Application of KWMTBOMO06644 gene in breeding of high cocoon silk variety

Abstract

The application relates to the field of biological gene technology, and particularly relates to application of a KWMTBOMO06644 gene in high cocoon silk yield variety breeding. The nucleotide sequence of the KWMTBOMO06644 gene is shown as SEQ ID NO:1, the application method is to improve the cocoon silk yield of silkworms by knocking out the KWMTBOMO06644 gene. The application discloses that the KWMTBOMO06644 gene is used as a target gene for improving the cocoon silk yield of male silkworms, the endonuclease reverse transcriptase KWMTBOMO06644 gene of the silkworm is targetedly knocked out through a CRISPR-Cas9 gene editing technology, the cocoon layer rate of the male silkworm can be obviously improved, and the cocoon silk yield is increased. Therefore, the KWMTBOMO06644 gene can be used for creating a high cocoon silk yield mutant and breeding a new variety of silkworm with high cocoon silk yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological gene technology, and particularly relates to application of KWMTBOMO06644 gene in high cocoon silk yield variety breeding. BACKGROUND

[0002] In silkworm production, cocoon layer rate of the silkworm is an important index, cocoon layer rate is higher, which means more silk spinning, and economic traits are better, so cocoon layer rate determines the silk yield and economic benefits in silkworm production. How to cultivate silkworm varieties with higher cocoon layer rate has been the main content in silkworm production. Although cocoon layer rates of different silkworm varieties fluctuate within a certain range due to different feeding conditions and growth conditions, there is no big difference in general; although traditional silkworm breeding techniques such as hybridization can improve silk production performance of the silkworm and increase cocoon layer rate to a certain extent, it still cannot meet the current development of silkworm industry, and a more efficient technology is needed to adapt to the current development of silkworm industry.

[0003] The emergence of gene editing technology greatly improves the process of modern breeding, which can precisely target from the inside, directionally modify the structure of the gene, and further more efficiently and conveniently change the gene function from the molecular level, regulate the growth and development process of the silkworm silk gland, and fundamentally improve the economic traits of silkworm silk.

[0004] Sericin secreted by the middle silk gland and fibroin secreted by the posterior silk gland are components of silk, so the size of the middle silk gland and the posterior silk gland determines the amount of silk production. Increasing the size of the middle silk gland or promoting the synthesis of related proteins can effectively improve the cocoon layer rate of the silkworm.

[0005] Based on this, the present application clones and identifies KWMTBOMO06644 gene of the silkworm endonuclease reverse transcriptase

[0006] KWMTBOMO06644 (abbreviated as: Bm6644) full-length sequence is cloned and identified, and a method for increasing silk amount of male silkworm by knocking out the gene is provided. SUMMARY

[0007] The present application provides application of KWMTBOMO06644 gene in high cocoon silk yield variety breeding, and through CRISPR-Cas9 gene editing technology, silkworm endonuclease reverse transcriptase KWMTBOMO06644 gene is knocked out, cocoon weight and cocoon layer rate can be improved, high cocoon silk mutant is created, and new silkworm variety with high silk yield is bred.

[0008] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0009] The application provides application of KWMTBOMO06644 gene in improving cocoon silk yield of silkworms, and the nucleotide sequence of the KWMTBOMO06644 gene is shown in SEQ ID NO:1.

[0010] Further, the application method is to improve the cocoon silk yield of silkworms by knocking out the KWMTBOMO06644 gene.

[0011] The application provides application of KWMTBOMO06644 protein in improving cocoon silk yield of silkworms, and the nucleotide sequence of the KWMTBOMO06644 protein is shown in SEQ ID NO:2.

[0012] Further, the application method is to improve the cocoon silk yield of silkworms by inhibiting expression of the KWMTBOMO06644 protein.

[0013] The application provides application of KWMTBOMO06644 gene or KWMTBOMO06644 protein in at least one of the following:

[0014] (1) improving cocoon weight of silkworms;

[0015] (2) improving pupa weight of silkworms;

[0016] (3) improving cocoon layer rate of silkworms;

[0017] (4) increasing silk glands of silkworms.

[0018] The application provides a method for improving cocoon silk yield of silkworms, which comprises knocking out KWMTBOMO06644 gene shown in SEQ ID NO:1 in silkworms, and / or inhibiting expression of KWMTBOMO06644 protein shown in SEQ ID NO:2.

[0019] Further, the silkworms are male silkworms.

[0020] Further, the KWMTBOMO06644 gene shown in SEQ ID NO:1 in silkworms is knocked out by gene editing technology.

[0021] Further, the KWMTBOMO06644 gene is knocked out by using ZFNs, TALENs, CRISPR / Cas9 or variants thereof.

[0022] Further, the KWMTBOMO06644 gene is knocked out by using CRISPR / Cas9, and the method comprises the following steps:

[0023] Step 1: construct sgRNA expression vector containing gRNA for knocking out KWMTBOMO06644 gene of the domestic silkworm, the gRNA is prepared by primers shown in SEQ ID NO: 4 and SEQ ID NO: 5; inject the sgRNA expression vector into silkworm eggs by microinjection technology, and then obtain sgRNA transgenic silkworm through fluorescence screening;

[0024] Step 2: construct SpyCas9 expression vector, inject the SpyCas9 expression vector into silkworm eggs by microinjection technology, and then obtain SpyCas9 transgenic silkworm through fluorescence screening;

[0025] Step 3: cross the sgRNA transgenic silkworm and the SpyCas9 transgenic silkworm, and then obtain the domestic silkworm with the KWMTBOMO06644 gene knocked out through screening.

[0026] The present application at least has the following beneficial effects:

[0027] The present application discloses that KWMTBOMO06644 is a gene target capable of improving the cocoon layer rate of male domestic silkworm and thus affecting economic benefits; after the KWMTBOMO06644 is knocked out in the domestic silkworm, the cocoon layer rate and cocoon silk yield of male silkworm are significantly improved, so that the KWMTBOMO06644 knockout can be used to create high-yield cocoon silk mutants and cultivate domestic silkworms of new varieties with high silk yield. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0029] Figure 1 The piggyBac[3xP3-EGFP, BmU6-KWMTBOMO06644-SV40] backbone map for successfully constructing sgRNA expression vector is shown in the figure;

[0030] Figure 2 The pictures of double transgenic positive individuals (A1 and A2 are the fluorescence of domestic silkworm successfully expressing gRNA; A3 and A4 are the fluorescence of domestic silkworm successfully knocking out Bm6644) are shown in the figure;

[0031] Figure 3 The figure for detecting the mutation site of the domestic silkworm with Bm6644 knocked out by CRISPR-Cas9 gene editing is shown in the figure;

[0032] Figure 4Figure 1 shows the phenotype observation and statistical analysis of economic traits of the silkworm with Bm6644 knocked out by gene editing technology (L5D5: the 5th day of the 5th instar; WT: wild type silkworm; Bm6644KO: silkworm with Bm6644 knocked out). DETAILED DESCRIPTION

[0033] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0034] The main purpose of the present application is to provide the application of KWMTBOMO06644 gene in the selection of high-yield cocoon silk varieties, which is specifically as follows:

[0035] Example 1 Construction of sgRNA expression vector

[0036] 1.1 Synthesis of target sequence

[0037] The CDS sequence full length of KWMTBOMO06644 gene was obtained by searching the Silkbase database, as shown in SEQ ID NO. 1. Based on the sequence full length, the sequence of sgRNA and the required primer sequence were designed using 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 primer was entrusted to Shanghai Shengong Bioengineering Co., Ltd. for synthesis. The amino acid sequence of the above Bm6644 gene is shown in SEQ ID NO. 2.

[0038] 1.2 Annealing experiment of sgRNA primer

[0039] First, preheat the metal bath at 95℃, then dissolve the above synthesized dry powder primer to 10uM with appropriate amount of ddH2O, take 10ul of forward primer and reverse primer respectively into the PCR tube, mix immediately, then place in the metal bath, incubate at 95℃ for 5min, then turn off the power, and wait for natural cooling to room temperature.

[0040] 1.3 Enzymatic digestion of p200 vector

[0041] In order to connect the annealed double-stranded sgRNA to the p200 vector, the p200 vector needs to be first digested; the enzyme digestion system and procedure are as follows: 10x Buffer 5ul, 50x Oligo 1ul, p200 2ug, AarI 5ul, dd H20 to make up the total system to 50ul. After the mixed system is centrifuged for a moment and mixed, it is incubated at 37℃ for 16h, and then 1.5% nucleic acid gel is used for electrophoresis detection and recovery of the digestion product.

[0042] 1.4, sgRNA is connected to p200 vector

[0043] A small amount of annealed sgRNA is taken for nucleic acid gel electrophoresis detection, the target band is bright and single, and after the concentration and purity are detected by the concentration spectrophotometer and meet the standards, the subsequent connection experiment can be carried out; the connection system and procedure are as follows: 10x T4 Buffer 1ul, T4 ligase 1ul, p200 1ul, sgRNA 7ul. The mixed system is centrifuged for a moment and mixed, and then incubated in a 16℃ metal bath overnight.

[0044] 1.5, transformation

[0045] Trans1-T1 competent cells purchased from Beijing Zison Biological Technology Co., Ltd. are taken out from the-80℃ refrigerator, placed on ice to melt, and another sterilized 1.5ml centrifuge tube is pre-cooled on ice, 10ul of the connection product is taken and added to 50ul of Trans1-T1 competent cells, and then mixed gently by blowing and sucking with a pipette gun, and then placed on ice for 30min; then placed in a preheated 42℃ metal bath for 90s, and then immediately placed in an ice bath for 2-3min; thereafter, 300ul of antibiotic-free LB liquid medium is added to the centrifuge tube in the clean bench, and placed in a 37℃ shaker for 1h; 100ul of the cultured bacterial liquid is evenly coated on the LB solid ampicillin culture medium plate (Ampicillin), and placed in a 37℃ incubator for overnight culture.

[0046] 1.6, picking bacteria and detection

[0047] In the clean bench, use the pipette tip to pick a single colony with moderate size and regular shape from the overnight culture plate into a 1.5 ml centrifuge tube containing 400 ul LB ampicillin liquid medium, and incubate at 37℃ for 4-6 h with shaking at 220 rpm. Then, take a small amount of bacterial solution for PCR detection. The detection system is as follows: 2x Taq Master Mix 7.5 ul, primer-F 0.25 ul, primer-R 0.25 ul, bacterial solution 1 ul, and ddH20 to make up the total system to 15 ul. The PCR program is as follows: Step 1: 95℃ pre-denaturation for 5 min, Step 2: 95℃ denaturation for 15 s; Step 3: 54℃ annealing for 15 s; Step 4: 72℃ extension for 90 s; Step 5: 72℃ extension for 5 min; Step 6: 12℃ storage. Step 2 to Step 4 are cycled for 30 cycles. After the reaction, use 1.0% agarose gel for nucleic acid electrophoresis detection. Select the strain with correct band size, take 100 ul for sequencing in Huada Gene, and store the remaining bacterial solution at 4℃ for standby.

[0048] 1.7, Extraction of plasmid

[0049] After the correct sequencing strain is inoculated into 10 ml LB ampicillin liquid medium and incubated overnight at 37℃ with shaking at 220 rpm, pour it into a 10 ml centrifuge tube, centrifuge at 12000 rpm at room temperature for 5 min, and discard the supernatant medium. Add 500 ul P1 solution in the super-pure plasmid extraction kit (QIAGEN Plasmid Midi Kit) to the precipitate, mix well by blowing with a pipette until there is no sterile spot, transfer all to a 2 ml centrifuge tube, then add 500 ul P2 solution, mix well by slowly inverting up and down, then add 700 ul N3 solution, mix well by slowly inverting up and down, then centrifuge at 13000 rpm at room temperature for 10 min. The centrifuged supernatant is absorbed into the DNA binding adsorption column in two times, centrifuge at 13000 rpm at room temperature for 1 min, and discard the effluent. Add 750 ul Washing Buffer PE containing 95% alcohol to the adsorption column, centrifuge at 12000 rpm at room temperature for 1 min, then discard the effluent, and repeat once. Finally, centrifuge the adsorption column at 13000 rpm for 2 min. Place the adsorption column on a 1.5 ml centrifuge tube, add 30 ul 65℃ preheated ddH20 to the center of the adsorption column, centrifuge at 12000 rpm at room temperature for 2 min to wash the DNA, and use a spectrophotometer to detect the plasmid concentration and plasmid. Store the qualified plasmid at -40℃.

[0050] Example 2: Obtaining of Bm6644 mutant silkworm edited by CRISPR-Cas9 editing technology

[0051] The sgRNA expression vector piggyBac [3xP3-EGFP, BmU6-KWMT BMO06644-SV40] plasmid (piggyBac [3xP3-EGFP, BmU6-KWMT BMO06644-SV40] backbone map see Figure 1 ) and the helper plasmid were mixed at a molar ratio of 1:1, and injected into non-diapausing silkworm eggs that were less than 2 h old using a microinjector, after which the injection needle hole was sealed with non-toxic glue; the injected silkworm eggs were incubated at 25°C, and after the larvae hatched, they were fed with mulberry leaves in a conventional manner. This batch of injected silkworm eggs was the G0 generation. After metamorphosis, the G1 generation was obtained by mating in the same circle; the G1 generation was fed conventionally until the 6th-7th day of egg laying, and green fluorescence was detected using excitation light under a fluorescence microscope, and transgenic positive individuals that specifically excited green fluorescence at the eyes were selected. This was the Bm6644-sgRNA positive G1 generation of transgenic silkworm individuals Figure 2 A1, the state of the silkworm egg); the positive G1 generation was fed conventionally until metamorphosis Figure 2 A2, after metamorphosis) and mated with the red light-emitting SpyCas9 adult to produce seeds, and the resulting eggs were the F1 generation. After the silkworm eggs hatched, the larvae were fed with mulberry leaves until the fifth instar, and larvae that emitted green and red fluorescence at the eyes were selected under a fluorescence microscope. This was the F1 generation of Bm6644 gene knockout positive individuals Figure 2 A3, A4). Further confirmation was carried out by fluorescence screening after metamorphosis.

[0052] Further, it was necessary to detect the mutation site of the Bm6644 silkworm knocked out by the CRISPR-Cas9 editing technology at the molecular level. Specifically, the silk gland tissue of the F1 generation of silkworms at the fifth day of the fifth instar was selected to extract its genome.

[0053] The extracted genome was used as the template for PCR amplification, and a pair of detection primers was designed with reference to the CDS sequence of Bm6644:

[0054] F: 5'-CTGAATGTCGATCGAAATTCCACC-3' (SEQ ID NO: 6)

[0055] R: 5'-CTCAATTGTTGAGTTAACCTCGCC-3' (SEQ ID NO: 7)

[0056] The silk gland tissue genome was amplified using the pair of primers, and the amplification product was recovered and then T-cloned and sent for sequencing. The sequencing results showed that the Bm6644 gene knockout positive individuals produced knock-in and deletion, two knockout forms Figure 3). It is worth noting that the detection primers in this embodiment must be specific, and no non-specific amplification phenomenon occurs, and the PCR product can be directly used for sequencing analysis.

[0057] Example 3 Phenotype observation of Bm6644 silkworm mutant knocked out by CRISPR-Cas9

[0058] The Bm6644 silkworm mutant knocked out obtained by screening was bred under the same conditions as the wild type silkworm, and it was found that the cocoon weight, pupa weight and cocoon layer rate of the Bm6644 silkworm mutant were greater than those of the wild type, and then the five-day-old silkworms were dissected, and it was found that the silk gland of the Bm6644 silkworm mutant was significantly larger than that of the wild type ( Figure 4 ). This shows that the deletion of Bm6644 expression in the silk gland of the silkworm caused by knocking out Bm6644 can increase the cocoon layer rate of the male silkworm.

[0059] In the embodiments of the present application, SpyCas9 is used to mediate the knockout of the target Bm6644 gene, and it is known to those skilled in the art that any means capable of knocking out the Bm6644 gene can achieve the purpose of the present application, such as ZFNs, TALENs and CRISPR / Cas9 or variants thereof to mediate the knockout of the target Bm6644 gene.

[0060] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection required by the present application is defined by the appended claims and their equivalents.

Claims

1. The application of KWMTBOMO06644 gene in improving cocoon silk yield of male silkworm, characterized in that, The nucleotide sequence of the KWMTBOMO06644 gene is shown in SEQ ID NO:1; The application method involves increasing silkworm cocoon production by knocking out the KWMTBOMO06644 gene.

2. The application of KWMTBOMO06644 protein in improving cocoon silk yield of male silkworm, characterized in that, The amino acid sequence of the KWMTBOMO06644 protein is shown in SEQ ID NO:2; The application method involves increasing silkworm cocoon production by inhibiting the expression of the KWMTBOMO06644 protein.

3. The use of knocking out the KWMTBOMO06644 gene with the nucleotide sequence shown in SEQ ID NO:1 or suppressing the expression of the KWMTBOMO06644 protein with the amino acid sequence shown in SEQ ID NO:2 in at least one of the following: (1) Increase the cocoon weight of male silkworms; (2) Increase the pupal weight of male silkworms; (3) Increase the cocoon layer rate of male silkworms; (4) Increase the size of the silk glands in male silkworms.

4. A method for improving cocoon yield of male silkworms, characterized by, The method involves 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.

5. The method of claim 4, wherein, The KWMTBOMO06644 gene, as shown in SEQ ID NO:1, was knocked out in silkworms using gene editing technology.

6. The method of claim 5, wherein, The KWMTBOMO06644 gene was knocked out using ZFNs, TALENs, and CRISPR / Cas9.

7. The method according to claim 6, characterized in that, Knocking out the KWMTBOMO06644 gene using CRISPR / Cas9 involves the following steps: 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. 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; Step 3: Hybridize and screen sgRNA transgenic silkworms and SpyCas9 transgenic silkworms to obtain silkworms with the KWMTBOMO06644 gene knocked out.

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