history and is one of the important economic insects. Transgenic technology can be used to explore the functions of silkworm genes and the regulatory mechanisms of silkworm growth and development, and is the main auxiliary means for studying silkworm genes. Transgenic silkworms can be used as bioreactors and have more advantages than other organisms. Specific genes are transferred into silkworms to produce recombinant proteins including monoclonal antibodies, cytokines, and collagen.

[0003] The subculture of transgenic silkworm lines is relatively cumbersome and complex, and a stable transgenic line needs to be established through operations such as hybridization and repeated screening. To obtain a silkworm variety with excellent traits, certain economic value, and production application prospects, long-term cultivation and selection are required after obtaining the initial transgenic individuals. In the current feeding methods, transgenic silkworms must be mated and reproduced at least once a year. Since the development of gametes is related to chromosome recombination, it is very difficult to reproduce and preserve transgenic silkworms for a long time without genetic changes. Therefore, the present invention proposes a method for inbreeding transgenic silkworms based on heat shock-induced parthenogenesis to solve the above problems. Summary of the Invention

[0004] (I) Technical Solution

[0005] The present invention provides the following technical solution: A method for inbreeding transgenic silkworms based on heat shock-induced parthenogenesis, comprising the following steps:

[0006] S1. Silkworm rearing and egg collection

[0007] The transgenic silkworms after hibernation are reared with conventional mulberry leaves in an environment with a temperature of 25 °C and a humidity of 80%-90%. After the female moths emerge, the emerged female moths are placed at 25 °C for 12 hours, protected at 5 °C for 3 days, and then placed on a gauze to lay eggs directly. After laying eggs for 1 day, they are collected. The female moths are then placed on a new gauze to lay eggs for another day, and then the second batch of eggs is collected.

[0008] S2. Parthenogenesis induction treatment and subculture

[0009] Immediately after obtaining the silkworm eggs, parthenogenetic induction was carried out using the "warm water method" in the heat shock induction method. The gauze bag containing the silkworm eggs was immersed in warm water at 46 °C in a digital display constant temperature water bath. The impregnation time was set at 16, 17, 18, 19, and 20 minutes, with a total of 5 treatment areas. 15 female moths were used in each treatment area. After immersion, they were transferred to room temperature water and placed for 5 minutes. After drying, they were placed in a constant temperature incubator at 16 °C and 80% humidity. After 3 days, they were taken out. The time when the eggs were taken out was equivalent to the oviposition time of the sexually reproductive moths of the conventional variety. This generation was designated as F1. The laid eggs were transferred to a constant temperature incubator at 25 °C and 80% humidity for subsequent observation. If newly hatched silkworm larvae emerged, they were reared until eclosion. The above steps were repeated for the successive subculture of parthenogenetic silkworms, designated as F2, F3, …, and so on.

[0010] S3. Investigation of parthenogenetic reproduction and data analysis

[0011] The situation of silkworm eggs in each generation was observed under a microscope, and the total number of silkworm eggs, the number of colored eggs, the number of hatched eggs, and the number of emerged moths were counted. The colored egg rate, hatching rate, and emerged moth rate were calculated. The development of the parthenogenetic silkworms was observed. A hand-held fluorescent lamp was used to observe the eye color of the transgenic silkworm moths during the moth stage. An electronic balance was used to weigh the cocoons of the parthenogenetic silkworms.

[0012] Preferably, in the step S1, the transgenic silkworm used is a hybrid obtained by cross-breeding a transgenic silkworm expressing the major ampullate silk protein of Nephila clavata and a silkworm variety with a fibroin gene defect.

[0013] Preferably, in the step S1, the transgenic silkworm is marked with red fluorescent protein, and its compound eyes are red.

[0014] Preferably, in the step S2, the temperatures for treatment by the warm water method are 46 °C, 43 °C, and 40 °C, and the treatment time is 10 minutes respectively.

[0015] Preferably, in the step S3, the coloring situation of the parthenogenetic induction eggs of the F1 generation of transgenic silkworms is statistically analyzed to verify the feasibility of the parthenogenetic induction method for transgenic silkworm varieties and explore the influence of different impregnation times on the incidence of parthenogenesis.

[0016] Preferably, in the step S3, the hatching situation of the parthenogenetic induction eggs of the F1 generation of transgenic silkworms is statistically analyzed to verify the feasibility of using the "warm water method" for the cultivation of transgenic silkworm varieties and explore the possibility of successive subculture of parthenogenetic silkworms of this variety.

[0017] Preferably, in the step S3, the F1 generation of emerged transgenic silkworm moths is allowed to lay eggs naturally, and then parthenogenetic induction is carried out to obtain F2 generation silkworm eggs. The coloring situation of the parthenogenetic induction eggs of the F2 generation of transgenic silkworms is statistically analyzed.

[0018] (2) Beneficial effects

[0019] Compared with the prior art, the present invention provides a method for purifying transgenic silkworm lines based on heat shock-induced parthenogenesis, which has the following beneficial effects:

[0020] The method for purifying transgenic silkworm lines based on heat shock-induced parthenogenesis uses the parthenogenesis method to breed animals with specific genetic modifications, such as animals that can produce heterologous gene proteins, and can be purified into pure lines faster than natural mating reproduction. The transgenic silkworms are subcultured using the parthenogenesis induction technique, avoiding the process of cross-breeding and selection, saving the time for purification, reducing the breeding cost, and laying a foundation for improving transgenic efficiency, genetic stability, and product safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a view showing the partial coloring of the F1 generation of parthenogenetic eggs induced by heat shock in the transgenic silkworm of the present invention;

[0022] Figure 2 It is a comparison chart of different periods of the F1 generation of transgenic silkworms induced by parthenogenesis in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] Please refer to Figure 1 and Figure 2 , the present invention provides a technical solution: a method for purifying transgenic silkworm lines based on heat shock-induced parthenogenesis, comprising the following steps:

[0025] S1. Silkworm rearing and egg collection

[0026] The transgenic silkworms after hibernation are reared with conventional mulberry leaves in an environment with a temperature of 25 °C and a humidity of 80%-90%. After the female moths emerge, the emerged female moths are placed at 25 °C for 12 hours, protected at 5 °C for 3 days, and then placed on a gauze to lay eggs directly. After laying eggs for 1 day, they are collected. The female moths are then placed on a new gauze to lay eggs for another day, and then the second batch of eggs is collected.

[0027] S2. Parthenogenesis induction treatment and subculture

[0028] Immediately after obtaining the silkworm eggs, parthenogenetic induction was carried out using the "warm water method" in the heat shock induction method. The gauze bag containing the silkworm eggs was immersed in warm water at 46 °C in a digital display constant temperature water bath. The immersion time was set at 16, 17, 18, 19, and 20 minutes for a total of 5 treatment areas. 15 female moths were used for each treatment area. After immersion, they were transferred to room temperature water and placed for 5 minutes. After drying, they were placed in a constant temperature incubator at a temperature of 16 °C and a humidity of 80%. After 3 days, they were taken out. The time when the eggs were taken out was equivalent to the oviposition time of sexually reproductive moths of conventional varieties. This generation was designated as F1. The eggs laid were transferred to a constant temperature incubator at a temperature of 25 °C and a humidity of 80% for subsequent observation. If newly hatched silkworm larvae emerged, they were reared until emergence. The above steps were repeated for the successive culture of parthenogenetic silkworms, designated as F2, F3, …, and so on.

[0029] S3. Investigation of parthenogenetic reproduction and data analysis

[0030] The situation of silkworm eggs in each generation was observed under a microscope. The total number of silkworm eggs, the number of colored eggs, the number of hatched eggs, and the number of emerged moths were counted. The colored egg rate, hatching rate, and emergence rate were calculated. The development of parthenogenetic silkworms was observed. A hand-held fluorescent lamp was used to observe the eye color of transgenic silkworm moths during the moth stage. An electronic balance was used to weigh the cocoons of parthenogenetic silkworms.

[0031] Furthermore, in step S1, the transgenic silkworms used were hybridized and selected from transgenic silkworms expressing the major ampullate silk protein of Nephila clavata and silkworms of a silk fibroin gene-deficient variety.

[0032] Furthermore, in step S1, the transgenic silkworms were marked with red fluorescent protein, and their compound eyes were red.

[0033] Furthermore, in step S2, the temperatures for treatment by the warm water method were 46 °C, 43 °C, and 40 °C, and the treatment time was 10 minutes respectively.

[0034] Furthermore, in step S3, the coloring situation of parthenogenetic induction eggs of the F1 generation of transgenic silkworms was counted to verify the feasibility of the parthenogenetic induction method for transgenic silkworm varieties and explore the influence of different immersion times on the incidence of parthenogenesis. The results are shown in the table.

[0035] The results show that the parthenogenesis induction method can cause parthenogenesis in transgenic silkworms. The incidence of parthenogenesis is the lowest at 16 minutes, being 73.31%; the highest at 18 minutes, being 95.00%; the incidences of parthenogenesis at 17 minutes, 19 minutes, and 20 minutes are also relatively high, being 91.26%, 93.27%, and 93.32% respectively. The reason for the lowest incidence of parthenogenesis at 16 minutes may be that the materials used in the research contain the bloodline of multivoltine silkworm breeds. Multivoltine silkworm breeds are in areas with relatively high temperatures in many places, have good heat resistance, and are less sensitive to heat stimulation. Therefore, heat stimulation for a shorter time cannot achieve good results.

[0036]

[0037] Furthermore, in step S3, the hatching situation of the parthenogenesis-induced eggs of the F1 generation of transgenic silkworms was statistically analyzed to verify the feasibility of the "hot water method" for the cultivation of transgenic silkworm varieties, and at the same time explore the possibility of the successive cultivation of parthenogenetic silkworms of this variety. The results are shown in the table.

[0038] The results show that the obtained parthenogenesis-induced eggs of transgenic silkworms can be successfully hatched. 6 newly hatched silkworm larvae were successfully hatched from the parthenogenesis-induced eggs, 3 were obtained with the dipping time of 18 minutes and 19 minutes respectively, and the hatching rates were 0.099% and 0.076% respectively. Among the 6 silkworms, 1 became a moth, and the moth emergence rate was 0.025%. 3 died during the newly hatched silkworm larva stage, 1 died during the 3rd instar stage, and 1 died during the 4th instar stage. Observing the parthenogenetic silkworm that became a moth, there was no deformity, the health condition was good, the cocoon layer weight was 0.88 g, and the compound eyes of this silkworm were red.

[0039]

[0040] Furthermore, in step S3, the F1 generation of adult silkworms was allowed to lay eggs naturally, and then parthenogenesis induction was carried out to obtain F2 generation silkworm eggs. The coloring situation of the parthenogenesis-induced eggs of the F2 generation of transgenic silkworms was statistically analyzed. The results are shown in the table.

[0041] The results show that the incidence of the first batch of parthenogenesis-induced eggs in the F2 generation is 81.17%, and the second batch is 64.00%. The parthenogenesis probability of the F2 generation has decreased compared with the F1 generation, and the parthenogenesis rate of the first batch is higher than that of the second batch. Since the moth emergence rate of the F1 generation is too low, further experiments are needed to determine the reliability of the data.

[0042]

[0043] The method for purifying transgenic silkworm lines based on heat shock-induced parthenogenesis conducts parthenogenesis experiments on transgenic silkworm varieties based on three common indicators: the coloring rate of silkworm eggs (the incidence of parthenogenesis), the hatching rate, and the adult emergence rate, and explores the influence of different impregnation times on the incidence of parthenogenesis in silkworm eggs. The results show that the parthenogenesis induction method adopted in the present invention can cause parthenogenesis in transgenic silkworms. Although the incidence of parthenogenesis and the hatching rate are relatively low, it confirms the possibility of heat shock-induced parthenogenesis in transgenic silkworms, and confirms the possibility of solving the problem of purifying transgenic silkworm lines by means of heat shock-induced parthenogenesis in transgenic silkworm eggs, laying a foundation for improving transgenic efficiency, genetic stability, and product safety.

[0044] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for pure-line transgenic silkworms based on heat shock-induced parthenogenesis, characterized in that: The following steps are involved: S1. Silkworm breeding and silkworm egg collection The transgenic silkworms after being accelerated were raised with regular mulberry leaves in an environment with a temperature of 25 degrees Celsius and a humidity of 80%-90%. After the female moths emerged, they were placed at 25 degrees Celsius for 12 hours, protected at 5 degrees Celsius for 3 days, and then placed on gauze to lay eggs directly. The eggs were collected one day after laying, and the female moths continued to lay eggs on new gauze for one day, and then the second batch of eggs were collected; S2. Parthenogenetic induction and subculture Immediately after obtaining silkworm eggs, use the "warm soup method" in the heat shock induction method to induce parthenogenesis. Soak the gauze bag containing silkworm eggs in 46 degrees Celsius warm water in a digital constant temperature water bath. The soaking time is set to 16, 17, 18, 19, and 20 minutes in 5 treatment areas. Each treatment area is tested with 15 female moths. After soaking, transfer them to room temperature water for 5 minutes. After drying, place them in a constant temperature incubator with a temperature of 16 degrees Celsius and a humidity of 80%. Take them out after 3 days. The time when the eggs are taken out is equivalent to the egg-laying time of conventional sexual reproduction moths. This generation is recorded as F1. The laid eggs are transferred to a constant temperature incubator with a temperature of 25 degrees Celsius and a humidity of 80% for subsequent observation. If ant silkworms are hatched, they are raised until they emerge. Repeat the above steps to carry out subculture of parthenogenetic silkworms, which are recorded as F2, F3, ..., and so on. S3. Parthenogenesis survey and data analysis Use a microscope to observe the condition of silkworm eggs of each generation, count the total number of silkworm eggs, the number of colored eggs, the number of hatched eggs, and the number of adult moths, calculate the colored egg rate, hatching rate, and adult moth rate, observe the development of silkworms obtained by parthenogenesis, use a handheld fluorescent lamp to observe the eye color of transgenic silkworms in the moth stage, and use an electronic balance to weigh the cocoons of silkworms obtained by parthenogenesis.

2. The method for pure-line transgenic silkworm based on heat shock-induced parthenogenesis according to claim 1, characterized in that: In the step S1, the transgenic silkworm used is a hybrid obtained by cross-breeding a transgenic silkworm expressing the Nephila clavata major ampullate silk protein with a silk fibroin gene-deficient variety of silkworm.

3. The method for pure-line transgenic silkworm based on heat shock-induced parthenogenesis according to claim 1, characterized in that: In the step S1, the transgenic silkworm is marked with red fluorescent protein, and the compound eyes of the silkworm are red.

4. The method for pure-line transgenic silkworm based on heat shock-induced parthenogenesis according to claim 1, characterized in that: In step S2, the temperatures of the warm water treatment are 46 degrees Celsius, 43 degrees Celsius and 40 degrees Celsius, and the treatment time is 10 minutes respectively.

5. The method for pure-line transgenic silkworm based on heat shock-induced parthenogenesis according to claim 1, characterized in that: In the step S3, the coloring of the F1 generation parthenogenesis induced eggs of the transgenic silkworm is statistically analyzed to verify the feasibility of the parthenogenesis induction method for transgenic silkworm varieties and explore the effects of different immersion times on the incidence of parthenogenesis.

6. The method for pure-line transgenic silkworm based on heat shock-induced parthenogenesis according to claim 1, characterized in that: In step S3, the hatching status of the F1 generation parthenogenetic induced eggs of transgenic silkworms is statistically analyzed to verify the feasibility of the "warm soup method" for breeding transgenic silkworm varieties, and to explore the possibility of subculture of parthenogenetic silkworms of this variety.

7. The method for pure-line transgenic silkworm based on heat shock-induced parthenogenesis according to claim 1, characterized in that: In step S3, the F1 generation adult silkworms are allowed to continue to lay eggs naturally, and then parthenogenesis is induced to obtain F2 generation silkworm eggs, and the coloring of the parthenogenesis induced eggs of the F2 generation of transgenic silkworms is statistically analyzed.

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