Application of specific knockout of p53 gene in improving silk yield and method thereof

Abstract

The application discloses application of specific knockout of a P53 gene in improvement of silk yield and a method thereof, and promotes development of a silkworm silk gland, increases expression of a silk protein gene and improves silk yield by specifically knocking out the P53 gene in the rear part of the silkworm silk gland through a CRISPR / Cas9 gene editing system, so as to provide a new breakthrough point and target for silkworm variety improvement aiming at improving a silk protein synthesis level and silk yield, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to the application of specific knockout of the P53 gene in increasing silk production, and also to a method for its preparation. Background Technology

[0002] As an important silk-producing insect, the silkworm synthesizes and secretes silk protein through its silk glands. During the embryonic stage, silk gland cells undergo approximately 10 mitotic divisions, differentiating into anterior, middle, and posterior silk glands. However, during the larval development of the silkworm, these cells cease mitosis and instead undergo 17-19 rounds of DNA replication via nuclear replication, ultimately increasing the DNA content within a single cell by approximately 300,000 times, resulting in polyploidy. This nuclear replication process in silk gland cells increases the genomic copy number of silk protein genes, which is undoubtedly the genetic basis for the rapid and efficient synthesis of silk protein in the final instar of the silkworm larva. Therefore, studying the regulatory mechanism of nuclear replication in silk gland cells will contribute to elucidating the molecular mechanisms of silk gland development and efficient silk protein synthesis in silkworms.

[0003] In recent years, using insect nuclear replication organs as a model to elucidate the regulatory mechanisms of intranuclear replication has become a research hotspot. Transcription factor p53 plays a crucial role in mitotic cell cycle regulation, DNA damage repair, and apoptosis; however, whether it affects the intranuclear replication process and silk yield in silkworms remains unclear. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide an application of specific knockout of the P53 gene in increasing silk production; another objective of the present invention is to provide a method for increasing silk production.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] 1. Application of specific knockout of the P53 gene in improving silk production, wherein the P53 gene sequence is shown in SEQ ID NO.1.

[0007] Preferably, the improvement in silk production in this invention is achieved by increasing cocoon weight and cocoon yield.

[0008] Preferably, the target site for knocking out the P53 gene is shown in SEQ ID NO.2.

[0009] 2. A method to increase silk production by knocking out the P53 gene in silkworms, screening for mutant individuals, and obtaining silkworm varieties with increased silk production.

[0010] Preferably, the method for knocking out the P53 gene in this invention is to knock out the silkworm P53 gene through CRISPR / Cas9-mediated knockout.

[0011] In a preferred embodiment of the present invention, the knockout of the P53 gene is achieved by first preparing Cas9 transgenic silkworms; then constructing an sgRNA expression vector for the target P53 gene and preparing sgRNA transgenic silkworms; and then hybridizing the sgRNA transgenic silkworms with Cas9 individuals that express red fluorescent protein in the eyes and have specific posterior silk gland characteristics to screen for mutant individuals, thereby obtaining a high-yielding silkworm variety.

[0012] The beneficial effects of this invention are as follows: This invention provides the application of specific knockout of the P53 gene in improving silk production. This invention promotes silk gland development, increases silk protein gene expression, and improves silk production by specifically knocking out the P53 gene in the posterior silk gland of silkworms using the CRISPR / Cas9 gene editing system. This provides a new entry point and target for improving silkworm varieties with the goal of increasing silk protein synthesis and silk production, and has good application prospects. Attached Figure Description

[0013] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0014] Figure 1 Design site and vector diagram of the gRNA of the silkworm P53 gene.

[0015] Figure 2 Results of the P53 gene knockout mutation in silkworm.

[0016] Figure 3 Knockout of the P53 gene in silkworms causes silk gland phenotype.

[0017] Figure 4 Changes in silk production caused by P53 gene knockout in silkworms. Detailed Implementation

[0018] 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 described are not intended to limit the present invention.

[0019] Example 1: Construction of gRNA for specific knockout of the p53 gene in the posterior silk gland of silkworm.

[0020] Based on the silkDB3.0, silkbase, and KAIKObase databases for silkworms, a specific knockout target site that can only recognize the P53 gene was selected in the first exon region of the P53 gene (SEQ ID NO.1). Its sequence is as follows: 5'-CGCAAAGACGATCCCGGGCA-3' (SEQ ID NO.2), and its structure is as follows: Figure 1As shown in Figure A. Using the target-specific primers 5'-AAGTGTGCCCGGGATCGTCTTTGCG-3' (SEQ ID NO.3) and 5'-AAACCGCAAAGACGATCCCGGGCAC (SEQ ID NO.4), the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 were annealed to form a double strand and ligated into a gRNA expression vector. Then, U6-gRNA-TTTTT replaced the PU6-gLMN-gRNA scaffold-T6 on the pBac[U6-gRNA-gLMN,3xp3-EGFP] vector to obtain the sgRNA expression vector. Using the transgenic vector containing the gRNA of the p53 gene, the green fluorescent protein (GFP) labeled for ocular expression was screened. Figure 1 B).

[0021] Example 2: Transgenic microinjection and screening of positive individuals

[0022] After multi-evolving silkworms were normally fed mulberry leaves until they pupated and emerged as moths, the male and female moths were mated for 4 hours, separated, and laid eggs in a dark environment. These eggs were neatly arranged on a clean glass slide, and the gRNA transgenic vector and the A3 helper plasmid were injected into 100 eggs using an Eppendorf microinjection system. After approximately 10 days of incubation, the G0 generation of silkworm larvae was obtained. Silkworms successfully reared to pupation and moth emergence were mated and laid eggs. Positive individuals with green fluorescent eyes were selected using a fluorescence microscope. Individuals with the gRNA of the silkworm P53 gene were hybridized with Cas9 individuals (Fzrregulates silk gland growth by promoting endoreplication and protein synthesis in the silkworm. PLoS Genet 2023,19,e1010602) that specifically expressed red fluorescent protein in their posterior silk glands. Individuals expressing both green and red fluorescent proteins in their eyes were selected, and genomic DNA from their posterior silk glands was extracted, amplified by PCR, and sequenced. The results showed that the P53 gene in the posterior silk glands of double-glossy individuals contained multiple mutations. Figure 2 A). RT-PCR and RT-qPCR results showed that knockout of the p53 gene specifically in the posterior silk gland led to a decrease in its expression level. Figure 2 (BC). The P53 gene was successfully knocked out in the posterior sepal gland.

[0023] Example 3: Knockout of the p53 gene in the posterior silk gland of silkworm promotes silk gland development and silk protein synthesis.

[0024] Dissection of the silk glands of silkworms in the upper clustering stage revealed that, compared with the control group, the specific knockout mutation P53 in the posterior silk glands of silkworms resulted in increased length and thickness of the posterior silk glands. Figure 3 Twenty silkworms of the P53 knockout strain and 20 silkworms of the control strain were randomly selected. The weight of the cocoon shell and the weight of the pupa were weighed and statistically analyzed. The cocoon shell ratio was calculated as the proportion of the silkworm shell weight to the total cocoon weight. The results showed that, compared with the control group, the P53 knockout strain produced larger cocoons, while the pupa weight showed no significant change. Figure 4 A), its cocoon weight and cocoon rate increased significantly ( Figure 4 ,BC).

[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 specific knockout of the P53 gene in improving silk production, characterized by: The P53 gene sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The increase in silk production refers to increasing cocoon weight and cocoon yield.

3. The application according to claim 1, characterized in that: The target site for knocking out the P53 gene is shown in SEQ ID NO.

2.

4. A method for increasing silk production, characterized in that: By knocking out the P53 gene in silkworms and screening for mutant individuals, a silkworm variety with increased silk production was obtained. The P53 gene sequence is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that: The method for knocking out the P53 gene is to knock out the silkworm P53 gene through CRISPR / Cas9.

6. The method according to claim 4, characterized in that: The knockout of the P53 gene involves first preparing Cas9 transgenic silkworms; then constructing an sgRNA expression vector targeting the P53 gene and preparing sgRNA transgenic silkworms; then crossing sgRNA transgenic silkworms and Cas9 transgenic silkworms to screen for mutant individuals and obtain silkworm varieties with high silk production; the Cas9 transgenic silkworms are silkworms that specifically express Cas9 in their posterior silk glands, which express red fluorescent protein in their eyes.

Images