Breeding method and application of bivoltine bombyx mori strain stably expressing cas9-gfp fusion protein

Abstract

The application discloses a breeding method of a bivoltine silkworm variety stably expressing a Cas9-gfp fusion protein and application, the method takes a multivoltine silkworm variety Nistari-Cas9gfp stably expressing a Cas9-gfp fusion protein as a female parent, a bivoltine silkworm as a male parent, and a F1 generation is obtained by hybridization; the F1 generation is backcrossed with the bivoltine silkworm to obtain a BC1 generation, the BC1 generation is sidecrossed with the Nistari-Cas9gfp silkworm, and the corresponding egg ring of the moth with green fluorescence is screened after rearing and subculturing; when economic traits are the same as those of the bivoltine male parent, a homozygous individual is selected by self-crossing.Compared with the prior art, the method has the advantages that (1) the method breeds the bivoltine silkworm variety capable of efficiently and stably expressing the Cas9-gfp fusion protein, so that the variety emits green fluorescence with a wavelength of 506nm under the excitation of light with a wavelength of 496nm; and (2) the method plays an important role in analyzing the diapause regulation molecular mechanism of the silkworm, gene function and excellent practical variety cultivation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for breeding superior varieties of economic insects, specifically a breeding method and application for bimorphic silkworm varieties that stably express the Cas9-gfp fusion protein. Background Technology

[0002] Insect diapause is an ecological adaptation strategy developed by insects over a long period of evolution in response to seasonal environmental changes such as temperature and photoperiod. Diapause insects often exhibit growth retardation and fat accumulation, which is crucial for their resistance to adverse environments such as low winter temperatures, high summer temperatures, and food scarcity. Therefore, by utilizing diapause, many agricultural and forestry pests can maintain their populations in harsh environments, which is a significant reason for pest outbreaks. The silkworm, as the model insect of Lepidoptera, is a typical egg-diapause insect. Studying the physiological and molecular regulatory mechanisms of silkworm diapause is not only of great scientific significance for understanding seasonal adaptation in insects, but also provides a theoretical basis for developing green pest control technologies based on diapause regulation.

[0003] Currently, CRISPR / Cas9-based silkworm gene editing technology has been widely applied in research on gene function, genetic improvement, and the creation of innovative materials. In particular, the construction of transgenic silkworm lines expressing Cas9-gfp has made the screening of gene-edited silkworms visible. However, the expression vector for Cas9-gfp is the Nistrali silkworm line, which is a dimorphic silkworm line without diapause. This line not only has small individuals, low cocoon layer ratio, and low silk yield, which is significantly different from the dimorphic varieties used in production, but also lacks a diapause period, making it unsuitable for research on the molecular mechanisms of diapause regulation in dimorphic silkworms. Summary of the Invention

[0004] Technical problem to be solved: In order to overcome the shortcomings of existing technologies, a bipolar silkworm variety that can stably express the Cas9-gfp fusion protein is to be bred, so as to play an important role in elucidating the molecular mechanism of silkworm diapause regulation, gene function and the breeding of superior and practical varieties; in view of this, the present invention provides a breeding method and application for a bipolar silkworm variety that stably expresses the Cas9-gfp fusion protein.

[0005] Technical Solution: A breeding method for a dimorphic silkworm variety that stably expresses the Cas9-gfp fusion protein. The method uses the dimorphic silkworm variety Nistrali-Cas9gfp, which stably expresses the Cas9-gfp fusion protein, as the female parent and the dimorphic silkworm as the male parent, to obtain the F1 generation. The F1 generation is backcrossed with dimorphic silkworms to obtain the BC1 generation. The BC1 generation is then sidecrossed with Nistrali-Cas9gfp silkworms. After rearing, the egg circles corresponding to moths with green fluorescence are selected, and the generations are continued. When the economic traits are the same as those of the dimorphic male parent, homozygous individuals are selected through self-pollination.

[0006] The aforementioned multi-evolving silkworm variety Nistrali-Cas9gfp is an Indian system multi-evolving four-molting variety. The silkworm eggs hatch uniformly, the cocoons are golden yellow, the cocoons are small and slender, the moths emerge in large numbers, and the mating performance is good. It expresses the Cas9-gfp fusion protein, and its larvae, pupae, and adults can all be observed to have green fluorescence under light excitation at a wavelength of 496 nm.

[0007] Preferably, the method specifically includes the following steps:

[0008] S1. Using the multi-evolving silkworm variety Nistrali-Cas9gfp, which stably expresses the Cas9-gfp fusion protein, as the maternal parent and the dimorphic silkworm for production as the paternal parent, the F1 generation was obtained by hybridization.

[0009] S2 and F1 generation backcrosses were used to produce bipolar silkworms to obtain BC1 generation;

[0010] S3 and BC1 generations were conventionally reared using the half-division method in moth areas. They were laterally crossed with the multi-evolving silkworm variety Nistrali-Cas9gfp, and the corresponding egg circles with green fluorescent moths were selected for rearing and sub-generation. Then, they were backcrossed with production-grade di-evolving silkworms to obtain the BC2 generation.

[0011] S4. Repeat steps S2 and S3 for 5 generations or more until the economic traits are the same as those of the dimorphic silkworm varieties used in production.

[0012] Individuals selected in S5 and S4 were self-crossed homozygous, and after 4-6 generations of selection, a variety of silkworms that can stably express the Cas9-gfp fusion protein and have the same economic traits as the dimorphic silkworms used in production were bred.

[0013] Preferably, the biphasic silkworm varieties include: Qiufeng, Baiyu, Jingsong, Haoyue, P33, P50, and Dazao. The aforementioned biphasic silkworm varieties, such as "Qiufeng," are Chinese biphasic, four-molting varieties.

[0014] Preferably, the green fluorescence is obtained by observation under excitation conditions of light with a wavelength of 496 nm.

[0015] Preferably, the wavelength of the green fluorescence is 506 nm.

[0016] Dimorphic silkworm varieties that stably express the Cas9-gfp fusion protein, obtained by any of the methods described above.

[0017] The above-mentioned silkworm varieties are used as models to analyze the molecular mechanisms of silkworm diapause regulation.

[0018] The above-mentioned silkworm varieties are used as models for studying the function of genes related to diapause in silkworms.

[0019] The above-mentioned silkworm varieties are used in the breeding of superior and practical varieties.

[0020] The application of any of the methods described above in constructing a green pest control system based on diapause regulation.

[0021] Beneficial effects: (1) The breeding method described in this invention has cultivated a bipolar silkworm variety that can express the Cas9-gfp fusion protein efficiently and stably, so that the variety emits green fluorescence with a wavelength of 506nm when excited by light with a wavelength of 496nm; at the same time, the variety has the practical and economical traits of a bipolar silkworm variety; (2) The breeding method described in this invention has played an important role in elucidating the molecular mechanism of silkworm diapause regulation, gene function and the cultivation of excellent and practical varieties. Attached Figure Description

[0022] Figure 1 This is a photo of the mating of a female Nistrali-Cas9gfp silkworm expressing the Cas9-gfp fusion protein and a male Qiufeng silkworm, a dimorphic silkworm.

[0023] Figure 2 These are photos of the pupa (left) and cocoon (right) of the polymorphic silkworm variety Nistrali-Cas9gfp.

[0024] Figure 3 These are photos of the pupa (top) and cocoon (bottom) of the biparietal silkworm variety Qiufeng;

[0025] Figure 4 The images show the silkworm moths of the "Autumn CZ" variety obtained in Example 1 under bright field (left) and fluorescence (right) conditions.

[0026] Figure 5 This is a picture of the larvae of "Autumn CZ" under a fluorescence microscope. Detailed Implementation

[0027] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are all within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0028] Example 1

[0029] The bipolar silkworm variety used in this embodiment is Qiufeng, which is preserved at the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences.

[0030] A breeding method for bipolar silkworm varieties stably expressing the Cas9-gfp fusion protein, the method comprising the following steps:

[0031] S1. Using the multi-variant silkworm variety Nistrali-Cas9gfp, which stably expresses the Cas9-gfp fusion protein, as the maternal parent (e.g., ... Figure 2 As shown), the bipolar silkworm Qiufeng is used as the male parent in production (e.g., Figure 3 As shown), crossbreeding yields the F1 generation (e.g. Figure 1 (as shown);

[0032] S2 and F1 generations were crossbred back into Qiufeng to obtain BC1 generation;

[0033] S3 and BC1 generations were conventionally reared using the half-division method in moth areas. They were laterally crossed with the multi-evolving silkworm variety Nistrali-Cas9gfp, and the corresponding egg circles with green fluorescent moths were selected for rearing and then backcrossed with Qiufeng to obtain the BC2 generation.

[0034] S4. Repeat steps S2 and S3 for 5 generations or more until the economic traits are the same as those of Qiufeng.

[0035] Individuals selected from S5 and S4 were self-crossed homozygous, and after 4-6 generations of selection, a variety capable of stably expressing the Cas9-gfp fusion protein and exhibiting economic traits similar to those of dimorphic silkworms used in production was bred. Specific traits include: total cocoon weight 1.4g-1.6g, cocoon layer ratio 20.41%, cocoon silk length 1100m, and good unwinding. Finally, through self-pollination and selection over 4-6 generations, a bimorphic silkworm variety named "Qiu CZ" was successfully bred, exhibiting stable expression of Cas9-gfp. Its main characteristics are: a Chinese bimorphic four-molting variety; uniform egg hatching with a usable hatching rate of over 90.00% for high-quality eggs; robust silkworms are bluish-white with limited mottled markings, feed quickly, and are active; all molting stages emerge uniformly, with mature silkworms emerging simultaneously; rapid cocooning, primarily producing mid-layer cocoons; whitish cocoons with a short, oval shape, moderate shrinkage, and a tight cocoon layer; total cocoon weight of 1.4-1.6g, cocoon layer ratio of 20.41%, cocoon silk length of 1100m, and good unwinding; simultaneous emergence of moths and good mating performance; good oviposition performance in female moths, with good egg adhesion and smooth egg attachment; approximately 500 high-quality eggs per moth; expressing the Cas9-gfp fusion protein; and green fluorescence (e.g., green fluorescence in larvae, pupae, and adults) can be observed under excitation at a wavelength of 496nm. Figure 4 , 5 (As shown).

Claims

1. A breeding method of a bivoltine Bombyx mori strain stably expressing a Cas9-gfp fusion protein, characterized by, The method uses the multi-evolving silkworm variety Nistrali-Cas9gfp, which stably expresses the Cas9-gfp fusion protein, as the maternal parent and the dimorphic silkworm as the paternal parent to obtain the F1 generation; the F1 generation is backcrossed with dimorphic silkworms to obtain the BC1 generation; the BC1 generation is then sidecrossed with Nistrali-Cas9gfp silkworms; after rearing, the egg circles corresponding to moths with green fluorescence are selected, and the generation is then raised for sub-generation. Once the economic traits are identical to those of the dimorphic parent, homozygous individuals are selected through self-crossing.

2. The breeding method for bipolar silkworm varieties stably expressing Cas9-gfp fusion protein according to claim 1, characterized in that, The method specifically includes the following steps: S1. Using the multi-evolving silkworm variety Nistrali-Cas9gfp, which stably expresses the Cas9-gfp fusion protein, as the maternal parent and the dimorphic silkworm for production as the paternal parent, the F1 generation was obtained by hybridization. S2 and F1 generation backcrosses were used to produce bipolar silkworms to obtain BC1 generation; S3 and BC1 generations were conventionally reared using the half-division method in moth areas. They were laterally crossed with the multi-evolving silkworm variety Nistrali-Cas9gfp, and the corresponding egg circles with green fluorescent moths were selected for rearing and sub-generation. Then, they were backcrossed with production-grade di-evolving silkworms to obtain the BC2 generation. S4. Repeat steps S2 and S3 for 5 generations or more until the economic traits are the same as those of the dimorphic silkworm varieties used in production. Individuals selected in S5 and S4 were self-crossed homozygous, and after 4-6 generations of selection, a variety of silkworms that can stably express the Cas9-gfp fusion protein and have the same economic traits as the dimorphic silkworms used in production were bred.

3. The breeding method for bipolar silkworm varieties stably expressing the Cas9-gfp fusion protein according to claim 1, characterized in that, The dimorphic silkworm varieties include: Qiufeng, Baiyu, Jingsong, Haoyue, P33, P50, and Dazao.

4. The breeding method for a dimorphic silkworm variety stably expressing the Cas9-gfp fusion protein according to claim 1, characterized in that, The green fluorescence was observed under excitation conditions of light with a wavelength of 496 nm.

5. The breeding method for a dimorphic silkworm variety stably expressing the Cas9-gfp fusion protein according to claim 1, characterized in that, The wavelength of the green fluorescence is 506 nm.

6. The application of the method described in any one of claims 1-5 in constructing a green pest control system based on diapause regulation.

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