Breeding method and application of bisexual silkworm variety capable of stably expressing Cas9-gfp fusion protein

Abstract

The invention discloses a breeding method and application of a bivolcanic silkworm variety capable of stably expressing Cas9-gfp fusion protein, the method comprises the following steps: taking a polymorphic silkworm variety Nistari-Cas9gfp capable of stably expressing Cas9-gfp fusion protein as a female parent and bivolcanic silkworms as a male parent, and hybridizing to obtain an F1 generation; backcrossing the F1 generation with the divalent silkworms to obtain a BC1 generation, laterally crossing the BC1 generation with Nistari-Cas9gfp silkworms, feeding, screening egg rings corresponding to moths with green fluorescence, and feeding for subculture; and when the male parent with the homozygous and divalent economic characters is obtained, selfing and selecting a homozygous individual. Compared with the prior art, the breeding method disclosed by the invention has the advantages that (1) the breeding method disclosed by the invention is used for breeding a bisexual silkworm variety capable of efficiently and stably expressing Cas9-gfp fusion protein, so that the variety emits green fluorescence with the wavelength of 506nm under the excitation of light with the wavelength of 496nm; meanwhile, the variety has practical and economic characters of a bigelous silkworm variety; and (2) the breeding method plays an important role in analyzing the diapause regulation and control molecular mechanism and gene functions of the silkworms and cultivating excellent practical varieties.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for breeding excellent varieties of economic insects, in particular to a breeding method and application of a bivoltine silkworm variety that stably expresses a Cas9-gfp fusion protein. Background Art

[0002] Insect diapause is an ecological adaptation strategy formed by insects in the long process of evolution in response to seasonal environmental changes such as temperature and photoperiod. Diapause insects often show growth stagnation and fat accumulation, which is of great significance for their resistance to adverse environments such as low temperatures in winter, high temperatures in summer and food shortages. Therefore, by utilizing the diapause characteristics, many agricultural and forestry pests are able to continue their populations in harsh environments, which has become an important cause of pest outbreaks. As a model insect of Lepidoptera, the silkworm is a typical egg-diapausing insect. Research on the diapause physiology and molecular regulation mechanism of the silkworm is not only of great scientific significance for understanding the seasonal adaptation of insects, but also provides a theoretical basis for the development of green pest control technology based on diapause regulation.

[0003] Currently, CRISPR / Cas9-based gene editing technology in silkworms has been widely used 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 enabled the visual screening of gene-edited silkworms. However, the Cas9-GFP expression vector is designed for the polyvoltine silkworm line Nistari, which lacks a diapause period. This line is not only small in size, exhibits a low cocoon layer rate, and produces little silk, significantly different from the bivoltine lines used in production, but also lacks a diapause period, making it unsuitable for research on the molecular mechanisms regulating diapause in bivoltine silkworms. Summary of the Invention

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

[0005] Technical solution: A method for breeding a bivoltine silkworm variety that stably expresses the Cas9-gfp fusion protein, comprising: using the polyvoltine silkworm variety Nistari-Cas9gfp that stably expresses the Cas9-gfp fusion protein as the female parent and the bivoltine silkworm as the male parent, hybridizing to obtain the F1 generation; backcrossing the F1 generation with the bivoltine silkworm to obtain the BC1 generation, and cross-crossing the BC1 generation with the Nistari-Cas9gfp silkworm; after rearing, selecting the egg circles corresponding to moths with green fluorescence, and rearing successive generations; and when the economic traits reach the same level as the bivoltine male parent, self-crossing to select homozygous individuals.

[0006] The polyvoltine silkworm variety Nistari-Cas9gfp is a polyvoltine four-dormant variety of the Indian system. Its eggs hatch evenly, its cocoons are golden yellow, its cocoons are small in shape, its moths emerge in large numbers, and its mating performance is good. It expresses the Cas9-gfp fusion protein, and its larvae, pupae, and adults can all be observed to emit green fluorescence under the excitation of light with a wavelength of 496nm.

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

[0008] S1, using the polyvoltine silkworm variety Nistari-Cas9gfp stably expressing the Cas9-gfp fusion protein as the female parent and the divoltine silkworm for production as the male parent, and hybridizing to obtain the F1 generation;

[0009] The S2 and F1 generations were backcrossed to produce bivoltine silkworms to obtain the BC1 generation;

[0010] The S3 and BC1 generations were reared conventionally using the moth-area half-split method, and then cross-crossed to the polyvoltine silkworm variety Nistari-Cas9gfp. The egg circles corresponding to the moths with green fluorescence were selected for rearing the next generation, and then backcrossed to produce divoltine silkworms to obtain the BC2 generation.

[0011] S4, repeating steps S2 and S3 for more than 5 generations until the economic traits are the same as those of the bivoltine silkworm varieties used in production;

[0012] The individuals screened by S5 and S4 were self-pollinated to homozygous, and after 4-6 generations of screening, a variety of silkworms that can stably express Cas9-gfp fusion protein and have the same economic traits as those of commercial divoltine silkworms was bred.

[0013] Preferably, the bivoltine silkworm varieties include: Qiufeng, Baiyu, Jingsong, Haoyue, P33, P50, Dazao. The above-mentioned bivoltine silkworm varieties, such as "Qiufeng", are Chinese bivoltine four-sleeping varieties.

[0014] Preferably, the green fluorescence is observed under the excitation condition of light with a wavelength of 496 nm.

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

[0016] A bivoltine silkworm variety stably expressing Cas9-gfp fusion protein bred by any of the above methods.

[0017] The application of the above-mentioned silkworm varieties as models for analyzing the molecular mechanisms of silkworm diapause regulation.

[0018] The application of the above-mentioned silkworm varieties as models for studying the functions of diapause-related genes in silkworms.

[0019] The application of the above-mentioned silkworm varieties in the breeding of excellent practical varieties.

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

[0021] Beneficial effects: (1) The breeding method of the present invention cultivates a bivoltine silkworm variety that can efficiently and stably express the Cas9-gfp fusion protein, so that the variety emits green fluorescence with a wavelength of 506 nm under the excitation of light with a wavelength of 496 nm; at the same time, the variety has the practical and economic traits of a bivoltine silkworm variety; (2) The breeding method plays an important role in analyzing the molecular mechanism of silkworm diapause regulation, gene function and the breeding of excellent and practical varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a fluorescence microscope photo of a female moth of the polyvoltine silkworm variety Nistari-Cas9gfp expressing the Cas9-gfp fusion protein mating with a male moth of the bivoltine silkworm variety Qiufeng.

[0023] Figure 2 Photos of pupae (left) and cocoons (right) of the polyvoltine silkworm strain Nistari-Cas9gfp.

[0024] Figure 3 Photos of the pupae (top) and cocoons (bottom) of the bivoltine silkworm variety Qiufeng.

[0025] Figure 4 The images of the silkworm moth "Autumn CZ" obtained by cultivation in Example 1 are obtained under bright field (left) and fluorescence (right);

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

[0027] The following examples further illustrate 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 substance of the present invention are intended to fall within the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional means well known to those skilled in the art.

[0028] Example 1

[0029] The production of the present embodiment uses the bivoltine silkworm variety Qiufeng as an example, which is stored in the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences.

[0030] A method for breeding a bivoltine silkworm variety that stably expresses a Cas9-gfp fusion protein, the method comprising the following steps:

[0031] S1, using the polymorphic silkworm variety Nistari-Cas9gfp stably expressing Cas9-gfp fusion protein as the mother parent (e.g. Figure 2 As shown), the production uses the bivoltine silkworm Qiufeng as the male parent (as shown Figure 3 As shown), hybridization to obtain F1 generation (as Figure 1 shown);

[0032] The S2 and F1 generations were backcrossed with Qiufeng to obtain the BC1 generation;

[0033] The S3 and BC1 generations were reared conventionally using the moth-area half-split method, and cross-crossed to the polyvoltine silkworm strain Nistari-Cas9gfp. The egg circles corresponding to the moths with green fluorescence were selected for rearing, and then backcrossed to Qiufeng to obtain the BC2 generation.

[0034] S4, repeat steps S2 and S3 for more than 5 generations until the economic traits are the same as autumn harvest;

[0035] Individuals selected from S5 and S4 were self-crossed to homozygous, and after 4-6 generations of screening, a strain was developed that stably expresses the Cas9-gfp fusion protein and exhibits economic traits comparable to those of commercial divoltine silkworms. Specific traits include: a cocoon weight of 1.4g-1.6g, a cocoon layer percentage of 20.41%, a silk length of 1100m, and good unwinding. Finally, the bivoltine silkworm "Qiu CZ" with stable expression of Cas9-gfp was bred after self-pollination and selection for more than 4-6 generations. Its main characteristics are: a Chinese bivoltine four-dorm variety; silkworm eggs hatch uniformly, with a practical hatching rate of more than 90.00% for good eggs; the body color of the strong silkworms is bluish-white, with limited spots, they eat mulberry quickly, and they are active; all instars sleep and wake up at the same time, and the mature ones emerge at the same time; they lay cocoons quickly, mostly in the middle layer; the cocoons are white, short and oval in shape, with moderate wrinkles and tight cocoon layers; the total cocoon weight is 1.4g-1.6g, the cocoon layer rate is 20.41%, the cocoon silk length is 1100m, and it is well unwinded; the moths emerge uniformly, with good mating performance; the female moths have good egg-laying performance, the eggs have good adhesion and are laid smoothly; the number of good eggs per moth is about 500, and the Cas9-gfp fusion protein is expressed. This variety of silkworm (larvae, pupae, and adults) can be observed to have green fluorescence (such as Figure 4 、 5 shown).

Claims

1. A method for breeding a bivoltine silkworm variety that stably expresses a Cas9-gfp fusion protein, characterized in that: The method uses a polyvoltine silkworm variety Nistari-Cas9gfp that stably expresses a Cas9-gfp fusion protein as a female parent and a bivoltine silkworm as a male parent, hybridizes to obtain an F1 generation; the F1 generation is backcrossed with the bivoltine silkworm to obtain a BC1 generation, the BC1 generation is cross-crossed with the Nistari-Cas9gfp silkworm, and after rearing, an egg circle corresponding to a moth with green fluorescence is selected, and rearing is continued; When the economic traits are the same as those of the dimorphic male parent, self-pollination is performed to select homozygous individuals.

2. The method for breeding a bivoltine silkworm variety that stably expresses the Cas9-gfp fusion protein according to claim 1, characterized in that: The method specifically comprises the following steps: S1, using the polyvalent silkworm variety Nistari-Cas9gfp that stably expresses the Cas9-gfp fusion protein as the female parent and the divalent silkworm used for production as the male parent, and hybridizing to obtain the F1 generation; The S2 and F1 generations were backcrossed to produce bivoltine silkworms to obtain the BC1 generation; The S3 and BC1 generations were reared conventionally using the moth-area half-division method, and cross-crossed with the polyvoltine silkworm variety Nistari-Cas9gfp. The egg circles corresponding to the moths with green fluorescence were selected for rearing the successive generations, and then backcrossed with divoltine silkworms to obtain the BC2 generation. S4, repeating steps S2 and S3 for more than 5 generations until the economic traits are the same as those of the bivoltine silkworm varieties used for production; The individuals selected from S5 and S4 were self-pollinated to be homozygous, and after 4-6 generations of screening, a variety of silkworms that can stably express Cas9-gfp fusion protein and have the same economic traits as those of commercial divoltinous silkworms was bred.

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

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

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

6. A bivoltine silkworm variety stably expressing Cas9-gfp fusion protein obtained by breeding by the method described in any one of claims 1 to 5.

7. Use of the silkworm variety described in claim 6 as a model for analyzing the molecular mechanism of silkworm diapause regulation.

8. Use of the silkworm variety according to claim 6 as a model for studying the function of silkworm diapause-related genes.

9. Use of the silkworm variety described in claim 6 in the breeding of excellent practical varieties.

10. Use of the method according to any one of claims 1 to 5 in constructing a green pest control system based on diapause regulation.

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