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Proc Natl Acad Sci U S A. May 22, 2007; 104(21): 8941–8946.
Published online May 11, 2007. doi:  10.1073/pnas.0702860104
PMCID: PMC1885607
From the Cover
Genetics

Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene

Abstract

Mechanisms for the uptake and transport of carotenoids, essential nutrients for humans, are not well understood in any animal system. The Y (Yellow blood) gene, a critical cocoon color determinant in the silkworm Bombyx mori, controls the uptake of carotenoids into the intestinal mucosa and the silk gland. Here we provide evidence that the Y gene corresponds to the intracellular carotenoid-binding protein (CBP) gene. In the Y recessive strain, the absence of an exon, likely due to an incorrect mRNA splicing caused by a transposon-associated genomic deletion, generates a nonfunctional CBP mRNA, resulting in colorless hemolymph and white cocoons. Enhancement of carotenoid uptake and coloration of the white cocoon was achieved by germ-line transformation with the CBP gene. This study demonstrates the existence of a genetically facilitated intracellular process beyond passive diffusion for carotenoid uptake in the animal phyla, and paves the way for modulating silk color and lipid content through genetic engineering.

Keywords: carotenoid transport, cocoon color, transgenic silkworm

Cocoon colors of the silkworm Bombyx mori vary, with naturally occurring shades of white, yellow, golden yellow, straw, salmon, pink, and green silk. The two main kinds of pigments responsible for cocoon color are ether-soluble yellowish carotenoids (1) and water-soluble green flavonoids (2, 3). These pigments are absorbed from dietary mulberry leaves, transferred from the midgut to the silk gland via the hemolymph, and accumulated in the silk fiber (4). Since the genetic research of Coutagne in 1903 (5), 15 genes (loci) that control cocoon color have been identified (6, 7) (also see www.shigen.nig.ac.jp/silkwormbase/index.jsp). To date, however, none of these genes has been cloned. Molecular elucidation of them will enhance our knowledge of the lipid/pigment transport system in the organism and pave the way for genetic manipulation of the color and lipid/pigment content of the silk, a practical natural fiber used in textile production for millennia and biomedical materials for centuries (8).

The Y (Yellow blood) gene (9) controls the uptake of carotenoids into the intestinal mucosa and the silk gland (10, 11). Larvae of mutants homozygous for the recessive +Y allele inadequately absorb dietary carotenoids, resulting in colorless hemolymph and white cocoons. At least six genes that control carotenoid-based color function only when they coexist with the dominant Y allele (6, 7), suggesting that the Y gene plays a central role in cocoon color pigmentation.

Carotenoid-binding protein (CBP) is an intracellular 33-kDa protein containing a lipid-binding domain named START (Fig. 1A) (12). Immunological analysis revealed that CBP expression is restricted to Y allele strains (12, 13), suggesting that the Y gene might control the expression of CBP.

Fig. 1.
Difference in the CBP gene structure between Y and +Y allele strains. (A) Schematic structure of the CBP cDNA sequence. CBP consists of exons 1–7. Arrows indicate the PCR-primers used in D. (B) A schematic structure of the CBP genomic sequence ...

While studying the BmStart1 gene, which produces an isoform of CBP that arises from alternative splicing, we found an insertion of a non-LTR retrotransposon, termed CATS, as well as a partial ORF deletion in the CBP gene of a colorless hemolymph strain (Fig. 1B, +Y) (14). This observation led us to postulate that the Y gene corresponds directly to the CBP gene.

In this article, we present evidence to support this hypothesis, and demonstrate the generation of a silkworm strain with colored silk by germ-line transformation of the CBP gene into a white cocoon strain.

Results

Genomic Structure of the CBP Gene in Y and +Y Allele Strains.

To address the link between the CBP gene and the Y gene, we characterized the CBP genomic sequence in four Y and +Y allele strains (for details, see Materials and Methods). The results are summarized in Fig. 1B. The Y allele strains had at least two copies of the CBP gene, all of which could be classified into two types: a Y-a sequence comprised of seven exons and six introns, and a Y-b sequence containing a CATS retrotransposon between exon 2 and exon 3 of the Y-a sequence. The homozygous +Y allele strains had a single copy: a +Y sequence containing a truncated CATS together with a 169-bp deletion of the 3′ terminus of exon 2 corresponding to the ORF of the CBP gene. Thus, the CBP gene of the +Y allele strains is considered a null allele, suggesting that the Y gene corresponds directly to the functional CBP gene. Deletion of the 4.5-kb region of the Y-b sequence containing the 3′ terminus of exon 2 and the 5′ terminus of CATS is likely to have produced the +Y sequence. The Y allele, rather than +Y, would therefore be the ancient form of B. mori.

Mechanism for the Loss of Expression of CBP Protein in the +Y Allele Strain.

To elucidate how these genomic alterations led to the absence of CBP immunoreactivity, we examined the expression and structure of CBP mRNA. We detected mRNA expression by Northern blot analysis in both the Y and +Y allele strains with exon 1–2 and 3–5 probes (Fig. 1C). The size of the major band was ≈3.8 kb and 3.5 kb for Y and +Y, respectively. RT-PCR analysis (Fig. 1D) and sequencing of the resulting PCR product revealed that the CBP cDNA from the +Y allele strain lacked exon 2, and that exon 1 was adjacent to exon 3. The length of exon 2 was 309 bp, consistent with the difference in size between the major mRNA detected in the Y and +Y allele strains by Northern blot analysis. We could not detect CBP mRNA in the +Y allele strain with an exon 2 probe (Fig. 1C). Based on these data, we propose that the absence of exon 2, likely due to incorrect mRNA splicing caused by the CATS-associated genomic alteration, generated a nonfunctional CBP mRNA lacking the true start methionine, resulting in an inability to produce CBP in the +Y allele strain (Fig. 1E).

Restoration of Carotenoid Uptake by Germ-Line Transformation of the CBP Gene.

To verify the function of CBP as the Y gene product, we examined the recovery of carotenoid uptake by transgenic expression of the CBP gene in the w1-pnd strain, a colorless hemolymph strain used for germ-line transformation (15). We used the binary GAL4/upstream activating sequence (UAS) system (16). We constructed an effector vector that carried the CBP gene linked to UAS and the EGFP reporter gene under the control of the artificial eye-specific 3xP3 promoter (Fig. 2A), and then generated effector (UAS-CBP-3xP3-EGFP) lines by germ-line transformation. After sib selection based on the presence of EGFP, G1 male moths of a UAS-CBP-3xP3-EGFP line were crossed with females of the Ser-1-GAL4-3xP3-DsRed line (Fig. 2B), which drives target gene expression in the middle silk gland (T.T., unpublished data). The progeny were screened for both EGFP (for UAS-CBP-3xP3-EGFP) and DsRed (for Ser-1-GAL4-3xP3-DsRed) fluorescence in the eyes. For the expression of CBP in the midgut where dietary carotenoids are absorbed, males of the resulting [Ser-1-GAL4-3xP3-DsRed, UAS-CBP-3xP3-EGFP] line, all of which produced colorless hemolymph and white cocoons, were then crossed with females of a BmA3-GAL4-3xP3-DsRed (193-2 or 52-2) line, which activates target gene expression ubiquitously, except in the silk gland where expression is weak (17), necessitating the introduction of the Ser-1-GAL4 driver. The progeny were screened for both EGFP (for UAS-CBP-3xP3-EGFP) and DsRed (for Ser-1-GAL4-3xP3-DsRed and/or BmA3-GAL4-3xP3-DsRed) fluorescence in the eyes (the details of the screening procedure are described in the legend of Table 1).

Fig. 2.
Recovery of carotenoid-rich yellow phenotype by transgenic expression of the CBP gene. (A) Organization of the pBacMCS[UAS-CBP-3xP3-EGFP] vector. ITR, inverted terminal repeats of piggyBac; term, SV40 terminator; 3xP3, eye-specific promoter. (B) The mating ...
Table 1.
Analysis of the transgenic lineages

We observed recovery of the yellow hemolymph phenotype in 52 of 114 larvae (Fig. 2C and Table 1). The concentration of lutein, a form of carotenoid found in the Y allele strain (10), was significantly higher in transgenic yellow larvae than in control colorless larvae (Fig. 2D and Table 1). We confirmed expression of CBP protein in the midgut of larvae that had yellow hemolymph by Western blot analysis (Fig. 2E). In the colored silk glands of the transgenic larvae, the pigmented region was restricted to the middle area (Fig. 2F) where CBP was expressed under the control of Ser-1-GAL4 (Fig. 2G).

Eighteen of 37 cocoons produced by recombinant larvae with yellow hemolymph exhibited weak yellowish color [Fig. 2H (lane 2) and Table 1]. Sib mating of the moths that emerged from the yellowish cocoons resulted in cocoons with increased color intensity (Fig. 2H, lane 3), which may reflect the increase of CBP expression by the homozygosity of transgenes such as UAS-CBP and Ser-1-GAL4.

Discussion

In this study, we investigated the relationship between the CBP gene and the Y gene. The CBP genomic sequence significantly differed between the Y and +Y allele strains (Fig. 1B). In the +Y allele strain, the splicing out of exon 2, which contains the true start methionine, resulted in the generation of a nonfunctional CBP mRNA (Fig. 1E). Transgenic expression of the CBP gene restored the carotenoid-based yellow phenotype in a colorless hemolymph strain (Fig. 2 and Table 1). In the Y allele strain, CBP protein is expressed in both midgut and middle silk gland (12, 13) where the Y gene has been shown to function (10). Transformation of a colorless strain with a GAL4 gene under the control of a middle silk gland promoter alone did not restore carotenoid uptake, as determined by larval and cocoon color; however, larvae carrying GAL4 transgenes expressed in both midgut under control of a whole-body promoter, and in the middle silk gland, produced yellow hemolymph and silk. All of these observations strongly support the conclusion that the Y gene corresponds directly to the CBP gene. Precise mapping at single-gene resolution with improvement of the B. mori genomic draft sequence (18, 19) will offer further support and elucidate whether the Y gene corresponds to Y-a, Y-b, or both.

The CBP gene was duplicated in the Y allele strains used in this study. Gene duplications associated with morphological characters may not be rare in the silkworm, e.g., two copies of the xanthine dehydrogenase (XDH) gene, which catalyses the synthesis of uric acid which is accumulated in larval epidermis to make the larval skin opaque (20), is a prime example. Our genomic analysis also demonstrate that the Y-b sequence is the likely progenitor of the +Y sequence, suggesting the Y allele strain is the ancient form of B. mori and the deletion of the Y-a sequence has occurred during the transition from the Y allele to the +Y allele. However, to draw the conclusion about the original form of the CBP gene of B. mori, analysis of the CBP gene in Bombyx mandarina, the putative wild ancestor of B. mori, and the oldest Chinese domesticated strains will be needed.

Carotenoids play important and various roles in diverse organisms, as pro-vitamin A, antioxidants, and colorants (21, 22). Their beneficial effects have also been reported in the prevention of coronary heart disease, certain kinds of cancer, and age-related macular degeneration in humans (23). Animals cannot synthesize carotenoids, which must be obtained from the diet and subsequently transported to proper tissues by plasma lipoproteins. Mechanisms for uptake and transport of carotenoids, however, are not well understood in any animal system. Although several earlier investigators have reported that the mucosa of the vertebrate intestine absorbs carotenoids by passive diffusion (24, 25), a recent study employing intestinal CaCo-2 cells (26) and differential carotenoid concentrations between tissues (27) suggested the existence of facilitated absorption mechanisms and the participation of a carotenoid binding protein(s) (for review, see refs. 28 and 29). Furthermore, a membrane receptor, NinaD, has been identified as a mediator of carotenoid uptake in Drosophila melanogaster (30). To our knowledge, CBP is the first functionally identified intracellular molecule involved in carotenoid uptake. This study, along with previous production of a hypomorphic phenotype of cocoon decoloration by putative RNA interference of the CBP/BmStart1 gene (31), demonstrates the existence of a genetically facilitated intracellular process for carotenoid uptake beyond passive diffusion. CBP may serve as a model for related physiological processes in other animals. It is noteworthy that CBP facilitated carotenoid uptake from both the diet in the intestinal mucosa (Fig. 2C) and the hemolymph lipoprotein in the silk gland (Fig. 2F).

The START domain-containing gene family is conserved in plants and animals and is thought to serve as a versatile binding interface for lipids and to function in intracellular lipid transport (3234). To date, mammalian StAR (35) and CERT (36) have been shown to be necessary for lipid transport. This study indicates that START domain-containing genes are indeed essential in organisms from insects to mammals for the transport of diverse lipids, ranging from cholesterol by StAR to ceramide by CERT to carotenoids by CBP.

To our knowledge, although the color intensity of the cocoons was still weak, this study represents the first example of coloration in a practical natural fiber by transport of a natural pigment based on molecular genetic engineering. The generation and use of GAL4 lines, which drive target genes with various temporality and intensity, should result in the ability to modulate yellowish color intensity and shade. We expect that the identification of other cocoon color determinant genes, such as the F gene (37), which creates a flesh color when the dominant F allele coexists with the Y allele, and the Pk gene (1), which causes a reddish color when the dominant Pk allele coexists with both the Y and F alleles, may lead to the ability to produce silks of various colors with a similar transgenic strategy. Expression of recombinant fluorescent proteins in the silk gland could also be a way to modify silk coloration through transgenics (38, 39).

The economic significance and ease of rearing have made silkworms the subject of genetic studies. More than 400 mutations, including 15 mutations in cocoon color, have been identified, and >1,000 strains are maintained as genetic resources (7, 40, 41). However, few of these mutants have been characterized by molecular cloning and restored by transgenic transformation. We are hopeful that this study will lead to further molecular genetic studies and silk creation with novel characteristics using available silkworm genetic resources and the transgenic technology.

Materials and Methods

Silkworm Strains.

Larvae were reared on an artificial diet containing mulberry leaves (Nihon Nosanko, Yokohama, Japan) under standard conditions (15). The CBP gene was analyzed in four silkworm strains: N4 (Y/Y), FL50 (Y/+Y or +Y/+Y), KINSYU × SHOWA (KxS; colorless hemolymph, presumably +Y/+Y), and w1-pnd (colorless hemolymph, presumably +Y/+Y). Because FL50 (Y/+Y) and FL50 (+Y/+Y) individuals are derived from the same mating, their genetic backgrounds are similar.

Genomic Characterization.

The CBP gene of the KxS strain and the Y-a sequence of the N4 strain were determined during the study of the BmStart1 gene (14). Southern blot analysis indicated that there was a single copy of the BmStart1/CBP gene in the KxS genome (14).

To clarify the difference between the CBP genomic sequence of the KxS and N4 strains, we sought to confirm the absence of the CATS insertion in the CBP locus of the N4 strain. We performed genomic PCR with two pairs of primers, both of which included one primer designed from the CATS sequence [Primer-40 (5′-TGCAGATGTCAGCAGTCAAACCATCCGC-3′) or Primer-33 (5′-GGTAGACTCCACACTCACACAG-3′)] and the other from exon 1 [Primer-43 (5′-GATCCCAAAAGCGATGTGTAGCTCCGTG-3′)] or exon 3 [Primer-18 (5′-GCCTTCAACTTTCCTTGACTCCACGACG-3′)]. The locations of the primers used are indicated by arrows in SI Fig. 3A. Unexpectedly, we observed amplification of the N4 genomic DNA with both primer pairs (SI Fig. 3B). The amplified DNA of the N4 strain with Primer-33 and Primer-18 was identical in size to that of the KxS strain. In contrast, the PCR product of the N4 strain amplified with Primer-43 and Primer-40 was significantly longer than that of the KxS strain. We then sequenced the N4 genomic DNA amplified with Primer-43 and Primer-40. We obtained a novel sequence containing the 5′ region of CATS including the ORF1 and the endonuclease domains, common structures on non-LTR type retrotransposons (SI Fig. 3A, Y-b) (42). A comparison of the CATS insertion site in these genomic sequences revealed a target site duplication (SI Fig. 3C), a common characteristic of a retrotransposon insertion (42). We therefore hypothesized the existence of two types of CBP genomic sequence in the N4 strain, a Y-a sequence lacking CATS and a Y-b sequence containing a full-length CATS insertion, which was supported by Southern blot analysis with a DIG-labeled DNA probe synthesized by PCR with Primer-1 (5′-ATGGCCGACTCTACGTCGAAAAGCG-3′) and Primer-8 (5′-CTCCTTGGTCGTGGTGTCCATATTG-3′) for exon 2 (SI Fig. 3 D–F). The intensity of the band for Y-b was significantly stronger than that for Y-a. We now speculate that the difference in the intensity of the band between for Y-a and Y-b was due to variation in their copy number.

To further address the link between the CBP gene and the Y gene, we next analyzed the FL50 (Y/+Y), FL50 (+Y/+Y), and w1-pnd strains. For the FL50 (Y/+Y) strain, the Y-a sequence was amplified by PCR with Primer-1 and Primer-202 (5′-TCTCGTTAGCCTGACTCTTGTACTC-3′) for exon 3, and the Y-b sequence was amplified with Primer-1 and Primer-204 (5′-GGATTGTGCAACGAGGGTCGACACAG-3′) for the CATS. For the FL50 (+Y/+Y) strain, the +Y sequence was amplified with Primer-203 (5′-AGCCCATACACATCTACGACGTAAATGACC-3′) for the sequence between exon 1 and exon 2 and Primer-206 (5′-GCGCTTACAAGCATATATTGCTATCTATCC-3′) for the sequence between the CATS and exon 3. The determined nucleotide sequences were supported by Southern blot analysis (SI Fig. 3 D and F). PCR analysis was used for genotyping the four strains, including the w1-pnd strain (SI Fig. 3G), using Primer-1 and Primer-18, Primer-43 and Primer-40, Primer-33 and Primer-2 (5′-CTAACCATCTCCTTGAGGGGCGGATACC-3′) for exon 5, and Primer-1 and Primer-112 (5′-AGCCTCTGTGACTACTTCTG-3′) for the CATS.

Northern Blotting.

mRNA was extracted from the midgut of fifth-instar day-3 larvae. As a hybridization probe, the CBP cDNA cloned into pGEM T-vector (Promega) was labeled with [α-32P]dCTP. The T-vector sequences for CBP exons 1–2, 3–5, and 2 contain nucleotides 4–370, 703–1018, and 254–371 of the CBP cDNA sequence, respectively. The exon 2 probe corresponds to the portion of exon 2 that is not truncated in the +Y sequence.

RT-PCR.

cDNA was synthesized from mRNA [poly(A)+] prepared from the midgut of fifth-instar day-3 larvae. Primers for exon 1 (5′-CAAGGCTAACAACTCTGGTTGG-3′) and exon 4 (5′-TGAACTCCTCGTATAGAAACCTGGC-3′) were used.

Construction of the pBacMCS[UAS-CBP-3xP3-EGFP] Vector.

The pBacMCS vector is a plasmid for germ-line transformation which contains multiple cloning sites between truncated ITRs (inverted terminal repeats) of piggyBac. First, the 3xP3-EGFP fragment was amplified from the pBac[3xP3-EGFPafm] vector (43) with the primers 5′-GGGGAATTCGCTTCGGTTTATATGAGAC-3′ and 5′-GGGGAATTCTGAGTTTGGACAAACCACAAC-3′. The PCR product was digested with EcoRI and inserted into the EcoRI site of the pBacMCS vector to generate pBacMCS[3xP3-EGFP]. Next, the UAS-EGFP-SV40 terminator fragment was amplified from the pBac[UAS-GFP] vector (16) with the primers 5′-ACGAACTAGTGCCGAGTCTCTGCACTGAAC-3′ and 5′-AGACGGACTAGTGCCCTTTGACGTTGGAGT-3′. The PCR product was digested with SpeI and inserted into the SpeI site of the pBluescriptII SK(−) vector. The EGFP fragment was removed from the UAS-EGFP-SV40 terminator fragment by inverse PCR with the primers 5′-TAGACCTAGGTCAGCCATACCACATTTGTA-3′ and 5′-CTGCACCTAGGCCAATTCCCTATTCAGAGT-3′. The inverse-PCR product was digested with BlnI and self-ligated to generate the pBlue[UAS] vector. The pBlue[UAS] vector was then digested with SpeI and inserted into the NheI site of the pBacMCS[3xP3-EGFP] vector to generate the pBacMCS[UAS-3xP3-EGFP] vector. Finally, to introduce the CBP gene into pBacMCS[UAS-3xP3-EGFP], the CBP cDNA sequence was amplified from the cDNA synthesized from the silk gland of the N4 strain with the primers 5′-ATGCTCTAGAGAAACCCTAAGCTCTTGAAAGTG-3′ and 5′-ATGCTCTAGAGGCCGATGGGTGAACATTGG-3′. The PCR product was digested with XbaI and inserted into the BlnI site of pBacMCS[UAS-3xP3-EGFP] located between the UAS and the SV40 sequence, giving rise to pBacMCS[UAS-CBP-3xP3-EGFP]. The nucleotide sequence of the resulting vector was confirmed by DNA sequencing.

Transgenesis and Screening of Silkworms.

The pBacMCS[UAS-CBP-3xP3-EGFP] vector was dissolved in 5 mM KCl and 0.5 mM phosphate buffer (pH 7.0) at a concentration of 0.2 mg/ml, and mixed with the helper plasmid pHA3PIG (15) dissolved in the same buffer and at the same concentration as the vector. About 15–20 nl of this mixture was injected individually into preblastoderm embryos of the w1-pnd strain at 2–8 h after oviposition as described in ref. 15. After the injection, the embryos were allowed to develop at 25°C. Screening of the silkworms by marker fluorescence was performed under a fluorescence stereomicroscope (MZ16FA; Leica) equipped with appropriate filter sets for the detection of EGFP and DsRed fluorescence. The yellow hemolymph phenotype in the wandering stage was obvious, permitting yellow hemolymph larvae to be distinguished from colorless hemolymph larvae in the rearing container (Fig. 2C). The CBP gene thus represents a useful transgenic marker for silkworms that does not require sophisticated equipment. Photographs and analyzed data of [193-2 × (Ser-1-GAL4-3xP3-DsRed, UAS-CBP-3xP3-EGFP), eye DsRed, body EGFP] are presented in Fig. 2 except for lanes 3 and 4 of Fig. 2F{[52-2 × (Ser-1-GAL4-3xP3-DsRed, UAS-CBP-3xP3-EGFP), eye DsRed, body EGFP]} and Fig. 2H{[52-2 × (Ser-1-GAL4-3xP3-DsRed, UAS-CBP-3xP3-EGFP), eye DsRed, eye EGFP]}.

Carotenoid Composition of the Hemolymph Analyzed by Reverse-Phase HPLC.

Each sample of hemolymph was collected from two or three individuals. Pigments in 20 μl of hemolymph were extracted three times with 80 μl of acetone and once with 80 μl of ether. Extracts were pooled, washed with water, evaporated to dryness, dissolved in methanol, filtered, and used for carotenoid analyses by HPLC. A reverse-phase column [PEGASIL-300 ODS-II (2 × 150 mm); Senshu Kagaku, Tokyo, Japan] was used under the following conditions: solvent, 100% methanol; flow rate, 0.17 ml/min. The carotenoid form was also confirmed as lutein by the absorbance spectrum of a peak fraction. Carotenoid standards were purchased from Sigma.

Detection of CBP Protein Expression by Immunoblotting.

Western blotting was carried out as described in ref. 14.

Supplementary Material

Supporting Figures:

Acknowledgments

We thank S. Takaichi, W. Hara, and K. Touhara for advice; T. Kanda and S. Kobayashi for technical assistance; and M. A. Wells (who died on May 23, 2006), M. R. Kanost, R. O. Ryan, J. J. Hull, and one anonymous reviewer for critical reading of the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research and the Research for the Future Program of Japan Society for the Promotion of Science; the National Bioresource Project (Silkworm) of the Ministry of Education, Culture, Sports, Science, and Technology (Japan); and the Teimei Empress Memorial Foundation (Japan).

Abbreviations

StAR
steroidogenic acute regulatory protein
START
StAR-related lipid transfer
UAS
upstream activating sequence
KxS
KINSYU × SHOWA.

Footnotes

The authors declare no conflict of interest.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession nos. AB263197AB263200).

This article contains supporting information online at www.pnas.org/cgi/content/full/0702860104/DC1.

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