CN112011540B - Silkworm binary transgenic system for knocking down Seroin1 gene and preparation method of silkworm pure naked pupa variety - Google Patents

Abstract

The invention discloses a silkworm binary transgenic system for knocking down Seroin1 gene and a preparation method of silkworm pure naked pupa variety, which comprises the steps of mediating the knockout expression of Seroin1 gene through CRISPR/Cas9 system to obtain silkworm strain with serin 1 gene function defect, leading the silkworm to become naked pupa due to loss of spinning and cocooning capabilities, shortening the growth cycle of larva, shortening the feeding time, reducing the consumption of mulberry leaves, feed and the like, enlarging pupa body, greatly increasing pupa, solving the problem of manpower and material resource consumption caused by silkworm pupa cocoon cutting by eating, improving the production benefit and having important application value.

Description

Silkworm binary transgenic system for knocking down Seroin1 gene and preparation method of silkworm pure naked pupa variety

Technical Field

The invention relates to the technical field of biology, in particular to a silkworm binary transgenic system for knocking down Seroin1 gene, and also relates to a preparation method of pure silkworm naked pupa varieties.

Background

Silkworm is one of the heavy lepidoptera insects, and undergoes four developmental stages of egg-larva-leap-imago in life. The silkworm has mild sexual conditions, small size, fast growth, easy feeding and high mulberry silk conversion rate, and is an important economic source for people in partial areas of China. In the initial domestication stage of silkworms, silkworms (pupas) are only used as food, and silks are used in the textile and clothing industry. Since silkworm cocoons have been an important economic income for silkworm farmers, many researchers are working on improving and increasing the silk yield of silkworms. But neglects the selling of silkworm chrysalis to be still used as economic support. The silkworm has a plurality of naked pupa strains, such as Nd-x, Nd-s and the like, but the varieties are not naked pupas in a strict sense, and can still spin and cocoon in the clustering period, only the cocoon layer is thinner than the normal strain, the silkworm pupa is not convenient to utilize due to the existence of the silkworm cocoon, manpower and material resources are greatly wasted, and the existing silkworm strain has a certain distance away from the development and the utilization of edible and medicinal silkworm strains.

The silkworm pupa has high nutritive value and is rich in protein, fatty acid, vitamins and the like. The silkworm pupa protein content is more than 50%, which is far higher than that of common food, and the silkworm pupa protein is composed of 18 amino acids, wherein 8 amino acids necessary for human body have high content, balanced nutrition and proper proportion, and is a high-quality insect protein. In addition, the efficacy and action of silkworm pupae are not considered in a small amount: 1. the pupa Bombycis can be used for culturing Cordyceps. Cordyceps militaris can enhance immunity of human body, and has anticancer, antifatigue, anoxia resisting, and antiaging effects; 2. silkworm pupa can kill parasite and treat malnutrition. The silkworm chrysalis can be eaten and used as a medicine, is a traditional Chinese medicine with excellent efficacy, has main efficacy of treating malnutrition by killing parasites, can enter spleen channels and stomach channels, is usually used for treating infantile malnutrition, fever and ascariasis of human beings, and has very obvious treatment efficacy. Can also promote the production of body fluid to quench thirst; 3. regulating metabolism. Can regulate sugar and fat metabolism of organism. 4. Preventing three highs. The pupa Bombycis oil has blood lipid reducing effect, and the pupa Bombycis oil purified product can be used for treating hypercholesterolemia, and has significant effects in reducing cholesterol and improving liver function. The silkworm chrysalis with proper amount has good auxiliary treatment effect on patients with hypertension, hyperlipidemia, chronic hepatitis and malnutrition. In addition, the silkworm pupa contains a broad-spectrum immune substance, and has certain efficacy on the treatment of cancer. Bombyx Batryticatus can also be used for treating infantile convulsion and night cry, spasm and wind evil, and pinching mouth.

The advent of the CRISPR/Cas9 system has revolutionized traditional fully protein-based genome editing tools. CRISPR/Cas9 recognizes specific sequences mainly through interaction with DNA, and Cas9 protein cleaves the target sequence. Compared with ZFN and TALEN, the CRISPR/Cas9 has the advantages of high specificity, simple design, convenient operation and the like. At present, it is most widely used, and is just like a sharp instrument for genome editing. Therefore, whether naked pupa strains can be obtained by knocking out the key genes of the cocoons by using the CRISPR/Cas9 system has important significance for developing edible silkworm strains.

Disclosure of Invention

In view of this, the Cas protein and the gRNA, which are two components of the CRISPR/Cas9 system, can be introduced into the body or cells in various ways, such as direct injection, chemical agent transfection, electrotransformation and the like, in the forms of DNA (expression vector or co-expression vector of each of them), RNA (in vitro transcribed into mRNA respectively) and protein complex (Cas9 protein and gRNA). Besides, transgenic strains of Cas9 protein and gRNA can be respectively constructed, the target gene can be knocked out through hybridization of the two strains, conditional knock-out of the target gene can be realized through expression of the Cas protein induced by a space-time specific expression promoter, and the homozygous strain with the seroin1 gene function defect is bred, silkworms cannot spin and cocoon, and can be used for eating, so that the problems of time pressure, manpower, material resources, energy consumption and the like caused by cocoon cutting required by eating silkworm chrysalis are solved.

The invention aims to provide a silkworm binary transgenic system for knocking down Seroin1 gene.

In order to achieve the purpose, the invention provides the following technical scheme:

a bombyx mori binary transgenic system for knocking down Seroin1 gene comprises a piggyBac-based Cas9 expression vector and a sgRNA expression vector of a target Seroin1 gene.

Preferably, the sgRNA has a site sequence shown in SEQ ID NO. 1.

Preferably, the sgRNA expression vector is prepared by the following method: annealing the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to form a double chain and connecting the double chain into a gRNA expression vector, and then replacing pBac [ U6-gRNA-gLMN,3xp3-EGFP by U6-gRNA-TTTTT]P on a support_U6gLMN-gRNA scaffold-T6 to obtain the sgRNA expression vector.

The second purpose of the invention is to provide a preparation method of pure naked pupa variety of silkworm.

In order to achieve the purpose, the invention provides the following technical scheme:

the method for preparing the silkworm pure naked pupa variety comprises the steps of knocking out Seroin1 gene expression in silkworms, and screening homozygous mutant individuals to obtain the silkworm pure naked pupa variety.

Preferably, the method for knocking out Seroin1 gene expression is to mediate target Seroin1 gene knock-out through ZFNs, TALENs and CRISPR/Cas9 or variants thereof.

Preferably, the method for knocking out Seroin1 gene expression is realized by knocking out silkworm Seroin1 gene mediated by CRISPR/Cas 9.

Preferably, the CRISPR/Cas9 mediated knockout of silkworm Seroin1 gene is mediated by a Cas9 expression vector and a binary transgene system of sgRNA of a target Seroin1 gene.

Preferably, the sgRNA of the target Seroin1 gene is annealed into a double chain by the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.

Preferably, a Cas9 transgenic silkworm is prepared; then constructing a sgRNA expression vector of a target serin 1 gene, and preparing a sgRNA transgenic silkworm; hybridizing the sgRNA transgenic silkworm and the Cas9 transgenic silkworm, performing fluorescence screening, performing PCR typing detection on individuals which can not continuously generate edits, screening a heterozygote, performing heterozygote selfing, and identifying homozygous mutant individuals in offspring through typing detection and clone sequencing of a mutant sequence to obtain a pure naked pupa variety of the silkworm.

Preferably, the sgRNA expression vector is prepared by the following method: annealing the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to form a double chain connected into a gRNA expression vector, and then replacing pBac [ U6-gRNA-gLMN,3xp3-EGFP by U6-gRNA-TTTTT]P on a support_U6gLMN-gRNA scaffold-T6 to obtain the sgRNA expression vector.

The invention has the beneficial effects that: the invention, by knocking out the serin 1 gene, develops a homozygous strain with serin 1 gene function defect, which can not spin and cocoon. The bred strain has the following characteristics:

1) because the silkworms lose spinning and cocooning capabilities, the silkworms become naked pupas, and the problem of consumption of manpower and material resources caused by cocoon cutting of edible silkworm pupas is solved from the source.

2) Because the spinning and the cocooning cannot be carried out, all energy is transferred to the pupa body, so that the pupa body becomes large and the pupa size is greatly increased.

3) The growth cycle of the larvae is shortened, the feeding time is shortened, and the consumption of mulberry leaves, feed and the like is reduced.

The variety can reduce the consumption of manpower and material resources, can improve the production benefit, and has important application value.

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 diagram of a successfully constructed gRNA expression vector backbone.

FIG. 2 is a picture of a binary transgenic positive individual (a is fluorescence of Cas 9; b is fluorescence of gRNA).

FIG. 3 is a graph of the sequencing peak of heterozygous individuals, with the PAM region shaded.

FIG. 4 is a sequence diagram and sequencing peak diagram of homozygous individuals (a is a mutant form of homozygous individual; b is a sequencing peak diagram of homozygous individual, and the shaded portion is a PAM region).

FIG. 5 is a table of homozygous phenotypes of individuals (a is a normal line; b is a mutant line).

Detailed Description

The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can implement the present invention, but the embodiments are not to be construed as limiting the present invention.

Example 1 acquisition of sgRNA Stable transgenic lines

The cDNA full-length sequence (SEQ ID NO.6) of silkworm gene seroin1 is obtained from NCBI, and sgRNA sites of the silkworm gene seroin1 are designed on a CRISPRdirect website according to the rule of GN19NGG, so that the sgRNA site sequence 5'-tgatattaaaactcacgtcgcgg-3' (CGG is PAM motif) (SEQ ID NO.1) is obtained. Synthesis of sequence F: 5'-aagtgtgatattaaaactcacgtcg-3' (SEQ ID NO.2) and R: 5'-aaaccgacgtgagttttaatatcac-3' (SEQ ID NO.3), annealing the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to form a double strand, ligating the double strand into a gRNA expression vector (see SanyuanMa. CRISPR/Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bomby mori.2014), and replacing U6-gRNA-TTTTT with pBac [ U6-gRNA-gLMN,3xp3-EGFP]P on a vector (see Yuanyuan Liu. tissue-specific genome editing of laminA/C in the porsterizer silk glandis of Bombyx mori.2017))_U6The sgRNA expression vector of the serin 1 gene is obtained from gLMN-gRNA scaffold-T6 and named as piggyBac- {3xp3-EGFP-sv40-TTTTT-gRNA-U6} expression vector, and the structure diagram of the expression vector is shown in figure 1.

The constructed piggyBac- {3xp3-EGFP-sv 40-TTT-gRNA-U6 } expression vector and the helper plasmid are mixed in a ratio of 1:1 and then injected into the silkworm eggs, which are non-diapause eggs, through microinjection, and are recorded as G0 generation, and the injections are performed within 2h of egg laying. The injected eggs are subjected to accelerated hatching and are raised until moths emerge, G0 generations are selfed to generate G1 generations, the G1 generations are screened for green fluorescence, and the transgenic individuals successfully expressing sgRNA are obtained when eyes express the green fluorescence.

Example 2 obtaining of hybrid mutants

Crossing the gRNA transgenic individual obtained in example 1 and the Cas9 transgenic individual obtained to obtain progeny F1. Cas9 transgenic individuals were prepared as follows: cloning HSP90 promoter, connecting the HSP90 promoter sequence into pUC57-Cas9 by using Xho I and Spe I enzyme cutting sites, performing AscI single enzyme cutting, inserting into piggyBac [3xp3-DsRed ] vector trunk, and obtaining Cas9 transgenic individual after silkworm egg injection (see Yuanyan Liu, tissue-specific genetic editing of laminA/C in the poster plasmid silk gland of Bombyx mori.2017 for pUC57-Cas9 vector). Depending on the presence of red and green fluorescence, there are 4 types in the F1 generation: cas9 transgenic individuals carrying red fluorescence only in the eye, gRNA transgenic individuals carrying green fluorescence only in the eye, binary transgenic individuals carrying red and green fluorescence in the eye, and individuals not carrying a fluorescent protein marker, wherein the binary transgenic individuals are chimeras of knockout of the seroin1 gene, as shown in fig. 2.

Heterozygous mutant individuals were obtained by: the binary transgenic individuals in the F1 generation are selfed to obtain offspring F2, and for convenience of subsequent screening and strain fixation, non-binary transgenic individuals in the F2 generation (editing cannot be continuously generated) are selected, namely individuals carrying only red fluorescence, only green fluorescence or no fluorescent protein marker are subjected to subsequent screening (in the embodiment, individuals carrying only red fluorescence are explained). Screening individuals carrying red fluorescence in the F2 generation, feeding the individuals with fresh mulberry leaves until the moths feather, extracting the genome of the silkworm skin after pupation, marking and conveniently producing seeds. With the detection primer F: 5'-gtaaattagatgaattagtaaattc-3' (SEQ ID NO.4) and R: 5'-aattaatgacttgatttaactttaa-3' (SEQ ID NO.5), and sequencing the amplified product. If the individual is heterozygous, the sequencing result of the amplification product is identified as heterozygous, and a nested peak appears near the PAM motif in a sequencing peak map, as shown in FIG. 3. It is noted that the detection primers in this example must be specific, no non-specific amplification occurs, and the PCR products can be directly used for sequencing analysis.

Example 3 obtaining of homozygous mutants

The silkmoth individuals (with consistent mutation forms) corresponding to the heterozygous samples identified in the example 3 are hybridized between males and females to obtain F3 generation, and the homozygous mutant individuals with serin 1 gene knockout can be identified. The identification method combines PCR product sequencing and clone sequencing, and comprises the following specific implementation steps: and (3) feeding the F3 generation individuals until the silkworm moths feather, collecting pupated silkworm skin, extracting genomes, marking and waiting for seed production. Amplifying and sequencing the sequence at the locus by using the detection primer in the embodiment 2 and taking the extracted genome as a template; and (3) carrying out T-A cloning on a sample of which the sequencing result is determined to be homozygous, picking a single colony for bacterial detection, and then sending positive bacteria to sequencing to determine whether the positive bacteria are heterozygous or homozygous. Following this approach, the sequence form of the homozygous mutant identified in this example for the seroin1 knockout was: the homozygous mutated sequence was deleted for 2bp near the PAM motif compared to the normal sequence, as shown in figure 4.

EXAMPLE 4 phenotypic Observation of homozygous mutants

The homozygous mutant strain was fed with fresh mulberry leaves, and observation during feeding revealed that the mutants clustered earlier than normal individuals, but none grew and cocooned normally, but could pupate, eclose, mate and lay eggs normally, as shown in fig. 5. The mutant individuals can normally grow and develop and have a shortened growth cycle, but cannot normally spin and cocoon because of the deletion of the seroin1 gene.

In the embodiment of the present invention, Cas9 mediates the knockout of the target seroin1 gene, and any means capable of knocking out the seroin1 gene can achieve the object of the present invention, as known to those skilled in the art, such as: ZFNs, TALENs and CRISPR/Cas9 or variants thereof mediate knock-out of the target seroin1 gene.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitutions or changes made by the person skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the invention is subject to the claims.

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Claims (10)

1. The application of the knockdown Seroin1 gene expression in silkworm in the preparation of pure naked pupa varieties of silkworm is characterized in that: the silkworm binary transgenic system for knocking down the Seroin1 gene is used for expressing the Seroin1 gene, and comprises a piggyBac-based Cas9 expression vector and a sgRNA expression vector of a target Seroin1 gene.

2. Use according to claim 1, characterized in that: the sequence of the sgRNA site is shown in SEQ ID NO. 1.

3. Use according to claim 1, characterized in that: the sgRNA expression vector is prepared by the following method: annealing the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to form a double chain connected into a gRNA expression vector, and then replacing pBac [ U6-gRNA-g ] with U6-gRNA-TTTTTLMN, 3xp3-EGFP]On a carrierP_U6-gLMN-gRNA scaffold-T6 to obtain sgRNA expression vector.

4. The preparation method of the silkworm pure naked pupa variety is characterized by comprising the following steps: the silkworm pure naked pupa variety is obtained by knocking out Seroin1 gene in silkworm and screening homozygous mutant individual.

5. The method for preparing silkworm pure naked pupae variety according to claim 4, wherein the method comprises the steps of: the method for knocking out Seroin1 gene expression is to mediate target Seroin1 gene knock-out through ZFNs, TALENs and CRISPR/Cas9 or variants thereof.

6. The method for preparing silkworm pure naked pupa variety according to claim 4, wherein the method comprises the following steps: the method for knocking out the expression of the Seroin1 gene is to knock out the silkworm Seroin1 gene through the mediation of CRISPR/Cas 9.

7. The method for preparing silkworm pure naked pupae variety according to claim 6, wherein the method comprises the steps of: the CRISPR/Cas9 mediated silkworm Seroin1 gene knockout is mediated by a Cas9 expression vector and a double-element transgene system of sgRNA of a target Seroin1 gene.

8. The method for preparing silkworm pure naked pupa variety according to claim 7, wherein the method comprises the following steps: the sgRNA of the target Seroin1 gene is annealed by nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to form a double chain.

9. The method for preparing silkworm pure naked pupa variety according to claim 4, wherein the method comprises the following steps: preparing a Cas9 transgenic silkworm; then constructing a sgRNA expression vector of a target serin 1 gene, and preparing a sgRNA transgenic silkworm; hybridizing the sgRNA transgenic silkworm and the Cas9 transgenic silkworm, performing fluorescence screening, performing PCR typing detection on individuals which can not continuously generate edits, screening a heterozygote, performing heterozygote selfing, and identifying homozygous mutant individuals in offspring through typing detection and clone sequencing of a mutant sequence to obtain a pure naked pupa variety of the silkworm.

10. The method for preparing silkworm pure naked pupa variety according to claim 9, wherein the method comprises the following steps: the sgRNA expression vector is prepared by the following method: annealing the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to form a double chain connected into a gRNA expression vector, and then replacing pBac [ U6-gRNA-g ] with U6-gRNA-TTTTTLMN, 3xp3-EGFP]P on a support_U6-gLMNThe sgRNA scaffold-T6 was used to obtain the sgRNA expression vector.

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