Application of knockout Seroin2 gene expression in preparation of silkworm pure naked pupa strain

Abstract

The invention discloses application of knockout Seroin2 gene expression in preparation of a silkworm pure naked pupa strain, and a homozygous silkworm strain with Seroin2 gene function defects is successfully cultivated by knocking out a Seroin2 gene. Due to the fact that the strain cannot spin and cocoon, the strain has the following remarkable characteristics that firstly, due to the fact that the capability of spinning and cocooning is lost, silkworms directly become naked pupae, and manpower and material resource input in the cocoon cutting link in the edible silkworm pupa processing process is fundamentally omitted; secondly, energy originally used for knotting silk is completely transferred to pupa bodies, the pupa bodies are promoted to be obviously enlarged, and the pupa weight is obviously increased. The strain has outstanding performance in the aspects of reducing resource investment and improving production efficiency, and has wide application prospects and important economic value.

Description

Technical Field

[0001] This invention relates to the field of silkworm breeding technology, specifically to the application of knocking out the Seroin2 gene expression in the preparation of pure naked silkworm pupae. Background Technology

[0002] The silkworm, a type economic insect of Lepidoptera, undergoes a complete metamorphosis from egg to larva to pupa to adult. However, due to its small size, short growth cycle, high silk conversion rate, and millennia of domestication with a clear genetic background, it is one of the very few agricultural animals possessing both a mature genetic manipulation system and a large-scale breeding platform. It is also a vital economic pillar for many regions in my country. The value of the silkworm lies primarily in its silk and pupae. Silk is a precious textile raw material, while pupae are rich in nutrients. However, researchers mostly focus on improving silk yield and quality through genetic modification and feeding management to directly increase silkworm farmers' income from cocoons and silk, neglecting the economic value of the pupae themselves. Silkworm pupae contain over 50% protein, have a complete amino acid profile, and are rich in active ingredients such as ecdysterone and antimicrobial peptides, making them highly promising for the functional food market. However, commercially available varieties are all "cocoon-spinning" types, with the pupae encased in thick cocoons, resulting in high dismantling costs. Furthermore, the denaturation of pupal proteins after silk reeling severely restricts the commercialization rate of pupal resources. Currently, some naked pupa strains (Nd-x, Nd-s, etc.) have been bred for silkworms, but these strains still secrete thin cocoons when they clump together, and there is still a genetic distance to go before achieving a silkworm strain that does not spin cocoons at all and is used exclusively for food.

[0003] The development of molecular biology has made precise gene manipulation possible, giving rise to genome editing technology. Its development can be traced back to 1987, when the first-generation system based on spontaneous homologous recombination in cells was established. However, this technology was extremely inefficient and heavily reliant on specific cellular mechanisms, limiting its application to only a few model organisms and hindering its widespread adoption. To overcome this limitation, zinc finger nuclease (ZFN) technology, which emerged around 2001, significantly improved efficiency by fusing artificially designed zinc finger proteins with endonuclease domains. However, ZFNs still face challenges such as complex design processes, high synthesis costs, cytotoxicity, and off-target risks, limiting their application. In 2009, transcription activator-like effector nuclease (TALEN) technology was introduced. Its DNA recognition module and base correspondence are simpler and more direct, allowing for greater flexibility in target design, and it has successfully achieved gene editing in various organisms such as silkworms and fruit flies. However, the large molecular weight of TALEN proteins, difficulties in in vivo delivery, and cumbersome module assembly processes still restrict its widespread application. The advent of the CRISPR / Cas9 system completely changed the landscape of genome editing. This system originates from the adaptive immune mechanism of bacteria. It can guide the Cas9 protein to precisely cut specific DNA sequences through a simple guide RNA (gRNA). It has outstanding advantages such as rapid design, low cost, high editing efficiency and the ability to perform multi-target editing in parallel, thus rapidly becoming the most mainstream tool in the current life science field.

[0004] The CRISPR / Cas9 system can be introduced into the body or cells through various delivery methods, including DNA, RNA, and proteins. Specific methods include direct injection, chemical transfection, and electroporation. Furthermore, gene editing can be achieved through genetic breeding strategies. For example, transgenic lines stably expressing Cas9 protein and transgenic lines expressing gRNA targeting the target gene can be constructed separately. By hybridizing the two lines, offspring can simultaneously possess both Cas9 and gRNA expression units, thus achieving continuous and heritable gene editing in vivo. Whether knocking out key cocooning genes using the CRISPR / Cas9 system can yield naked pupa strains is crucial for developing edible silkworm strains. To knock out the Seroin2 gene in silkworms, transgenic silkworm lines stably expressing Cas9 and gRNA targeting the Seroin2 gene can be established separately. Through hybridization, offspring can simultaneously carry both components, achieving efficient, stable, and easily trackable gene knockout. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide an application of knocking out the Seroin2 gene expression in the preparation of pure naked silkworm pupae; another objective of the present invention is to provide a method for preparing pure naked silkworm pupae by knocking out the Seroin2 gene. That is, by obtaining a homozygous strain with the Seroin2 gene knocked out, the silkworm cannot spin cocoons, and the mutant strain can be used for consumption. This alleviates the time pressure and the consumption of manpower, material resources, and energy caused by the need to remove cocoons for consuming silkworm pupae. To achieve the above objectives, the present invention provides the following technical solution: 1. Application of knocking out Seroin2 gene expression in the preparation of pure naked silkworm pupae, wherein the knockout of Seroin2 gene expression uses the CRISPR / Cas9 transgenic system, including a piggyBac-based Cas9 expression vector and an sgRNA expression vector targeting the Seroin2 gene.

[0006] Preferably, the site sequence of the sgRNA is shown in SEQ ID NO.2.

[0007] Preferably, the sgRNA expression vector of the present invention is prepared by the following method: annealing the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 to form a double strand and linking it into the gRNA expression vector, and then replacing the P on the pBac[U6-gRNA-gLMN, 3xp3-EGFP] vector with U6-gRNA-TTTTT. U6- The sgRNA expression vector was obtained by using gLMN-gRNA scaffold-T6.

[0008] 2. A method for preparing a pure naked silkworm pupa variety by knocking out the Seroin2 gene in silkworms, comprising the following steps: constructing a Cas9 expression vector based on piggyBac and preparing Cas9 transgenic silkworms; then constructing an sgRNA expression vector targeting the Seroin2 gene and preparing sgRNA transgenic silkworms; hybridizing sgRNA transgenic silkworms and Cas9 transgenic silkworms, fluorescently screening individuals that can no longer produce edits, performing PCR typing to screen for heterozygotes, self-crossing the heterozygotes, identifying homozygous mutant individuals in the offspring through typing and cloning sequencing of the mutant sequences, expanding the rearing scale, and thus obtaining a pure naked silkworm pupa variety.

[0009] Preferably, the site sequence of the sgRNA targeting the Seroin2 gene is shown in SEQ ID NO.2.

[0010] Preferably, the sgRNA expression vector of the present invention is prepared by the following method: annealing the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 to form a double strand and linking it into the gRNA expression vector, and then replacing the P on the pBac[U6-gRNA-gLMN, 3xp3-EGFP] vector with U6-gRNA-TTTTT. U6 The sgRNA expression vector was obtained by using -gLMN-gRNA scaffold-T6.

[0011] In a preferred embodiment of the present invention, the method for screening individuals that can no longer be edited is to screen for binary transgenic individuals with red and green fluorescence in their eyes, self-cross the binary transgenic individuals in the F1 generation to obtain their offspring F2, and select individuals in the F2 generation that carry only red fluorescence, only green fluorescence, or no fluorescent protein marker.

[0012] Preferably, in this invention, the PCR typing detection involves raising individuals that cannot be further edited until they emerge as moths, extracting the genome from the silkworm skin, performing PCR amplification using the sequences shown in SEQ ID NO.5-6 as primers, and sequencing the genome. If a peak appears near the PAM motif in the sequencing graph, it indicates a heterozygote. The heterozygotes are then self-crossed, and after cloning and identification of the Seroin2 gene sequence, homozygous mutant individuals with the Seroin2 gene knockout are obtained. These individuals are then raised on a large scale to obtain a pure naked pupa silkworm strain.

[0013] The beneficial effects of this invention are as follows: By knocking out the Seroin2 gene, this invention cultivates a homozygous strain of silkworms that is unable to spin cocoons due to the functional defect of the Seroin2 gene. The cultivated strain possesses the following characteristics: 1. Due to the loss of the ability to spin cocoons, the silkworms become naked pupae, thus fundamentally solving the problem of manpower and material resources consumed in consuming silkworm pupae and processing cocoons. 2. Because they cannot spin cocoons, all energy is transferred to the pupa, resulting in a larger pupa and a significantly increased pupal weight. This variety can reduce the consumption of manpower and material resources, improve production efficiency, and has significant application value. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Here is a structural diagram of the Seroin2gRNA expression vector; Figure 2 Image of a binary transgenic positive individual (a is the fluorescence of Cas9, b is the fluorescence of gRNA). Figure 3 The sequencing peak diagrams are as follows: (a) is the sequencing peak diagram of a normal individual (WT); (b) is the sequencing peak diagram of a knockout homozygous individual (Seroin2). Ko (Sequencing peak diagram); Figure 4 Phenotypic diagrams of homozygous individuals (a is the normal strain; b is the mutant strain); Figure 5 The graph shows a comparison of pupal weight; a) shows the change in pupal weight after Seroin1 knockout; b) shows the change in pupal weight after Seroin2 knockout; c) shows a comparison of the proportion of pupal weight increase after Seroin1 and Seroin2 knockout. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments are not intended to limit the present invention. Experimental methods not specified in the embodiments are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (3rd Edition, by J. Sambrook et al.), or as recommended by the manufacturer.

[0016] Example 1: Obtaining a stable sgRNA transgenic line The full-length cDNA sequence of the silkworm Seroin2 gene was obtained from NCBI (SEQ ID NO.1), and its sgRNA site was designed on the CRISPRdirect website according to the GN19NGG rule. The obtained sgRNA site sequence is 5'-TGGCAGGACGATAACTTCCCGGG-3' (CGG is the PAM motif) (SEQ ID NO.2).

[0017] The synthesized sequences F: 5'-aagtTGGCAGGACGATAACTTCCC-3' (SEQ ID NO.3) and R: 5'-aaacGGGAAGTTATCGTCCTGCCA-3' (SEQ ID NO.4) were annealed to form a double strand, which was then ligated into a gRNA expression vector (see Sanyuan Ma. CRISPR / Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bombyx mori. 2014). Then, the pBac[U6-gRNA-gLMN, 3xp3-EGFP] vector (see Yuanyuan Liu. Tissue-specific genome editing of laminA / C in the posterior silk glands of Bombyx mori. 2017) was replaced with U6-gRNA-TTTTT. U6The sgRNA expression vector piggyBac{3xp3-EGFP-sv40-TTTTT-gRNA-U6} of the Seroin2 gene was obtained using the scaffold-T6 method with GLMN-gRNA. This vector is hereinafter referred to as the Seroin2-gRNA expression vector. Figure 1 This is a diagram illustrating the structure of the carrier.

[0018] The successfully constructed Seroin2-gRNA expression vector was mixed with the helper plasmid at a 1:1 ratio and injected into silkworm eggs via microinjection, designated as generation G0. The silkworm eggs were non-diapause eggs, and the injection was performed within 2 hours of egg laying. The injected eggs were then induced to regrow and reared until moth emergence. Generation G0 self-pollinated to produce generation G1. Generation G1 was screened for green fluorescence; those exhibiting green fluorescence in their eyes were transgenic individuals successfully expressing sgRNA.

[0019] Example 2: Obtaining Heterozygous Mutants The gRNA transgenic individuals obtained in Example 1 were hybridized with the previously obtained Cas9 transgenic individuals (exhibiting red fluorescence in the eyes) to obtain the F1 generation. Based on the presence of red and green fluorescence, the F1 generation contained four types: Cas9 transgenic individuals carrying only red fluorescence in the eyes, gRNA transgenic individuals carrying only green fluorescence in the eyes, binary transgenic individuals with both red and green fluorescence in the eyes, and individuals without fluorescent protein markers. The binary transgenic individuals were chimeras with the Seroin2 gene knocked out, such as... Figure 2 As shown.

[0020] Heterozygous mutant individuals were obtained as follows: F1 generation binary transgenic individuals were self-crossed to obtain F2 offspring. For ease of subsequent screening and strain fixation, non-binary transgenic individuals (who could not be further edited) were selected from the F2 generation, i.e., individuals carrying only red fluorescence, only green fluorescence, or no fluorescent protein marker, for subsequent screening (this example uses individuals carrying only red fluorescence). Individuals carrying only red fluorescence from the F2 generation were screened and fed with fresh mulberry leaves until pupation. The genome of the pupated silkworm skin was extracted and labeled for easy breeding. The sequences at the loci were genotyped using primers F: 5'-TCTCTCAGGTTGGGCTTGTG-3' (SEQ ID NO. 5) and R: 5'-AGGACATGTCATAGCGTGCAA-3' (SEQ ID NO. 6), and the amplified products were sequenced. If the individual was heterozygous, the sequencing results of its amplified product identified it as heterozygous, with overlapping peaks appearing near the PAM motif in the sequencing peak diagram. It is worth noting that the detection primers in this embodiment must be specific, with no non-specific amplification, and their PCR products can be directly used for sequencing analysis.

[0021] Example 3: Obtaining homozygous mutants The silkworm moths (with the same mutation type) corresponding to the heterozygous samples identified in Example 2 were crossbred between males and females to obtain the F3 generation. Homozygous individuals with the Seroin2 gene knockout could then be identified from this F3 generation. The identification method combined PCR product sequencing and cloning sequencing. The specific steps are as follows: F3 generation individuals were reared until the silkworm moths emerged, and the pupalized silkworm skins were collected to extract the genome. The genome was then labeled and prepared for breeding. Using the detection primers from Example 2, the extracted genome was used as a template to amplify and sequence the sequence at the target site. Samples identified as homozygous by sequencing results were subjected to TA cloning. Single colonies were picked for bacterial testing, and positive bacteria were sent for sequencing to determine whether they were heterozygous or homozygous. Following this method, the sequence form of the homozygous Seroin2 knockout mutant identified in this example is as follows: Compared to the normal sequence, the homozygous mutant sequence has a base mutation near the PAM motif, resulting in a stop codon that prematurely terminates translation, such as... Figure 3 The sequencing peak diagram of b is shown.

[0022] Example 4: Phenotypic observation of homozygous mutants Homozygous mutant strains were fed with fresh mulberry leaves and observed during the rearing process. It was found that the mutants could not spin cocoons normally, but they could pupate, emerge as adults, mate, and lay eggs normally. Figure 4 As shown, this indicates that the mutant individuals can grow and develop normally, but due to the absence of the Seroin2 gene, they cannot spin silk and form cocoons normally.

[0023] The Seroin2 gene knockout naked silkworm pupa mutant of this invention was compared with the Seroin1 gene knockout naked silkworm pupa mutant in the method for preparing pure naked silkworm pupa varieties of the Seroin1 gene knockdown binary transgenic system published by our research group in CN112011540 B. The results are as follows: Figure 5 As shown, compared to knocking out the Seroin1 gene, knocking out the Seroin2 gene resulted in a higher proportion of upregulated pupal weight in naked pupae.

[0024] In this embodiment of the invention, Cas9 is used to mediate the knockout of the target Seroin2 gene. As is known to those skilled in the art, any means that can knock out the Seroin2 gene can achieve the purpose of this invention, such as ZFNs, TALENs, and CRISPR / Cas9 or their variants mediating the knockout of the target Seroin2 gene.

[0025] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of knocking out Seroin2 gene expression in the preparation of pure naked silkworm pupae, characterized in that, The knockout of the Seroin2 gene expression uses the CRISPR / Cas9 transgenic system, including a piggyBac-based Cas9 expression vector and an sgRNA expression vector targeting the Seroin2 gene.

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

2.

3. The application according to claim 1, characterized in that, The sgRNA expression vector was prepared by the following method: the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 were annealed to form a double strand and ligated into the gRNA expression vector, and then the P on the pBac[U6-gRNA-gLMN, 3xp3-EGFP] vector was replaced with U6-gRNA-TTTTT. U6 The sgRNA expression vector was obtained by using -gLMN-gRNA scaffold-T6.

4. A method for preparing a pure naked pupa variety of silkworm by knocking out the Seroin2 gene, characterized in that, The process includes the following steps: constructing a Cas9 expression vector based on piggyBac and preparing Cas9 transgenic silkworms; then constructing an sgRNA expression vector targeting the Seroin2 gene and preparing sgRNA transgenic silkworms; hybridizing sgRNA transgenic silkworms and Cas9 transgenic silkworms, fluorescently screening individuals that can no longer produce edits, performing PCR typing to screen for heterozygotes, self-pollinating the heterozygotes, identifying homozygous mutant individuals in the offspring through mutant sequence typing and cloning sequencing, expanding the rearing scale, and finally obtaining a pure naked pupa silkworm variety.

5. The method according to claim 4, characterized in that, The site sequence of the sgRNA targeting the Seroin2 gene is shown in SEQ ID NO.

2.

6. The method according to claim 4, characterized in that, The sgRNA expression vector was prepared by the following method: annealing the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 to form a double strand and linking it into the gRNA expression vector; then replacing the P on the pBac[U6-gRNA-gLMN, 3xp3-EGFP] vector with U6-gRNA-TTTTT. U6- The sgRNA expression vector was obtained by using gLMN-gRNA scaffold-T6.

7. The method according to claim 4, characterized in that, The method for screening individuals that can no longer be edited is to screen for binary transgenic individuals with red and green fluorescence in their eyes, self-cross the binary transgenic individuals in the F1 generation to obtain their offspring F2, and select individuals in the F2 generation that carry only red fluorescence, only green fluorescence, or no fluorescent protein label.

8. The method according to claim 4, characterized in that, The PCR typing detection involved raising individuals that could no longer be edited until they emerged as moths, extracting the genome from their skins, performing PCR amplification using the sequences shown in SEQ ID NO.5-6 as primers, and sequencing. If a peak appeared near the PAM motif in the sequencing graph, it was a heterozygote. The heterozygotes were then self-crossed, and after cloning and identification of the Seroin2 gene sequence, homozygous mutant individuals with the Seroin2 gene knockout were obtained. These individuals were then raised on a large scale to obtain a pure naked pupa silkworm strain.

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