Application of specific knockout P53 gene in increasing yield of silk and method of specific knockout P53 gene in increasing yield of silk

Abstract

The invention discloses an application of a specific knockout P53 gene in improving the yield of silk and a method thereof, the P53 gene is specifically knocked out in the posterior silk gland of bombyx mori through a CRISPR / Cas9 gene editing system to promote the development of the silk gland of bombyx mori, increase the silk protein gene expression and improve the yield of the silk. A new entry point and a new target are provided for silkworm variety improvement aiming at improving the silk protein synthesis level and the silk yield, and the method has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to the application of specifically knocking out P53 gene in improving silk yield, and also to a preparation method. Background Art

[0002] As an important silk-producing economic insect, the silkworm's silk glands synthesize and secrete silk protein. Silkworm silk gland cells undergo about 10 mitosis during the embryonic period, and then differentiate into anterior, middle and posterior silk glands. However, silk gland cells no longer undergo mitosis during the growth and development of silkworm larvae, but instead undergo 17-19 rounds of DNA replication in the form of endoreduplication, which eventually increases the DNA content in a single cell by about 300,000 times, forming polyploidy. The endoreduplication process in silkworm silk gland cells increases the genomic copy number of silk protein genes, which is undoubtedly the genetic material basis for the rapid and efficient synthesis of silk protein in the late instar stage of silkworm larvae. Therefore, studying the regulatory mechanism of endoreduplication in silkworm silk gland cells will help to analyze the molecular mechanism of silk gland development and efficient synthesis of silk protein in silkworms.

[0003] In recent years, using insect endoreduplication organs as a model to analyze the regulatory mechanism of cell endoreduplication has become one of the research hotspots. Transcription factor P53 plays a key role in mitotic cell cycle regulation, DNA damage repair and cell apoptosis, but it is still unclear whether it affects the process of silkworm endoreduplication and silk production. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide an application of specifically knocking out the P53 gene in increasing silk production; a second object of the present invention is to provide a method for increasing silk production.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

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

[0007] Preferably, the method of increasing silk production is to increase cocoon layer weight and cocoon layer rate.

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

[0009] 2. A method for increasing silk production, wherein the P53 gene is knocked out in silkworms, mutant individuals are screened, and silkworm varieties with increased silk production are obtained.

[0010] Preferably, the method for knocking out the P53 gene is to knock out the silkworm P53 gene through CRISPR / Cas9.

[0011] Preferably, the knockout of P53 gene is performed by first preparing Cas9 transgenic silkworm; then constructing an sgRNA expression vector of the target P53 gene, and preparing sgRNA transgenic silkworm; hybridizing the sgRNA transgenic silkworm and the Cas9 individuals specific to the posterior silk gland expressing red fluorescent protein in the silkworm eyes to screen mutant individuals, and obtaining a silkworm variety with high silk yield.

[0012] The beneficial effects of the present invention are as follows: the present invention provides an application of specifically knocking out the P53 gene in improving silk production. The present invention promotes the development of the silk gland of the silkworm by specifically knocking out the P53 gene in the posterior silk gland of the silkworm through the CRISPR / Cas9 gene editing system, increases the expression of silk protein genes, and improves silk production. This provides a new entry point and target for silkworm variety improvement aimed at improving the level of silk protein synthesis and silk production, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0014] Figure 1 Map of gRNA design sites and vectors for the silkworm P53 gene.

[0015] Figure 2 The results of knockout mutation of P53 gene in silkworm.

[0016] Figure 3 Knockout of the silkworm P53 gene causes silk gland phenotypes.

[0017] Figure 4 Changes in silk production caused by knockout of the silkworm P53 gene. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0019] Example 1: Construction of gRNA for specifically knocking out the P53 gene in the posterior silk gland of silkworm

[0020] Based on the silkDB3.0, silkbase and KAIKObase databases of Bombyx mori, a specific knockout target that can only recognize the P53 gene was selected in the first exon region of the P53 gene (SEQ ID NO.1), and its sequence is as follows: 5'-CGCAAAGACGATCCCGGGCA-3' (SEQ ID NO.2), and its structure is as follows Figure 1As shown in A. Using this target-specific primer 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 connected to the gRNA expression vector, and 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 obtained with the gRNA of the P53 gene, the green fluorescent protein ( Figure 1 , B).

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

[0022] After the polymorphism silkworm is raised to pupation and moth, the male and female silkworm moths are mated for 4 hours, and the pairs are separated and the eggs are laid in a dark environment. The silkworm eggs are neatly arranged on a clean glass slide, and the transgenic vector of gRNA and the auxiliary plasmid A3 Helper are injected into 100 silkworm eggs together with the Eppendorf microinjection system. The G0 generation of ant silkworms is obtained for about 10 days of expediting. The silkworms successfully raised to pupation and moth, mate and lay eggs, and the positive individuals with green fluorescence in the eyes are obtained by fluorescence microscopy screening. The gRNA individual of the silkworm P53 gene is hybridized with the Cas9 individual (Fzrregulates silk gland growth by promoting endoreplication and proteinsynthesis in the silkworm.PLoS Genet 2023,19,e1010602) of the rear silk gland specificity of the silkworm eye expressing red fluorescent protein, and the silkworm individuals expressing green and red fluorescent proteins in the eyes are screened out, and the genomic DNA of the rear silk gland is extracted and PCR amplified and sequenced. The results showed that there were multiple mutation forms of P53 gene in the posterior silk gland of diphotic individuals ( Figure 2 , A). RT-PCR and RT-qPCR results showed that specific knockout of P53 gene in the posterior silk gland resulted in a decrease in its expression level ( Figure 2 , BC). It was confirmed that the P53 gene was successfully knocked out in the posterior silk gland.

[0023] Example 3: Specific knockout of the P53 gene in the posterior silk gland of the silkworm promotes silk gland development and silk protein synthesis

[0024] Dissecting the silk glands of silkworms at the tuft stage revealed that the posterior silk gland-specific knockout mutation of P53 caused the posterior silk glands to grow and thicken compared with the control group ( Figure 3 ). 20 male and female cocoons of P53 knockout strain and 20 control strain were randomly selected, and their cocoon shell weight and pupa weight were weighed and counted, and the cocoon layer rate was calculated by the ratio of silkworm layer weight to total cocoon weight. The results showed that compared with the control group, the cocoons of P53 knockout strain silkworms were larger, and there was no significant change in pupae ( Figure 4 , A), its cocoon layer weight and cocoon layer rate increased significantly ( Figure 4 , BC).

[0025] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Application of specific knockout of P53 gene in increasing silk yield, characterized in that: The P53 gene sequence is shown as SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The method of increasing silk production is to increase cocoon layer weight and cocoon layer rate.

3. The use 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 mutant individuals, silkworm varieties with improved silk yield can be obtained.

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 mediation.

6. The method according to claim 4, characterized in that: The method of knocking out the P53 gene is to first prepare Cas9 transgenic silkworms; then construct an sgRNA expression vector for the target P53 gene, and prepare sgRNA transgenic silkworms; the sgRNA transgenic silkworms and the Cas9 individuals specific to the posterior silk gland that express red fluorescent protein in the silkworm eyes are hybridized to screen mutant individuals, and a silkworm variety with high silk yield is obtained.

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