A method for controlling the amount of silkworm wing scales by inhibiting the expression of the ILRUN gene.

Abstract

We provide silkworms with fewer scales. [Solution] This invention discloses the application of the ILRUN gene to the control of silkworm wing scale quantity. This invention is the first to propose and verify that the amount of wing scales in adult silkworms can be reduced by inhibiting ILRUN, an inflammation and lipid regulatory factor gene having UBA-like and NBR1-like domains. The sgRNA according to this invention can effectively achieve ILRUN gene knockout, making it usable in actual production and enabling the development of silkworm varieties with "low scales".

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to the use of the ILRUN gene in controlling the amount of wing scales in silkworms.

Background Art

[0002] Bombyx mori is a fully domesticated economic insect and the only source of raw material for the silk industry, whose cocoons can be reeled and processed into various silk products. In large-scale silkworm egg production, the thick and dense scales covering the body surface of adult commercial varieties easily peel off during behaviors such as eclosion and mating, tending to form a large amount of floating "scales dust". Inhalation of fine, scattered scales dust can cause respiratory discomfort and allergic symptoms in workers and nearby residents, easily threatening their health. Furthermore, peeled scales may carry spores of pathogens such as *Nosema bombycis*, acting as a vector for disease transmission between silkworm rearing rooms, greatly increasing the risk of cross-infection of devastating epidemics such as pebrine disease, and directly threatening the safety of silkworm eggs. At the same time, settled scales dust seriously pollutes the environment of silkworm rearing rooms, silkworm rearing equipment and silkworm eggs, increases cleaning burdens, and adversely affects production efficiency.

[0003] Currently, production mainly relies on physical and environmental management measures to address this problem. For example, before and after silkworm rearing, thorough cleaning of silkworm rearing rooms and equipment and drug disinfection are carried out to reduce the base number of pathogenic bacteria. Meanwhile, protection is achieved by means such as enhancing ventilation in silkworm rearing rooms and requiring workers to wear masks. However, all these methods belong to post-event treatment or passive protection, only alleviating superficial symptoms, and cannot fundamentally reduce the formation and peeling of scales. Therefore, the control effect is limited, and it is impossible to completely eliminate the risk.

Summary of the Invention

Problem to be Solved by the Invention

[0004] The object of the present invention is to provide an application of the ILRUN gene in controlling the amount of silkworm wing scales, and more particularly in reducing the amount of silkworm wing scales. [Means for solving the problem]

[0005] This invention relates to the application of the ILRUN gene described in the present invention to the control of silkworm wing scale quantity.

[0006] Preferably, the application involves inhibiting the expression of the ILRUN gene to reduce the amount of wing scales in adult silkworms.

[0007] Preferably, the application steps include constructing an expression inhibitor based on the silkworm ILRUN gene, introducing silkworm eggs into it, hatching them, and obtaining adult silkworms with reduced wing scales.

[0008] Preferably, the exon sequence of the silkworm ILRUN gene is shown in SEQ ID NO:1.

[0009] Preferably, the expression inhibitor is a) siRNA, shRNA, miRNA, dsRNA, antisense nucleotide, or b) Nucleic acid molecules that express a), c) sgRNA, and, d) A composition of Cas9 protein, or a nucleic acid molecule expressing Cas9 protein, and c), or e) c) is one of the following nucleic acid molecules that express the Cas9 protein.

[0010] Preferably, the expression inhibitor is a composition of Cas9 protein and sgRNA, and more preferably, the molar ratio of Cas9 protein to sgRNA is 1:0.8 to 1.2.

[0011] Preferably, the target sequence of the sgRNA is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0012] Preferably, the sgRNA is EnGen (登録商標) It is prepared using an sgRNA synthesis kit.

[0013] Preferably, the Cas9 protein is EnGen (登録商標) SpyCas9 is an NLS nuclease. [Effects of the Invention]

[0014] Compared to conventional technologies, the present invention has the following remarkable advantages. The present invention is the first to propose and verify that the amount of wing scales in adult silkworms can be reduced by inhibiting the inflammation and lipid regulator with UBA-like and NBR1-like domains (ILRUN) gene. The sgRNA according to the present invention can effectively achieve ILRUN gene knockout, making it usable in actual production and enabling the development of silkworm varieties with "fewer scales". [Brief explanation of the drawing]

[0015] [Figure 1] This is a schematic diagram of the silkworm ILRUN gene structure. [Figure 2] These are representative images of adult silkworms with the ILRUN gene knocked out. Image A is a comparison of the wild-type silkworm Nistari and the silkworm Nisnlw with the ILRUN gene knocked out. Image B is a comparison of the left forewing (LF), right forewing (RF), left hindwing (LH), and right hindwing (RH) of the wild-type silkworm Nistari and the silkworm Nisnlw with the ILRUN gene knocked out. [Figure 3] This figure shows the sequencing analysis results of silkworms in which the ILRUN gene was knocked out. [Modes for carrying out the invention]

[0016] The technical solutions of the present invention will be described further below.

[0017] In the examples, the polyvoltine silkworm variety Nistari is used as the experimental subject, and the variety is provided by the Institute of Sericulture, Jiangsu University of Science and Technology, China.

[0018] Example 1: Design and Synthesis of sgRNA Based on ILRUN 1. Cloning of Silkworm ILRUN (BmILRUN) Gene Primers that specifically amplify BmILRUN were designed and synthesized. The sequence of the forward primer is 5'-ATGGACGTTGATGGCGCCT-3', and the sequence of the reverse primer is 5'-TCAACACATTTGATCGTCTTGT-3'.

[0019] Total RNA was extracted from the wings of emerged Nistari silkworms using RNAiso Plus (Takara, Catalog No. 9767). With this RNA as a template, PrimeScript TM Reverse transcription was performed using the RT Master MIX kit (Takara, Catalog No. RR036A) to synthesize cDNA. Then, PCR amplification was carried out with LA Taq DNA polymerase (Takara, Catalog No. RR002M) using the synthesized cDNA and the above-mentioned BmILRUN primers. The amplification program was as follows: after pre-denaturation at 95°C for 5 min, 30 cycles of the steps of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 50 s were repeated, and a final extension was further performed at 72°C for 10 min to obtain an amplification product.

[0020] The obtained amplification product was cloned into pMD TM 19-T (TaKaRa, Catalog No. 3271), and the correct expression plasmid pMD TM 19-T-BmILRUN was obtained through sequencing verification.

[0021] 2. Design and Synthesis of sgRNA The sequence of the BmILRUN gene obtained by sequencing validation is shown as SEQ ID NO:1, and as shown in Figure 1, two knockout target sites located in the first and third exons were selected. The target site (Site 1) located in the first exon is 5'-GTTGATGGCGCCTCAGTACC TGG -3' (SEQ ID NO:2), the target site (Site 2) located in the third exon has a 5'- CCA The sequence was CCCCCTTCCTGTTGGACATTC-3' (SEQ ID NO:3).

[0022] The oligonucleotides for sgRNA synthesis, whose sequences are indicated by SEQ ID NO:4 and SEQ ID NO:5, were synthesized by commissioning Shanghai Biotechnology Co., Ltd. Subsequently, EnGen was synthesized using the oligonucleotides for sgRNA synthesis. (登録商標) In vitro transcription was performed using an sgRNA synthesis kit (NEB, catalog number E3322V) to synthesize sgRNA-1 targeting Site 1 and sgRNA-2 targeting Site 2.

[0023] Example 2: Preparation of ILRUN knockout silkworms sgRNA-1, sgRNA-2, and Cas9 protein (EnGen (登録商標) Spy Cas9 NLS nuclease (NEB, catalog number M0646T) was mixed at a final concentration of sgRNA-1 + sgRNA-2: 330 ng / μL (equal amounts of sgRNA-1 and sgRNA-2 mixed) and Cas9 protein: 10 μM, i.e., a molar ratio of sgRNA to Cas9 protein of 1:1. 10 nL of each mixture was injected into silkworm eggs. After injection, the silkworm eggs were sealed with a non-toxic adhesive to prevent contamination, and the eggs were incubated at 25°C until hatching.

[0024] Silkworms injected with a mixture of sgRNA and Cas9 protein were reared to adulthood on mulberry leaves, and their wing scales were observed.

[0025] Representative images of adult silkworms are shown in Figure 2. The wing scales of silkworms Nisnlw, in which the ILRUN gene was knocked out, were significantly reduced compared to those of the wild-type silkworm Nistari.

[0026] Tissue from two legs was used as material from each adult that emerged, and genomic DNA was extracted using a cell / tissue genomic DNA extraction kit (Bioteke, catalog number DP1902). Using the genomic DNA as a template, the genomic region was amplified using the ExTaq enzyme (Takara, catalog number RR001C) with the primers shown in Table 1. The amplification program consisted of 30 cycles of preliminary denaturation at 95°C for 5 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes.

[0027] [Table 1]

[0028] The amplified genomic region is pMD TM Each cell was cloned into a 19-T vector, transformed into DH5α-competent cells, plated onto ampicillin-resistant LB plates, and inverted cultured at 37°C for 16 hours. Single colonies were picked and sequenced. The presence or absence of mutations was detected by sequence alignment, and the type of mutation was determined. Silkworms with BmILRUN somatic cell displacement were selected. The gene mutations induced by CRISPR / Cas9 are shown in Figure 3. [Table 2] TIFF0007917240000003.tif255148

Claims

1. A method for controlling the amount of silkworm wing scales by inhibiting the expression of the ILRUN gene.

2. The method according to claim 1, characterized in that the method involves reducing the amount of wing scales of adult silkworms by inhibiting the expression of the ILRUN gene.

3. The method according to claim 2, characterized in that the steps of the method include constructing an expression inhibitor based on the silkworm ILRUN gene, introducing it into silkworm eggs, hatching them, and obtaining silkworms in which the amount of adult wing scales is reduced.

4. The method according to claim 3, characterized in that the silkworm ILRUN gene sequence is shown in SEQ ID NO:

1.

5. The aforementioned expression inhibitors are a) siRNA, shRNA, miRNA, dsRNA, antisense nucleotide, or b) Nucleic acid molecules that express a), c) sgRNA, and, d) A composition of Cas9 protein, or a nucleic acid molecule expressing Cas9 protein, and c), or The method according to claim 3, characterized in that it is one of the following: e) a nucleic acid molecule containing c) and expressing Cas9 protein.

6. The method according to claim 5, characterized in that the expression inhibitor is a composition of Cas9 protein and sgRNA.

7. The method according to claim 6, characterized in that the expression inhibitor has a molar ratio of Cas9 protein to sgRNA of 1:0.8 to 1.

2.

8. The method according to 6, characterized in that the target sequence of the sgRNA is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

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