Use of kwmtbomo06644 gene in improving silkworm cocoon shell ratio and silk yield

Abstract

The present invention relates to a use of a KWMTBOMO06644 gene in improving the silkworm cocoon shell ratio and silk yield. A nucleotide sequence of the KWMTBOMO06644 gene is as shown in SEQ ID NO: 1. The use method relates to improving the silk yield of silkworms by knocking out the KWMTBOMO06644 gene. By using CRISPR-Cas9 gene editing technology to perform targeted knockout of the silkworm endonuclease reverse transcriptase KWMTBOMO06644 gene, the cocoon shell ratio of male silkworms can be significantly improved, and the silk yield can be increased. Therefore, the knockout of the KWMTBOMO06644 gene can be used for creating high silk‑yield mutants and breeding new silkworm varieties having high silk yield.

Description

Application of KWMTBOMO06644 gene in improving silkworm cocoon layer ratio and silk yield Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of the KWMTBOMO06644 gene in improving the cocoon layer ratio and cocoon silk yield of silkworms. Background Technology

[0002] In sericulture, the cocoon shell ratio is a crucial indicator. A higher cocoon shell ratio means more silk is produced, resulting in better economic traits. Therefore, the cocoon shell ratio determines the silk yield and economic benefits of sericulture. Developing silkworm varieties with higher cocoon shell ratios has always been a major focus in sericulture. While the cocoon shell ratio of different silkworm varieties fluctuates within a certain range due to variations in rearing conditions and growth status, the overall differences are not significant. Although traditional silkworm breeding techniques such as hybridization can improve silk production performance and increase cocoon shell ratio to some extent, they still cannot meet the current needs of sericulture development. A more efficient technology is urgently required to adapt to the current state of sericulture.

[0003] The emergence of gene editing technology has greatly improved and accelerated the progress of modern breeding science. This technology can precisely target and modify the structure of genes from the inside, thereby changing gene function at the molecular level more efficiently and conveniently, regulating the growth and development of silkworm silk glands, and fundamentally improving the silk economic traits of silkworms.

[0004] The sericin secreted by the central silk gland and the fibroin secreted by the posterior silk gland are components of silk, therefore the size of the central and posterior silk glands determines the silk yield. Increasing the size of the central silk gland or promoting the synthesis of related proteins can effectively improve the cocoon layer ratio of silkworms. Technical issues

[0005] 1. Lack of breeding techniques to efficiently increase silkworm cocoon layer ratio and silk yield.

[0006] Traditional silkworm breeding methods (such as hybridization) can improve silk production traits to some extent, but the improvement is limited and the breeding cycle is long, which cannot meet the modern sericulture's demand for rapid breeding of high-silk-producing varieties.

[0007] 2. Lack of breeding technology targeting the silk production performance of male silkworms

[0008] In traditional breeding, male silkworms typically produce less cocoon silk than female silkworms, and there is a lack of efficient and targeted breeding methods to significantly improve the development of silk glands and the yield of cocoon silk in male silkworms.

[0009] 3. Lack of highly efficient molecular breeding targets related to cocoon and silk yield.

[0010] Previously, no endonuclease reverse transcriptase gene directly related to male silkworm cocoon silk production had been discovered, resulting in a lack of reliable gene editing targets in molecular breeding. Technical solutions

[0011] The purpose of this invention is to provide an application of the KWMTBOMO06644 gene in improving the cocoon layer ratio and silk yield of silkworms. By knocking out the silkworm reverse transcriptase KWMTBOMO06644 gene through CRISPR-Cas9 gene editing technology, the cocoon layer ratio and silk yield can be improved, high-yield cocoon and silk mutants can be created, and new high-yield cocoon and silk varieties of silkworms can be bred.

[0012] This invention provides the following technical solution:

[0013] This invention provides the application of the KWMTBOMO06644 gene in improving the cocoon layer ratio and cocoon silk yield of silkworms, and the nucleotide sequence of the KWMTBOMO06644 gene is shown in SEQ ID NO:1;

[0014] Furthermore, the application method is to increase silkworm cocoon production by knocking out the KWMTBOMO06644 gene;

[0015] This invention provides the application of KWMTBOMO06644 protein in increasing silkworm cocoon yield, the nucleotide sequence of which is shown in SEQ ID NO:2;

[0016] Furthermore, the application method is to increase silkworm cocoon production by inhibiting the expression of KWMTBOMO06644 protein;

[0017] This invention provides the use of the KWMTBOMO06644 gene or KWMTBOMO06644 protein in at least one of the following:

[0018] (1) Increase the weight of silkworm cocoons;

[0019] (2) Increase the weight of silkworm pupae;

[0020] (3) Increase the cocoon layer rate of silkworms;

[0021] (4) Increase the size of the silkworm's silk glands;

[0022] This invention provides a method for increasing silk production in silkworms, wherein the method comprises knocking out the KWMTBOMO06644 gene in silkworms as shown in SEQ ID NO:1, and / or inhibiting the expression of the KWMTBOMO06644 protein as shown in SEQ ID NO:2;

[0023] Furthermore, the silkworm in question is a male silkworm;

[0024] Furthermore, the KWMTBOMO06644 gene, as shown in SEQ ID NO:1, was knocked out in silkworms using gene editing technology;

[0025] Furthermore, the KWMTBOMO06644 gene was knocked out using ZFNs, TALENs, CRISPR / Cas9 or their variants.

[0026] Furthermore, knocking out the KWMTBOMO06644 gene using CRISPR / Cas9 includes the following steps:

[0027] Step 1: Construct an sgRNA expression vector containing gRNA that knocks out the KWMTBOMO06644 gene in silkworms. The gRNA is prepared using primers shown in SEQ ID NO:4 and SEQ ID NO:5. The sgRNA expression vector is injected into silkworm eggs using microinjection technology, and then sgRNA transgenic silkworms are obtained by fluorescence screening.

[0028] Step 2: Construct the SpyCas9 expression vector, inject the SpyCas9 expression vector into silkworm eggs using microinjection technology, and then obtain SpyCas9 transgenic silkworms through fluorescence screening;

[0029] Step 3: The sgRNA transgenic silkworm and the SpyCas9 transgenic silkworm were hybridized and screened to obtain silkworms with the KWMTBOMO06644 gene knocked out. Beneficial effects

[0030] 1. Significantly increases cocoon and silk production.

[0031] Knocking out the KWMTBOMO06644 gene significantly increased the cocoon layer ratio in male silkworms, directly leading to an increase in cocoon silk production. Experiments showed that both cocoon weight and pupa weight were improved.

[0032] 2. High-efficiency breeding of male silkworms

[0033] In traditional breeding, male silkworms produce relatively low silk yields. This technology is particularly suitable for breeding high-silk-yielding male silkworm varieties and has a sex-specific advantage.

[0034] 3. Gene editing technology is precise and efficient.

[0035] Using the CRISPR-Cas9 system, which offers strong targeting and high editing efficiency, high-yield mutants can be created rapidly. Compared with traditional hybridization breeding, it has a shorter cycle and significant effects.

[0036] 4. Provides new targets for silkworm breeding.

[0037] The KWMTBOMO06644 gene is the first reported endonuclease reverse transcriptase gene associated with male silkworm cocoon silk production, providing a new direction for subsequent functional research and breeding applications.

[0038] 5. Compatible with a variety of gene editing tools

[0039] In addition to CRISPR-Cas9, gene editing technologies such as ZFNs and TALENs can also be used, offering flexibility and adaptability.

[0040] 6. Possesses potential for industrial application.

[0041] This method has a clear operation procedure and strong repeatability, making it suitable for large-scale, industrialized breeding of high-yield silkworm varieties.

[0042] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0044] Figure 1 shows the skeleton of the successfully constructed sgRNA expression vector piggyBac[3×P3-EGFP,BmU6-KWMTBOMO06644-SV40];

[0045] Figure 2 shows images of binary transgenic positive individuals (A1 and A2 are fluorescence images of silkworms that successfully expressed gRNA; A3 and A4 are fluorescence images of silkworms that successfully knocked out Bm6644).

[0046] Figure 3 shows the detection of the Bm6644 mutation site in silkworms after CRISPR-Cas9 gene editing.

[0047] Figure 4 shows the phenotypic observation and economic trait statistical analysis of the gene-edited Bm6644 knockout silkworm (L5D5: 5th day of the fifth instar; WT: wild-type silkworm; Bm6644KO: Bm6644 knockout silkworm). Embodiments of the present invention

[0048] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0049] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0050] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0051] Example 1: Construction of sgRNA expression vector

[0052] 1.1 Synthesis of the target sequence

[0053] The full-length CDS sequence of the KWMTBOMO06644 gene was obtained by searching the Silkbase database, as shown in SEQ ID NO.1. Based on this full-length sequence, the sgRNA sequence and its required primer sequences were designed using the CCTop - CRISPR / Cas9 target online predictor website, as shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5. The designed primers were synthesized by Shanghai Sangon Biotech Co., Ltd. The amino acid sequence of the Bm6644 gene is shown in SEQ ID NO.2.

[0054] 1.2 Annealing experiment of sgRNA primers

[0055] First, preheat the metal bath to 95°C. Then, add an appropriate amount of ddH2O to dissolve the synthesized dry primers to 10 μM. Add 10 μL of the forward and reverse primers to the PCR tubes, mix briefly, and place in the metal bath. Incubate at 95°C for 5 minutes, then immediately turn off the power and allow it to cool naturally to room temperature.

[0056] 1.3. Enzyme digestion of p200 vector

[0057] To ligate the annealed double-stranded sgRNA into the p200 vector, the p200 vector must first be digested with enzymes. The digestion system and procedure are as follows: 10x Buffer 5 μL, 50x Oligo 1 μL, p200 2 μg, AarI 5 μL, and dd H2O to bring the total volume to 50 μL. After briefly centrifuging the mixture, incubate it at 37°C for 16 h. Then, perform electrophoresis on a 1.5% nucleic acid gel and recover the digested products.

[0058] 1.4. sgRNA ligated into p200 vector

[0059] A small amount of the annealed sgRNA was subjected to nucleic acid gel electrophoresis. The target band was bright and uniform. The concentration and purity were confirmed to be within acceptable limits using a concentration spectrophotometer before proceeding with the subsequent ligation experiment. The ligation system and procedure were as follows: 10xT4 Buffer 1 μL, T4 ligase 1 μL, p200 1 μL, and sgRNA 7 μL. This mixture was briefly centrifuged and then incubated overnight at 16°C in a metal bath.

[0060] 1.5 Transformation

[0061] Take Trans1-T1 competent cells purchased from Beijing TransGen Biotech Co., Ltd. from a -80℃ freezer and thaw them on ice. Take another sterile 1.5ml centrifuge tube and pre-cool it on ice. Add 10ul of ligation product to 50ul of Trans1-T1 competent cells, gently mix with a pipette, and let it stand on ice for 30min. Then heat shock it on a preheated 42℃ metal bath for 90s, and immediately return it to ice for 2-3min. After that, add 300ul of antibiotic-free LB liquid medium to the centrifuge tube in a clean bench and incubate it at 37℃ for 220rpm for 1h. Take 100ul of the cultured bacterial solution and spread it evenly on LB solid ampicillin agar plates and incubate it overnight at 37℃.

[0062] 1.6. Bacterial Picking and Detection

[0063] Using a pipette tip in a clean bench, pick single colonies of appropriate size and regular morphology from overnight culture plates and transfer them to 1.5 ml centrifuge tubes containing 400 μL LB ampicillin broth. Incubate at 37°C and 220 rpm for 4-6 hours. Then, take a small amount of the bacterial culture for PCR detection. The detection system is as follows: 7.5 μL 2xTaq Master Mix, 0.25 μL primer-F, 0.25 μL primer-R, 1 μL bacterial culture, and bring the total volume to 15 μL using ddH2O. The PCR program is as follows: Step 1: 95°C pre-denaturation for 5 min; Step 2: 95°C denaturation for 15 s; Step 3: 54°C annealing for 15 s; Step 4: 72°C extension for 90 s; Step 5: 72°C extension for 5 min; Step 6: 12°C storage. Steps 2 to 4 are repeated 30 times. After the reaction, nucleic acid electrophoresis was performed using 1.0% agarose gel. Strains with the correct band size were selected, and 100 μL was sent to BGI for sequencing. The remaining bacterial culture was stored at 4°C for later use.

[0064] 1.7 Plasmid Extraction

[0065] After inoculating the correctly sequenced strain into 10 ml of LB ampicillin broth, incubate overnight at 37°C and 220 rpm on a shaker. Then, transfer the culture to a 10 ml centrifuge tube and centrifuge at 12000 rpm for 5 min, discarding the supernatant. Add 500 μL of P1 solution from the QIAGEN Plasmid Midi Kit to the pellet, and mix thoroughly by pipetting until no plaques remain. Transfer the entire volume to a 2 ml centrifuge tube, then add 500 μL of P2 solution, gently invert to mix, and then add 700 μL of N3 solution, gently invert to mix, and centrifuge at 13000 rpm for 10 min. Divide the supernatant into two portions and transfer them to a DNA binding adsorption column, centrifuge at 13000 rpm for 1 min, discarding the eluent. Add 750 μL of Washing Buffer containing 95% ethanol to the adsorption column. Centrifuge at 12000 rpm at room temperature for 1 min and discard the eluent. Repeat once. Finally, centrifuge the adsorption column at 13000 rpm for 2 min. Place the adsorption column onto a 1.5 ml centrifuge tube, add 30 μL of preheated ddH2O (65℃) to the center of the column, centrifuge at 12000 rpm at room temperature for 2 min to wash off the DNA, and use a spectrophotometer to detect the plasmid concentration and plasmid. Store qualified plasmids at -40℃.

[0066] Example 2: Obtaining the Bm6644 silkworm mutant by CRISPR-Cas9 editing technology

[0067] The sgRNA expression vector piggyBac[3×P3-EGFP,BmU6-KWMTBOMO06644-SV40] plasmid (see Figure 1 for the backbone diagram of piggyBac[3×P3-EGFP,BmU6-KWMTBOMO06644-SV40]) and helper plasmid were mixed at a 1:1 molar ratio and injected into non-diapause silkworm eggs less than 2 hours after being laid using a microinjector. The injection needle holes were then sealed with non-toxic glue. The injected silkworm eggs were incubated at 25°C until hatching. After hatching, the silkworms were fed with mulberry leaves as usual. This batch of injected silkworm eggs is the G0 generation. After metamorphosis, the silkworms were mated in the same enclosure to obtain the G1 generation individuals. The G1 generation individuals were incubated as usual until 6-7 days after egg laying. Green fluorescence was detected under a stereofluorescence microscope using excitation light to screen for transgenic positive individuals that specifically emitted green fluorescence at the eyes. This refers to the Bm6644-sgRNA-positive G1 generation transgenic silkworm individuals (A1 in Figure 2, silkworm egg state). These positive G1 generation individuals were routinely reared until they emerged as moths (A2 in Figure 2, after moth emergence). They were then mated with SpyCas9 adults whose eyes emitted red fluorescence, and the resulting eggs were the F1 generation. After hatching, the queen larvae were fed on mulberry leaves until the fifth instar. The larvae were then screened under a fluorescence microscope. Larvae whose eyes emitted both green and red fluorescence simultaneously were identified as F1 generation Bm6644 gene knockout positive individuals (A3 and A4 in Figure 2). Further confirmation was made through fluorescence screening after moth emergence.

[0068] Further investigation is needed at the molecular level to detect the mutation site in the CRISPR-Cas9 editing technology that knocked out Bm6644 silkworms. Specifically, the genome was extracted from the silk gland tissue of the fifth-instar F1 generation silkworms on the fifth day.

[0069] Using the extracted genome as a template for PCR amplification, a pair of detection primers was designed based on the CDS sequence of Bm6644:

[0070] F: 5'-CTGAATGTCGATCGAAATTCCACC-3' (SEQ ID NO:6)

[0071] R: 5'-CTCAATTGTTGAGTTAACCTCGCC-3' (SEQ ID NO:7)

[0072] The selected genomic DNA from the silk gland tissue was amplified using this primer pair. The amplification products were recovered, T-cloned, and then sampled for sequencing. The sequencing results showed that Bm6644 gene knockout positive individuals produced both knock-in and deletion knockout forms (Figure 3). It is worth noting that the detection primers used in this example must be specific, with no non-specific amplification, and the PCR products can be directly used for sequencing analysis.

[0073] Example 3: Phenotypic observation of the CRISPR-Cas9 knockout Bm6644 silkworm mutant

[0074] The Bm6644 knockout mutants obtained through screening were reared under the same conditions as wild-type silkworms. It was found that the male silkworms of the Bm6644 knockout type had greater cocoon weight, pupa weight, and cocoon layer ratio than the wild type. Further dissection of fifth-instar, fifth-day-old silkworms revealed that the silk glands of the male larvae of the Bm6644 knockout type were significantly larger than those of the wild type (Figure 4). This indicates that the absence of Bm6644 expression in the silk glands of silkworms caused by the Bm6644 knockout type leads to an increase in the cocoon layer ratio of male silkworms.

[0075] In this embodiment of the invention, SpyCas9 is used to mediate the knockout of the target Bm6644 gene. As is known to those skilled in the art, any means that can knock out the Bm6644 gene can achieve the purpose of this invention, such as ZFNs, TALENs, and CRISPR / Cas9 or their variants mediating the knockout of the target Bm6644 gene.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of KWMTBOMO06644 gene in improving cocoon shell ratio and cocoon silk yield, characterized in that, The nucleotide sequence of the KWMTBOMO06644 gene is shown as SEQ ID NO: 1; The application method is to improve cocoon rate and cocoon silk yield by knocking out KWMTBOMO06644 gene.

2. Use of KWMTBOMO06644 protein in improving cocoon shell rate and cocoon silk yield, characterized in that, The amino acid sequence of the KWMTBOMO06644 protein is shown as SEQ ID NO: 2; The application method is to improve cocoon rate and cocoon silk yield by inhibiting KWMTBOMO06644 protein expression.

3. The application of knocking out KWMTBOMO06644 gene with nucleotide sequence shown as SEQ ID NO: 1 or inhibiting KWMTBOMO06644 protein expression with amino acid sequence shown as SEQ ID NO: 2 in at least one of the following: (1) increasing cocoon weight of male silkworm; (2) increasing pupa weight of male silkworm; (3) increasing cocoon rate of male silkworm; (4) increasing silk gland of male silkworm.

4. A method for improving cocoon shell rate and silk yield of male silkworms, characterized by, The method is to knock out KWMTBOMO06644 gene shown as SEQ ID NO: 1 in silkworm, and / or, inhibit KWMTBOMO06644 protein expression shown as SEQ ID NO:

2.

5. The method of claim 4, wherein, Knocking out KWMTBOMO06644 gene shown as SEQ ID NO: 1 in silkworm by gene editing technology.

6. The method of claim 5, wherein, Knocking out KWMTBOMO06644 gene by ZFNs, TALENs, CRISPR / Cas9.

7. The method of claim 6, wherein, Knocking out KWMTBOMO06644 gene by CRISPR / Cas9 includes the following steps: Step 1: Constructing sgRNA expression vector containing gRNA for knocking out KWMTBOMO06644 gene of silkworm, the gRNA is prepared by primers shown as SEQ ID NO: 4 and SEQ ID NO: 5; injecting sgRNA expression vector into silkworm eggs by microinjection technology, then screening sgRNA transgenic silkworm by fluorescence; Step 2: Constructing SpyCas9 expression vector, injecting SpyCas9 expression vector into silkworm eggs by microinjection technology, then screening SpyCas9 transgenic silkworm by fluorescence; Step 3: Crossing sgRNA transgenic silkworm and SpyCas9 transgenic silkworm, screening to obtain silkworm with knocked out KWMTBOMO06644 gene.

Images