Technology for removing flavonoids from silkworm cocoon shells

Abstract

To provide a technique for whitening colored silkworm cocoons.SOLUTION: The present invention provides a silkworm variant that exhibits enhanced whiteness of a cocoon layer when compared to the parent strain through the functional modification of specific glycosidase-like genes and / or specific ABC transporter-like genes within the genes of the silkworm.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a technique for bleaching colored cocoons of, for example, silkworms. [Background technology]

[0002] Although white silk is commercially preferred in silk production, some silkworm strains accumulate mulberry-derived pigments in the silk glands, which remain strongly in the raw silk after reeling. Since the pigments accumulate in the sericin layer, if the sericin is completely removed, the silk will be white after degumming, but if the silk is not completely degummed, a thin layer of pigment will remain. Even when using silk proteins as biomaterials other than fibers, the accumulation of impurities such as pigments is undesirable. On the other hand, many of the strains that form colored cocoons have their roots in tropical regions, and many of them show excellent characteristics in terms of heat resistance and robustness. In recent years, there has been concern about a decline in cocoon yields due to global warming, so there is expected to be demand for modifying the cocoon color of such strains as a technology to create practical, superior strains.

[0003] The presence or absence of pigment accumulation in sericin is genetically determined (Non-Patent Document 1). Therefore, in the past, the trait of white cocoon was introduced into other lines by cross breeding using an already established white cocoon line.

[0004] However, there was a problem that regions of the genome of the white cocoon strain that are not related to the cocoon color were also introduced because of the crossing. In addition, although it is possible to genetically approximate the original strain by successive backcrossings, this requires many generations of crossing. Furthermore, in order to identify the introduced genomic regions in detail, it is very costly to perform whole genome sequencing, etc. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takashi Sakudo et al., "How are cocoons colored?", Seikagaku, January 2009, Vol. 81, No. 1, pp. 27-31 Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above-mentioned circumstances, an object of the present invention is to provide a technique for bleaching colored silkworm cocoons. [Means for solving the problem]

[0007] As a result of intensive research conducted to solve the above problems, it was discovered that colored silkworm cocoons can be bleached by modifying the function of specific glycosidase-like genes and / or specific ABC transporter-like genes among silkworm genes, and this led to the completion of the present invention.

[0008] That is, the present invention includes the following. [1] Compared to the parent strain, the activity of a protein having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and having glycosidase activity and / or the function of a protein having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 and having ABCC transporter function is reduced; A silkworm mutant that has a higher whiteness of the cocoon layer compared to the parent strain. [2] A method for producing a silkworm, comprising the steps of: subjecting a gene encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 3 and having glycosidase activity, and / or a gene encoding a protein having an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 6 and having an ABCC transporter function to a mutation treatment; As a result of the mutation treatment, the activity of the protein having glycosidase activity and / or the function of the protein having ABCC transporter function is reduced compared to a parent strain. A method for breeding a silkworm mutant that has a higher whiteness of the cocoon layer compared to the parent strain. Effect of the Invention

[0009] According to the present invention, colored cocoons of silkworms can be whitened by modifying specific gene functions without crossing with existing white cocoon strains. In addition, since only specific genes are modified, there is no change in genomic regions not related to cocoon color and the associated traits, as compared to conventional techniques. Furthermore, there is no need for successive backcrossings, and cocoon color can be modified with as few as two crosses. [Brief description of the drawings]

[0010] [Figure 1] As a result of the QTL analysis, three QTLs (chromosome 15: qtl15, chromosome 20: qtl20, and chromosome 27: qtl27) were found to be significant at the upper 5% level. [Figure 2-1] The following shows knockout mutants (KO1: 5-base deletion, KO2: 2-base deletion) in which a frameshift mutation was introduced into the 5th exon of BmLQGH1 (KWMTBOMO12227), as well as the CDS sequence (sequence number 1), amino acid sequence (sequence number 3), and genomic sequence (sequence number 2) of BmLQGH1 (KWMTBOMO12227). [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-2. [Figure 2-4] This is a continuation of Figure 2-3. [Figure 2-5] This is a continuation of Figure 2-4. [Figure 2-6] This is a continuation of Figure 2-5. [Figure 2-7] This is a continuation of Figure 2-6. [Figure 2-8] This is a continuation of Figure 2-7. [Figure 2-9] This is a continuation of Figure 2-8. [Figure 3-1]The target sequence of BmABCC4 (KWMTBOMO08988) (TALEN-L: 19 bases, TALEN-R: 17 bases) and the sequences of the corresponding regions of the established mutant lines (KO3: 4 bp deletion, KO4: 11 bp deletion), as well as the CDS sequence (sequence number 4), amino acid sequence (sequence number 6) and genomic sequence (sequence number 5) of BmABCC4 (KWMTBOMO08988) are shown. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 3-3] This is a continuation of Figure 3-2. [Diagram 3-4] This is a continuation of Figure 3-3. [Figure 3-5] This is a continuation of Figure 3-4. [Diagram 3-6] This is a continuation of Figure 3-5. [Diagram 3-7] This is a continuation of Figure 3-6. [Diagram 3-8] This is a continuation of Figure 3-7. [Diagram 3-9] This is a continuation of Figure 3-8. [Figure 3-10] This is a continuation of Figure 3-9. [Figure 3-11] Continuation of Figure 3-10. [Figure 3-12] This is a continuation of Figure 3-11. [Figure 3-13] This is a continuation of Figure 3-12. [Figure 3-14] This is a continuation of Figure 3-13. [Figure 3-15] This is a continuation of Figure 3-14. [Figure 3-16] Continuation of Figure 3-15. [Figure 3-17] This is a continuation of Figure 3-16. [Figure 3-18] This is a continuation of Figure 3-17. [Figure 4] The results of quantifying the color of the cocoons of each strain are shown below (A: lightness (L*), B: chromaticity (green-red, a*), C: chromaticity (blue-yellow, b*), D: whiteness (W = 100-sqr [(100-L)^2+(a^2+b^2)])). [Diagram 5]The results of the prediction of the transmembrane domain for the amino acid sequence of BmLQGH1 using SOSUI (Hirokawa et al., 1998) are shown below. A highly hydrophobic transmembrane domain was predicted at the N-terminus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below. The present invention relates to a silkworm mutant in which the whiteness of the cocoon layer is increased by reducing the amount of flavonoids in the cocoon layer compared to the parent strain through reducing the activity of a glycosidase-like protein called "LPH-like Quercetin Glycosides Hydrolase 1 (BmLQGH1)" and / or the function of a transporter-like protein called "ATP-binding cassette transporter sub-family C (ABCC)4 (BmABCC4)."

[0012] In the present invention, examples of silkworms that are the subject of the present invention include silkworm strains that have colored cocoons, such as Daizo (p50), Aohaku, Tenryu Aohaku, Hekiren, Mysore, Pure Mysore, Ringetsu, 209tei, and Kansanmin.

[0013] The silkworm mutant according to the present invention is a silkworm mutant obtained by subjecting the above-mentioned silkworm as a parent strain to a method for reducing the activity of the BmLQGH1 protein and / or the function of the BmABCC4 protein. In particular, reducing both the activity of the BmLQGH1 protein and the function of the BmABCC4 protein is preferable because it can significantly reduce the amount of flavonoids in the cocoon layer and significantly increase the whiteness of the cocoon layer, compared to reducing the activity or function of each protein alone.

[0014] In the present invention, the BmLQGH1 protein includes, for example, a protein consisting of an amino acid sequence having at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, and having glycosidase activity.

[0015] Examples of the BmLQGH1 gene include genes encoding the BmLQGH1 protein. Examples of the BmLQGH1 gene include genes that are composed of a base sequence having at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the coding region (CDS) shown in SEQ ID NO: 1 or the genomic DNA sequence shown in SEQ ID NO: 2, and that encode a protein having glycosidase activity.

[0016] In the present invention, "glycosidase activity" refers to the activity of hydrolyzing, for example, quercetin glycosides such as quercetin-3-rutinoside (rutin), quercetin 3-O-(6''-O-malonyl)-β-D-glucoside (Q3MG) and / or quercetin-3-β-glucoside (isoquercitrin).

[0017] On the other hand, in the present invention, examples of the BmABCC4 protein include a protein consisting of an amino acid sequence having at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6 and having ABCC transporter function.

[0018] Examples of the BmABCC4 gene include genes encoding the BmABCC4 protein. Examples of the BmABCC4 gene include genes that have a base sequence with at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the coding region (CDS) shown in SEQ ID NO: 4 or the genomic DNA sequence shown in SEQ ID NO: 5, and that encode a protein having an ABCC transporter function.

[0019] In the present invention, the "ABCC transporter function" refers to the function of transporting quercetin glycosides, such as quercetin-5-glucoside, from inside to outside of a cell.

[0020] In the following, the BmLQGH1 gene and the BmABCC4 gene may be collectively referred to as "the genes according to the present invention."

[0021] In the present invention, a silkworm mutant of the present invention can be obtained by subjecting a silkworm having the gene of the present invention described above to a method for reducing the activity or function of the protein encoded by the gene of the present invention.

[0022] Specifically, in the present invention, examples of methods for reducing the activity or function of a protein include the following method of subjecting the gene of the present invention to a mutation treatment (a method of introducing a mutation into the gene of the present invention as a target to destroy the gene).

[0023] As a method for introducing a mutation by targeting the gene of the present invention, a gene knockout method called ZFN, TALEN, or CRISPR / Cas, which is a genome editing technology, can be used to create a silkworm mutant lacking the gene.

[0024] Alternatively, the activity or function of a protein can be reduced by (i) a method of introducing a mutation or a marker gene into the promoter region of the gene of the present invention to suppress the transcription of the gene of the present invention and reduce the expression of the gene, or (ii) a method of suppressing the translation of the gene of the present invention by RNA interference or antisense technology to reduce the translation efficiency of the gene.

[0025] As described above, according to the breeding method of the present invention, which includes a step of subjecting the gene of the present invention to mutation treatment, by growing the silkworm mutant in which the gene of the present invention has been subjected to mutation treatment in a conventional manner, it is possible to obtain a silkworm mutant in which the amount of flavonoids in the cocoon layer is significantly reduced and the whiteness of the cocoon layer is significantly higher compared to the parent strain.

[0026] In the present invention, examples of flavonoids in the cocoon shell include quercetin triglucoside, quercetin diglucoside, quercetin-3,7-diglucoside, quercetin-5-glucoside, quercetin-5,4'-diglucoside, and quercetin-3-glucoside (isoquercitrin).

[0027] A method for growing silkworm mutants includes, for example, rearing them from eggs to adults at 25°C and feeding them with raw mulberry leaves or artificial feed (Silkmate, Nosan Corporation). For example, feeding is stopped on the 7th day of the 5th instar when metamorphosis into a pupa is approaching, and the silkworms are allowed to spin cocoons in plastic nets. Mating can be easily initiated, for example, by bringing a pair of newly emerged males and females close together, leaving them in a mated state for 2 to 3 hours, separating them, and placing the female on paper or the like to initiate egg-laying.

[0028] The amount of flavonoids in the cocoon layer can be quantified using a simple measurement method based on absorbance (Hirayama, Chikara and Okada, Eiji (2014) Simple analysis method for silkworm flavonoids using a spectrophotometer. Japanese Society of Silk Science 22, pp. 65-70).

[0029] On the other hand, the whiteness of the cocoon layer can be calculated by, for example, using a color difference meter to quantify the color of the cocoon based on the Lab color system, and calculating the whiteness (W = 100-sqr [(100-L)^2+(a^2+b^2)]) in terms of lightness (L*), chromaticity (green-red, a*) and chromaticity (blue-yellow, b*). EXAMPLES

[0030] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0031] 〔the purpose〕 Some strains accumulate pigments (flavonoids, mostly quercetin glycosides) in the cocoons, which remain in the raw silk after reeling. When using silk as a fiber or other biomaterial, the accumulation of impurity pigments is undesirable. In this example, the aim was to establish a new breeding technique for producing a strain in which flavonoids have been removed from the cocoon layer by modifying genes involved in flavonoid metabolism in silkworms using genome editing technology.

[0032] [Methods and Results] (1) Sample The strains used were "p50", "p50T" (same as p50, but previously passed repeatedly in a single pair at the University of Tokyo to reduce genetic diversity within the population), and "Nichi-01" stored at the National Agriculture and Food Research Organization. The silkworm strains used in the analysis were reared at 25°C under a 12-hour / 12-hour light / dark cycle using mulberry leaves or an artificial feed, Silkmate Original Silkworm Seed for 1st to 3rd Instars (Nosan Corporation).

[0033] (2)QTL analysis The loci involved in the amount of flavonoids in the cocoon shell were identified by QTL analysis using the F2 progeny of a cross between "p50," a line that produces green cocoons with high flavonoid content, and "Hino 01," a line that produces white cocoons with low flavonoid content. The amounts of flavonoids in the cocoon shell of p50 and Hiino 01 grown on Mulberry Leaves are shown in Table 1 below. The amount of flavonoids was quantified by a simple method based on absorbance (Hirayama and Okada, 2014).

[0034] [Table 1]

[0035] DNA was extracted from pupae of one individual from each parent line and 102 female individuals from the cross progeny using the DNeasy Blood & Tissue Kit (QIAGEN) and used as samples for double-digested restriction-site associated DNA sequencing (ddRAD-seq). Two restriction enzyme pairs, EcoRI-HF / MspI and PstI-HF / MspI (New England Biolabs), were used to construct the ddRAD-seq library. Library construction was performed as described in Uchibori-Asano et al. 2019. Sequencing was performed by paired-end sequencing with a read length of 101 bp using Hiseq X (Illumina).

[0036] The sequence reads were redistributed by individual using the script “process_radtags” in Stacks v1.48 (Catchen et al. 2011), and the reads were mapped to the silkworm reference genome published by Kawamoto et al. (2019) using BWA v0.7.17 (Li and Durbin 2009). Using the mapping data as input, comprehensive genotyping of each individual of the parents and the cross progeny was performed using the script “ref_map.pl” in Stacks v1.48 (Catchen et al. 2011). Of the extracted SNP markers, only those that were successfully genotyped in 80% or more of the cross progeny were used, and only the data of individuals that were successfully genotyped in 80% or more of those markers were used for subsequent analysis. The genetic distance between markers was estimated using Onemap v2.8.2 (Margarido et al. 2007) for combinations of markers that satisfied an LOD score of 3 or more. The constructed linkage map contained a total of 1038 markers and was organized into 28 linkage association groups, consistent with the number of chromosomes in the silkworm.

[0037] The amount of flavonoids in the cocoon shell was measured as phenotypic information for QTL analysis by a simple analysis method based on extraction of flavonoids in the cocoon shell with methanol and absorbance measurement (Hirayama and Okada, 2014). Using the genetic coordinates of 1038 markers and the genotypes and phenotypes of the cross progeny as inputs, QTL analysis was performed by Component Interval Mapping (CIM) using r / qtl (Broman et al., 2003). Random permutation tests were performed and three QTLs (chromosome 15: qtl15, chromosome 20: qtl20, and chromosome 27: qtl27) that were significant at the upper 5% level were detected (Figure 1). The effect size of each QTL and their interactions was estimated by analysis of variance using the function “fitqtl” in r / qtl. It was found that qtl15, qtl20, and qtl27 explained 7.04%, 24.57%, and 56.05% of the phenotypic variance, respectively. Interactions were also found between qtl15 and qtl27, and between qtl20 and qtl27, explaining 1.93% and 5.47% of the phenotypic variance, respectively.

[0038] (3) Mutation introduction using the CRISPR-Cas9 system and identification of the target gene on chromosome 20 Referring to the gene models provided by Kawamoto et al. (2019), we found a glycosidase cluster in qtl20 (gene model id: KWMTBOMO12222, KWMTBOMO12223, KWMTBOMO12224, KWMTBOMO12225, KWMTBOMO12227, KWMTBOMO12229, KWMTBOMO12230, KWMTBOMO12233, KWMTBOMO12236). Lactase / Phlorizin Hydrolase (LPH), one of the glycosidases in mammals, is expressed in the mammalian intestine and is known to hydrolyze quercetin glycosides (Day et al. 2000). The released quercetin aglycone is absorbed through the intestinal epithelium, which is an important process in quercetin metabolism in mammals (Nemeth et al., 2003). (Note: The glycosidase on qtl20 has a sequence homologous to LPH, but is not orthologous, and there are more than 20 LPH-like glycosidases in the silkworm genome, making it difficult to identify quercetin glycoside glycosidases by simple reverse genetic analysis.) We therefore selected these as candidate genes and attempted to perform functional analysis by mutagenesis using the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Proteins 9) (Jinek et al. 2012) system and microinjection.

[0039] The target sequence was designed using CHOPCHOP (Labun et al. 2019: https: / / chopchop.cbu.uib.no / ). Guide RNA was transcribed using MEGAshortscript, using PCR products amplified by a pair of primers with complementary sequences on the 3' side as templates, according to Kistler et al. 2015. TMThe T7 Transcription Kit (ThermoFisher Scientific) was used. The primers used for template preparation are shown in Sequence 1 (SEQ ID NO: 7) and Sequence 2 (SEQ ID NO: 8). Purification was performed using Guide-it TM The IVT RNA Clean-Up Kit (Takara Bio) was used. Alt-R (registered trademark) Sp HiFi Cas9 Nuclease V3 (Integrated DNA Technologies) was mixed with an equimolar ratio of guide RNA in distilled water to a concentration of 0.5 μg / μL, and then left at room temperature for 30 minutes to form a Cas9 protein guide RNA complex. This was introduced into p50T strain fertilized eggs 4-10 hours after laying by microinjection. The p50T strain was made non-diapause by changing the environmental conditions during the embryonic stage to 17°C and a 12-hour / 12-hour light / dark cycle. Microinjection was performed according to Tamura et al. (2000). After emergence, the injected larvae were mated with wild-type larvae to obtain G1 eggs. After pupation, the exuviae were ground in a lysis buffer containing 1M Tris-HCl (pH = 8.0), 5M NaCl, 0.5M EDTA, 10% SDS, and ultrapure water in a ratio of 4:1:1:13:1, and the supernatant was collected by centrifugation, and DNA was extracted by isopropanol precipitation and ethanol precipitation. Simple genotyping was performed by High Resolution Melting (HRM) analysis using primers designed to sandwich the target sequence and the KAPA HRM Fast PCR Kit (Kapa Biosystems), and samples that were different from the wild type but showed the same melting curve were crossed to obtain G2 eggs including individuals homozygous for the mutation. Sequence determination was performed by direct sequencing. The primers used for HRM analysis and direct sequencing are shown in Sequence 3 (SEQ ID NO: 9) and Sequence 4 (SEQ ID NO: 10).

[0040] Among the candidate genes for which mutations were introduced using the above method, the cocoon color was confirmed to be decolorized in knockout mutants (KO1: 5-base deletion, KO2: 2-base deletion, Figure 2) in which a frameshift mutation was introduced into the 5th exon of gene model id = KWMTBOMO12227. The primers used to prepare the PCR template for guide RNA synthesis are shown in Sequence 1 (SEQ ID NO: 7) and Sequence 2 (SEQ ID NO: 8). In the following, this glycosidase-like gene is provisionally named "LPH-like Quercetin Glycosides Hydrolase 1 (BmLQGH1)" based on the homology of its amino acid sequence with LPH and the hydrolysis activity of quercetin glycosides revealed by subsequent analysis.

[0041] (4) TALEN-mediated mutagenesis and identification of the target gene on chromosome 15 Although it was thought that the effect size of qtl15 was small and it was very difficult to identify the responsible gene, in a project unrelated to this study, a knockout strain of the ATP-binding cassette transporter sub-family C (ABCC) transporter-like gene (gene model ID: KWMTBOMO08988) located in qtl15 was produced, and the cocoon color was bleached, which led to the identification of the target gene by chance. Hereafter, this ABCC transporter-like gene is referred to as "BmABCC4" according to Wang et al. (2021).

[0042] Mutations were introduced using Transcription Activator-Like Effector Nuclease (TALEN) (Cermak T, et al. 2011). Construction of the TALEN expression plasmid, preparation of TALEN mRNA, and microinjection were performed by the same method as Takasu et al. (Takasu et al., 2013) (https: / / patents.google.com / patent / JP2015100326A / ja). Establishment of knockout mutant strains was performed by the same method as the above-mentioned CRISPR-Cas9-based mutagenesis. The primers used for HRM analysis are shown in Sequence 5 (SEQ ID NO: 11) and Sequence 6 (SEQ ID NO: 12). The primers used for direct sequencing are shown in Sequence 7 (SEQ ID NO: 13) and Sequence 8 (SEQ ID NO: 14). The target sequence (TALEN-L: 19 bases, TALEN-R: 17 bases) and the sequences of the corresponding regions of the established mutant strains (KO3: 4 bp deletion, KO4: 11 bp deletion) are shown in Figure 3.

[0043] (5) Generation of BmLQGH1 and BmABCC4 double knockout strains This double knockout line was established by crossing KO2 and KO4. Hereafter, this double knockout line is referred to as "KO5."

[0044] (6) Changes in cocoon color and flavonoid content due to mutations The color of the cocoons was quantified based on the Lab color system using a color difference meter CR-400 (Konica Minolta). The average of the values ​​at five different points on the cocoon surface was used as the measured value. Female individuals reared on mulberry leaves were used as samples. The results are shown in Figure 4 (A: lightness (L*), B: chromaticity (green-red, a*), C: chromaticity (blue-yellow, b*), D: whiteness (W = 100-sqr [(100-L)^2+(a^2+b^2)]). No difference was observed in lightness between the lines, but knockout of BmLQGH1 and BmABCC4 reduced green and yellow and increased whiteness. The effect of the double knockout was synergistic, with KO5 showing the highest whiteness.

[0045] Next, the amount of flavonoids in the cocoon shell was quantified using a simple method based on absorbance (Hirayama and Okada, 2014). The results for p50T and the knockout mutant KO1-5 are shown in Table 2 below.

[0046] [Table 2]

[0047] Consistent with the bleaching of cocoon color, the knockout mutants showed reduced levels of flavonoids in the cocoon shell. Estimation of the effect size of each qtl on phenotypic variation showed that the effect of qtl20 was greater than that of qtl15, whereas the effect of BmABCC4 knockout was greater than that of BmLQGH1 knockout in increasing cocoon whiteness and reducing flavonoid content.

[0048] (7) Quercetin glycoside degradation activity in the midgut BmLQGH1 may be involved in the intestinal absorption of quercetin by hydrolyzing quercetin glycosides, similar to LPH in mammals. We therefore measured the hydrolysis activity of three major quercetin glycosides in mulberry leaves, quercetin-3-rutinoside (rutin), quercetin-3-(6''-O-malonyl)-β-D-glucoside (Q3MG), and quercetin-3-β-glucoside (isoquercitrin) (Sugiyama et al., 2013) in midgut tissue. LPH has a hydrophobic transmembrane domain at the C-terminus, which localizes it to the brush border cell membrane (Panzer et al., 1998), and the amino acid sequence of BmLQGH1 predicted a highly hydrophobic transmembrane domain at the N-terminus using SOSUI (Hirokawa et al., 1998) (Figure 5).

[0049] Therefore, the localization of BmLQGH1 was also evaluated by investigating the hydrolysis activity of quercetin glycosides in suspensions and cell membrane fractions obtained by grinding silkworm midgut samples. The midguts of 5th instar larvae on the 5th day of instar were sampled and washed with PBS buffer, then homogenized in 4 volumes of 10 mM phosphate, 1 mM PMSF buffer (pH 7.5). The suspensions or cell membrane fractions obtained by repeating centrifugation and washing twice were used as samples. The reaction was carried out at 37°C for 4 hours in a 20 mM phosphate (pH 5.5) solution containing quercetin glycosides at a concentration of 2 mM. Quercetin, rutin, and isoquercitrin were purchased from Extrasynthese, and Q3MG was purchased from Sigma-Aldrich. The reaction was stopped by adding methanol, and the amount of quercetin produced was quantified by HPLC. HPLC was performed according to Hirayama et al. (2009). The results for the suspensions and cell membrane fractions are shown in Tables 3 and 4, respectively.

[0050] [Table 3]

[0051] [Table 4]

[0052] The suspension showed hydrolytic activity for rutin, Q3MG, and isoquercitrin. The knockout of BmLQGH1 reduced these hydrolytic activities, and in particular, the activity of rutin was almost completely lost. The hydrolytic activity of the cell membrane fraction was higher for rutin than the suspension, and was almost completely lost by the knockout. The activity of Q3MG and isoquercitrin was lower than that of the suspension. The decrease in activity of these two glycosides by the knockout was approximately 0.80-fold and 0.85-fold, respectively, in the suspension, whereas the decrease was greater at approximately 0.22-fold and 0.54-fold, respectively, in the cell membrane fraction.

[0053] [Consideration] Here, we identified genes involved in flavonoid accumulation in the cocoon layer in silkworms and developed a technology to suppress the accumulation of flavonoids in the cocoon layer by knocking out these genes. QTL analysis identified three loci involved in the amount of flavonoids in the cocoon layer (Figure 1). Knocking out the glycosidase-like gene BmLQGH1 located on chromosome 20 and the ABCC transporter-like gene BmABCC4 located on chromosome 15 by genome editing both reduced the amount of flavonoids in the cocoon layer and increased the whiteness of the cocoon, and double knockout of these genes produced a synergistic effect (Figure 4, Table 2).

[0054] Comparison of the hydrolytic activity of midgut tissues between the original strain and the knockout mutant revealed that BmLQGH1 exhibited hydrolytic activity against all of the major quercetin glycosides in mulberry leaves, rutin, Q3MG, and isoquercitrin (Tables 3 and 4). In particular, the hydrolytic activity of rutin was almost completely lost by the knockout, making it clear that BmLQGH1 is the only gene in silkworms that has hydrolytic activity against rutin. Furthermore, the cell membrane fraction showed higher rutin hydrolytic activity than the suspension, suggesting that BmLQGH1 is localized in the cell membrane. Similar to LPH in mammals, BmLQGH1 is localized on the cell surface of the midgut lumen and is thought to play a major role in mediating quercetin absorption through hydrolysis of quercetin glycosides. Although the hydrolytic activity of Q3MG and isoquercitrin was reduced by the knockout, it was not lost in either the suspension or the cell membrane fraction. The hydrolytic activity of these glycosides was higher in the suspension than in the control, suggesting that, in addition to BmLQGH1, there exist membrane-localized enzymes and cytoplasmic enzymes that degrade these glycosides.

[0055] Although the detailed function of BmABCC4 in flavonoid metabolism is unknown, in plants, ABC transporters are thought to be responsible for the transport of flavonoids from the cytoplasm to the vacuole and their extracellular excretion (Pucker and Selmar, 2022; Zhao and Dixon, 2010). In particular, the Arabidopsis ABCC2 transporter exhibits isoquercitrin transport activity in a yeast vesicle transport assay (Behrens et al., 2019). In addition, since BmABCC4 is highly expressed in the silkworm midgut (Wang et al., 2021), it is possible that it plays a role in excreting quercetin glycosides from midgut cells into the body fluid via vesicular transport or direct transport.

[0056] Surprisingly, in the QTL analysis using the cross progeny of the p50 line and the Hi-01 line, the effect sizes of qtl20 and qtl15 predicted by the former were larger (Fig. 1), but the knockout of BmABCC4 had a greater effect on phenotypic changes than the knockout of BmLQGH1 (Fig. 4, Table 2). This indicates that the frameshift mutation introduced by genome editing has a greater effect on gene function than the natural mutation conserved in the p50 line and that the loss of function by genome editing can achieve a greater effect on flavonoid removal in the cocoon shell than the introduction of the already conserved mutation in BmABCC4 by traditional cross breeding.

[0057] These results demonstrate that knocking out BmLQGH1 and BmABCC4 by genome editing can significantly reduce the amount of flavonoids that accumulate in the cocoon layer. Until now, the only way to change the cocoon color of a variety was through traditional cross breeding with a white cocoon strain, but this requires significant costs for trait research and backcrossing, as well as the introduction of genomic regions that are not related to cocoon color. This technology makes it possible to change cocoon color easily and without modifying unnecessary genomic regions.

[0058] [References] Behrens, C.E. et al. (2019) ‘Transport of Anthocyanins and other Flavonoids by the Arabidopsis ATP-Binding Cassette Transporter AtABCC2’, Scientific Reports, 9(1), p. 437. Broman, K.W. et al. (2003) ‘R / qtl: QTL mapping in experimental crosses’, Bioinformatics, 19(7), pp. 889-890. Catchen, J.M. et al. (2011) ‘Stacks: Building and Genotyping Loci De Novo From Short-Read Sequences’, G3 Genes|Genomes|Genetics, 1(3), pp. 171-182. Cermak, T. et al. (2011) ‘Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting’, Nucleic Acids Research, 39(12), p. e82. Day, A.J. et al. (2000) ‘Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase’, FEBS Letters, 468(2-3), pp. 166-170. Hirokawa, T., Boon-Chieng, S. and Mitaku, S. (1998) ‘SOSUI: classification and secondary structure prediction system for membrane proteins’, Bioinformatics, 14(4), pp. 378-379. Jinek, M. et al. (2012) ‘A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity’, Science, 337(6096), pp. 816-821. Kawamoto, M. et al. (2019) ‘High-quality genome assembly of the silkworm, Bombyx mori’, Insect Biochemistry and Molecular Biology, 107, pp. 53-62. Labun, K. et al. (2019) ‘CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing’, Nucleic Acids Research, 47(W1), pp. W171-W174. Li, H. and Durbin, R. (2009) ‘Fast and accurate short read alignment with Burrows-Wheeler transform’, Bioinformatics, 25(14), pp. 1754-1760. Margarido, G.R.A., Souza, A.P. and Garcia, A. a. F. (2007) ‘OneMap: software for genetic mapping in outcrossing species’, Hereditas, 144(3), pp. 78-79. Nemeth, K. et al. (2003) ‘Deglycosylation by small intestinal epithelial cell β-glucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans’, European Journal of Nutrition, 42(1), pp. 29-42. Panzer, P. et al. (1998) ‘Protein Domains Implicated in Intracellular Transport and Sorting of Lactase-Phlorizin Hydrolase*’, Journal of Biological Chemistry, 273(22), pp. 13861-13869. Sugiyama, M. et al. (2013) ‘Varietal Differences in the Flavonol Content of Mulberry (Morus spp.) Leaves and Genetic Analysis of Quercetin 3-(6-Malonylglucoside) for Component Breeding’, Journal of Agricultural and Food Chemistry, 61(38), pp. 9140-9147. Takasu, Y. et al. (2013) ‘Efficient TALEN Construction for Bombyx mori Gene Targeting’, PLOS ONE, 8(9), p. e73458. Tamura, T. et al. (2000) 'Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector', Nature Biotechnology, 18(1), pp. 81-84. Wang, Y. et al. (2021) 'ATP-binding cassette transporter subfamily C members 2, 3 and cadherin protein are susceptibility-determining factors in Bombyx mori for multiple Bacillus thuringiensis Cry1 toxins', Insect Biochemistry and Molecular Biology, 139, p. 103649. Zhao, J. and Dixon, RA (2010) 'The “ins” and “outs” of flavonoid transport’, Trends in Plant Science, 15(2), pp. 72-80. Hirayama, R. (2009) Analysis of flavonoids in the cocoon shell of regional silkworm varieties by LC-MS. Sericulture and Insect Biotech, 78(1), pp. 57-63. Hirayama, R. and Okada, E. (2014) A simple method for analyzing flavonoids in silkworms using a spectrophotometer. Journal of the Japanese Society of Silk Science 22, pp. 65-70.

Claims

1. Compared to the parent strain, the activity of a protein having an amino acid sequence with at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and having glycosidase activity and / or the function of a protein having an amino acid sequence with at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 and having ABCC transporter function is reduced. A silkworm mutant that has a higher whiteness of the cocoon layer compared to the parent strain.

2. The method includes subjecting a gene encoding a protein having an amino acid sequence with at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and having glycosidase activity in a silkworm and / or a gene encoding a protein having an amino acid sequence with at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 and having an ABCC transporter function to a mutation treatment, As a result of the mutation treatment, the activity of the protein having glycosidase activity and / or the function of the protein having ABCC transporter function is reduced compared to a parent strain. A method for breeding a silkworm mutant that has a higher whiteness of the cocoon layer compared to the parent strain.

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