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CN-119162212-B - Application of specific knockout P53 gene in improving silk yield and method thereof

CN119162212BCN 119162212 BCN119162212 BCN 119162212BCN-119162212-B

Abstract

The invention discloses an application of specifically knocking out a P53 gene in improving silk yield and a method thereof, wherein the specifically knocking out the P53 gene in the silk gland at the rear part of silkworms through a CRISPR/Cas9 gene editing system promotes the development of the silk gland of the silkworms, increases the expression of silk protein genes, improves the silk yield, provides a new entry point and a new target for improving the silkworm varieties aiming at improving the silk protein synthesis level and the silk yield, and has good application prospect.

Inventors

  • Qian Wenliang
  • CHENG DAOJUN
  • ZHANG XING
  • Dai Zongcai
  • YUAN DONGQIN

Assignees

  • 西南大学
  • 西部(重庆)科学城种质创制大科学中心

Dates

Publication Date
20260512
Application Date
20241016

Claims (6)

  1. 1. The application of the specific knockout P53 gene in improving the silk yield is characterized in that the sequence of the P53 gene is shown as SEQ ID NO. 1.
  2. 2. The method according to claim 1, wherein the increase in silk yield is an increase in cocoon layer weight and cocoon layer rate.
  3. 3. The method according to claim 1, wherein the target point of the knocked-out P53 gene is shown in SEQ ID NO. 2.
  4. 4. A method for improving silk yield is characterized in that a mutant is screened by knocking out a P53 gene in silkworms to obtain a variety of silkworms with improved silk yield, wherein the sequence of the P53 gene is shown as SEQ ID NO. 1.
  5. 5. The method of claim 4, wherein the method of knocking out the P53 gene is by CRISPR/Cas9 mediated knocking out of the silkworm P53 gene.
  6. 6. The method of claim 4, wherein the P53 gene knockout is performed by preparing a Cas9 transgenic silkworm, constructing an sgRNA expression vector of the target P53 gene, preparing an sgRNA transgenic silkworm, crossing the sgRNA transgenic silkworm and the Cas9 transgenic silkworm to obtain a silkworm variety of high-yield silk, and the Cas9 transgenic silkworm is a silkworm with the rear silk gland of the eye-expressed red fluorescent protein specifically expressed Cas 9.

Description

Application of specific knockout P53 gene in improving silk yield and method thereof Technical Field The invention relates to the field of genetic engineering, in particular to application of a specific knockout P53 gene in improving silk yield and a preparation method. Background Silkworm is used as an important silk-producing economic insect, and the synthesis and secretion of fibroin are completed by silk glands. The silk gland cells undergo about 10 mitoses during embryonic stages, and differentiate to form anterior, middle and posterior silk glands. However, the silk gland cells do not undergo mitosis during the growth and development of silkworm larvae, but undergo 17-19 rounds of DNA replication in the form of nuclear replication, and finally the DNA content in single cells is increased by about 30 ten thousand times, so that polyploids are formed. The nuclear replication process in the silk gland cells of the silkworm increases the genome copy number of the silk protein genes, which is undoubtedly the genetic material basis for the rapid and efficient synthesis of silk proteins at the final stage of silkworm larvae. Therefore, research on the regulation and control mechanism of the nuclear replication of silk gland cells of a family is helpful to analyze the molecular mechanism of silk gland development and efficient synthesis of silk protein of the family. In recent years, a regulatory mechanism of nuclear replication has been analyzed in the mode of insect nuclear replication, and one of the hot spots has been studied. The transcription factor P53 plays a key role in the processes of mitotic cell cycle regulation, DNA damage repair, apoptosis and the like, but whether the transcription factor P53 affects the nuclear replication process of silkworms and the silk yield is not clear at present. Disclosure of Invention In view of the above, one of the objectives of the present invention is to provide an application of specifically knocking out the P53 gene in improving silk yield, and another objective of the present invention is to provide a method for improving silk yield. In order to achieve the above purpose, the present invention provides the following technical solutions: 1. The specific knockout P53 gene is applied to the improvement of silk yield, and the sequence of the P53 gene is shown as SEQ ID NO. 1. Preferably, the method for improving silk yield is to improve cocoon layer weight and cocoon layer rate. Preferably, the target point of the knockout P53 gene is shown as SEQ ID NO. 2. 2. A method for increasing silk yield includes knocking out P53 gene from silkworm, screening mutant individual, and obtaining silkworm variety with increased silk yield. Preferably, the method for knocking out the P53 gene is to mediate knocking out the silkworm P53 gene through CRISPR/Cas 9. The invention preferably discloses that the P53 gene knockout is that Cas9 transgenic silkworms are firstly prepared, then a sgRNA expression vector of a target P53 gene is constructed, the sgRNA transgenic silkworms are prepared, and Cas9 individuals with rear silk gland specificity for expressing red fluorescent protein in the eyes of the sgRNA transgenic silkworms and silkworms are subjected to hybridization screening mutation individuals, so that silkworm varieties of high-yield silkworms are obtained. The invention has the beneficial effects that the invention provides the application of the specific knockout P53 gene in improving the silk yield, the invention promotes the growth of the silk gland of the silkworm by specifically knockout the P53 gene in the silk gland at the rear part of the silkworm through a CRISPR/Cas9 gene editing system, increases the silk protein gene expression, improves the silk yield, provides a new entry point and a new target for improving the silkworm variety aiming at improving the silk protein synthesis level and the silk yield, and has good application prospect. Drawings In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description: FIG. 1 shows the gRNA design site and vector diagram of silkworm P53 gene. FIG. 2 results of the silkworm P53 gene knockout mutant sequences. FIG. 3 silkworm P53 gene knockout causes a silk gland phenotype. FIG. 4 variation in silk yield due to silkworm P53 gene knockout. Detailed Description The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it. EXAMPLE 1 construction of a silkworm rear Silk gland-specific knockout gRNA of the P53 Gene Based on three databases of silkdb3.0, silkbase and KAIKObase of silkworm, a specific knockout target capable of recognizing only P53 gene was selected in the first exon region of P53 gene (SEQ ID No. 1), and the sequ