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Expression of GFP and RFP in different tissues. A: Em- bryo injected with pMLC-EGFP/pCK-RFP and viewed under a blue excitation light for GFP observation. B: The same embryo viewed under a yellow excitation light for RFP observation.
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Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 po...
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... into the same chromosome locus. More likely, the 2 constructs had been concatenated and integrated. Un- like the expression in transient transgenic embryos, where weak GFP expression was also detected in muscle ( Figure 2, C), no GFP expression was detected in muscles from stable transgenic embryos and fry. Using the gfp reporter gene under zebrafish fish gene promoters, faithful expression of the transgene has been re- peatedly demonstrated (Higashijimas et al., 1997, 2000; Long et al., 1997, 2000; Meng et al., 1997, 1999; Ju et al., 1999; Muller et al., 1999). In the present study, using 2 different fluorescent reporter genes, we have further demonstrated the faithful expression of the 2 transgenes simul- taneously in the same fish and thus proved the feasibility of generating 2-color transgenic animals. Multicolor transgenic animals should be useful in examination and tracing of the development of 2 or more tissues and organs simul- taneously in the same animal, thus providing a valuable tool for a better and closer comparison. In particular, the ...
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... is valuable for 2 adjacent tissues or cell types. For example, expression of the 2 fluorescent proteins can be targeted to endocrine and exocrine cells of pancreas under 2 suitable promoters, and thus a useful transgenic model could be created for detailed study of pancreas development and the interaction of different types of cells in the pancreas. When the 2 fluorescent proteins were expressed in the same tissues, GFP generally appeared several hours earlier than RFP. This was not likely to be due to the timing of transcription since the same promoter was used. It seems that GFP is more sensitive for detection than RFP and re- quires a lower concentration for visualization. The gfp DNA used in the present study was a mutant form, encoding a GFP variant containing a critical amino acid substitution in the chromophore region, and the codons have also been modified based on preferred human codon usage (Cormack et al., 1996). The resultant GFP is called enhanced GFP, or EGFP, which is 35-fold stronger in fluorescence intensity than the wild-type GFP in human cells and presumably in other vertebrate cells as well (Yang et al., 1996). At later stages, however, it seemed that the fluorescence intensity of RFP in both muscle and skin cells was stronger than that of GFP. It is thus likely that RFP is more stable than GFP and that a higher steady level of RFP can be reached within the cells. Under our conditions, GFP could only be detected under its excitation light (blue) but not under the excitation light for RFP (yellow); while RFP, when it was expressed at a high level, could be detected as orange under the blue light and frequently interfered with the detection of GFP. However, strong expression of GFP seemed to have no effect on detection of RFP. Despite the potential interference of strong RFP with GFP detection, the simultaneous visualization of both GFP and RFP under the same excitation light could be beneficial under certain cir- cumstances as no switch of light or double exposure is required for real-time observation. The reporter genes are commonly used to determine temporal and spatial patterns of gene expression. For temporal expression, based on our unpublished data in stable gfp transgenic zebrafish lines, detection of GFP fluorescence is generally 2 to 3 hours later than detection of gfp messen- ger RNA in developing embryos because of the delay of the translation event and accumulation of sufficient GFP for detection. According to the current study, the appearance of RFP fluorescence is delayed by about 10 hours compared with that of GFP fluorescence, and thus it is likely that the detection of RFP is 12 to 13 hours later than the detection of its mRNA. Therefore, gfp reporter gene should be a better choice than rfp if the timing of gene activation is critical in the analysis. Although stable transgenic animals have been made for many species, the fate of exogenously introduced DNA and how it is integrated into the host chromosomes remain unclear. It has been reported that the foreign DNA, once injected into fertilized eggs, forms DNA concatemers and undergoes a rapid amplification and subsequent degrada- tion (Flytzanis et al., 1985; Stuart et al., 1988; Chong and Vielkind, 1989). Because of this, as previously demonstrated, analysis of DNA cis -elements can be achieved by coinjection of a promoter-reporter gene construct and a testing DNA cis -element. Conceivably, the cis -element will be ligated to the promoter in vivo, thus eliminating the laborious process of constructing a proper test DNA plasmid in vitro. This approach has proved useful in zebrafish (Muller et al., 1997, 1999). In our present study, as shown in Figure 2, C, indeed muscle expression of GFP was observed due to the presumable concatemerization of a muscle element from a heterologous DNA construct. However, the expression of GFP in this way was relatively weak, and probably only effective in early embryos in which a large amount of amplified exogenous DNA remains available. Consistent with this, the muscle expression of GFP in embryos injected with pCK-EGFP/pMLC-RFP became weak and even undetectable at late embryonic stages when most of the exogenously DNA was presumably degraded. Furthermore, we never observed muscle expression of RFP in embryos injected with pCK-RFP/pMLC-EGFP because more accumulated RFP is required for fluorescence visualization. The “ ectopic ” expression of GFP and RFP was never observed in skin cells, probably because the skin-specific CK promoter is not as strong as the muscle-specific MLC promoter. The lack of muscle expression was further confirmed in stable transgenic F 1 embryos in which only one or a few copies of transgenes are integrated. Thus, our observation indicated that a reliable analysis of DNA cis -element remains the approach to introduce in vitro ligated DNA constructs. In summary, in the present study, we have generated 2-color transgenic zebrafish using gfp and rfp reporter genes, and both genes were correctly expressed in the targeted tissues according to the specificity of the promoters used. The 2-color transgenic model should facilitate com- parative studies of development of multiple tissues or organs and differentiation of different cell types in the same ...
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... 2 promoters used in the present study have been previously characterized in transient transgenic zebrafish embryos (Ju et al., 1999) or by direct injection of naked DNA into adult muscles (Xu et al., 1999). When pCK-EGFP was injected into zebrafish embryos, GFP was specifically expressed in the surface skin cells in early embryos ( Figure 1, A and B), consistent with our earlier observation (Ju et al., 1999). In embryos injected with pMLC-EGFP, GFP expression was specifically detected in trunk skeletal muscles (Figure 1, C and D). These expression patterns have been confirmed in stable transgenic zebrafish lines by expression of GFP under the 2 promoters (our unpublished observations). Therefore, the 2 promoters used in the present study were highly tissue-specific. To determine whether the 2 fluorescent proteins driven by different tissue-specific promoters can be expressed correctly in targeted tissues, pMLC-EGFP/pCK-RFP and pCK- EGFP/pMLC-RFP were respectively coinjected into zebrafish embryos. As shown in Figure 2, both fluorescent proteins were presented faithfully in the expressing tissues according to the respective promoters. In embryos injected with pMLC-EGFP/pCK-RFP, GFP and RFP were correctly expressed in muscle and skin cells, respectively (Figure 2, A and B). RFP was first detected in skin cells at 16 hpf, while GFP was first expressed in muscle fibers of somites at 22 hpf. It is interesting to note that strong expression of RFP can be detected as orange color under the excitation light for GFP (Figure 2, A); however, no GFP fluorescence can be observed under the excitation light for RFP (B). In embryos injected with pCK-EGFP/pMLC-RFP (Figure 2, C – F), GFP was first detected at the superficial layer of injected embryos at 6 hpf (shield stage), and the expression continued in the outmost layer of skin cells during embryogenesis. This is in contrast to the earliest detection of RFP expression at 16 hpf in embryos injected with pMLC-EGFP/pCK-RFP. In muscle cells, RFP was first observed at 30 hpf, also later than the earliest detection of GFP (22 hpf) under the same MLC promoter. Thus, GFP is detected earlier than RFP in the same tissue under the same promoter. Unexpectedly in the embryos injected with pCK-EGFP/ pMLC-RFP, GFP was also observed in muscle fibers at the same stage as RFP detection. It is apparent that all GFP- expressing muscle fibers also express RFP (Figure 2, C). This phenomenon is likely due to the formation of a het- eroconcatemer of the 2 injected plasmid DNAs, and thus the muscle-specific elements from pMLC-RFP affected the expression of gfp , as previously reported by Muller et al. (1997). During embryogenesis, RFP accumulation increased steadily in muscle fibers. In comparison, GFP fluorescence in muscle fibers became relatively weak and seemed to dis- appear after 72 hpf (Figure 2, E). Despite the observation of “ ectopic ” expression of GFP in muscles of the embryos injected with pCK-EGFP/pMLC- RFP, we never observed the expression of RFP in the muscles of the embryos injected with the reciprocal combination, pCK-RFP/pMLC-EGFP. Neither was “ ectopic ” expression of GFP or RFP observed in skin cells from embryos injected with either combination, pCK-EGFP/pMLC- RFP or pCK-RFP/pMLC-EGFP. To investigate whether there is any interference between the 2 fluorescent proteins if they are expressed in identical cells, coinjection of pCK-EGFP/pCK-RFP or pMLC-EGFP/ pMLC-RFP was carried out. In both cases, GFP and RFP were correctly expressed in the same tissues and presented in identical sets of cells (Figure 3). However, as observed from injection experiments with 2 heterogenous promoter constructs, GFP generally appears about 10 hours earlier than RFP. The first detection of GFP in skin and muscle was around 5 hpf and 22 hpf, respectively; in comparison, the appearance of RFP in the 2 tissues was 16 hpf and 30 hpf, respectively. The timing of GFP and RFP appearance in embryos injected with different combinations of DNA constructs is summarized in Figure 4. Although RFP was detected later than GFP, the intensity of its fluorescence increased steadily during development. By 72 hpf, only RFP fluorescence was detected in skin cells in embryos injected with pCK-EGFP/pCKRFP (Figure 3, C and D). Similarly, under the MLC promoter, RFP fluorescence became predominant after 5 dpf and GFP fluorescence was greatly overshadowed, even under the optimal excitation light for GFP (Figure 3, G and ...
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... 2 promoters used in the present study have been previously characterized in transient transgenic zebrafish embryos (Ju et al., 1999) or by direct injection of naked DNA into adult muscles (Xu et al., 1999). When pCK-EGFP was injected into zebrafish embryos, GFP was specifically expressed in the surface skin cells in early embryos ( Figure 1, A and B), consistent with our earlier observation (Ju et al., 1999). In embryos injected with pMLC-EGFP, GFP expression was specifically detected in trunk skeletal muscles (Figure 1, C and D). These expression patterns have been confirmed in stable transgenic zebrafish lines by expression of GFP under the 2 promoters (our unpublished observations). Therefore, the 2 promoters used in the present study were highly tissue-specific. To determine whether the 2 fluorescent proteins driven by different tissue-specific promoters can be expressed correctly in targeted tissues, pMLC-EGFP/pCK-RFP and pCK- EGFP/pMLC-RFP were respectively coinjected into zebrafish embryos. As shown in Figure 2, both fluorescent proteins were presented faithfully in the expressing tissues according to the respective promoters. In embryos injected with pMLC-EGFP/pCK-RFP, GFP and RFP were correctly expressed in muscle and skin cells, respectively (Figure 2, A and B). RFP was first detected in skin cells at 16 hpf, while GFP was first expressed in muscle fibers of somites at 22 hpf. It is interesting to note that strong expression of RFP can be detected as orange color under the excitation light for GFP (Figure 2, A); however, no GFP fluorescence can be observed under the excitation light for RFP (B). In embryos injected with pCK-EGFP/pMLC-RFP (Figure 2, C – F), GFP was first detected at the superficial layer of injected embryos at 6 hpf (shield stage), and the expression continued in the outmost layer of skin cells during embryogenesis. This is in contrast to the earliest detection of RFP expression at 16 hpf in embryos injected with pMLC-EGFP/pCK-RFP. In muscle cells, RFP was first observed at 30 hpf, also later than the earliest detection of GFP (22 hpf) under the same MLC promoter. Thus, GFP is detected earlier than RFP in the same tissue under the same promoter. Unexpectedly in the embryos injected with pCK-EGFP/ pMLC-RFP, GFP was also observed in muscle fibers at the same stage as RFP detection. It is apparent that all GFP- expressing muscle fibers also express RFP (Figure 2, C). This phenomenon is likely due to the formation of a het- eroconcatemer of the 2 injected plasmid DNAs, and thus the muscle-specific elements from pMLC-RFP affected the expression of gfp , as previously reported by Muller et al. (1997). During embryogenesis, RFP accumulation increased steadily in muscle fibers. In comparison, GFP fluorescence in muscle fibers became relatively weak and seemed to dis- appear after 72 hpf (Figure 2, E). Despite the observation of “ ectopic ” expression of GFP in muscles of the embryos injected with pCK-EGFP/pMLC- RFP, we never observed the expression of RFP in the muscles of the embryos injected with the reciprocal combination, pCK-RFP/pMLC-EGFP. Neither was “ ectopic ” expression of GFP or RFP observed in skin cells from embryos injected with either combination, pCK-EGFP/pMLC- RFP or pCK-RFP/pMLC-EGFP. To investigate whether there is any interference between the 2 fluorescent proteins if they are expressed in identical cells, coinjection of pCK-EGFP/pCK-RFP or pMLC-EGFP/ pMLC-RFP was carried out. In both cases, GFP and RFP were correctly expressed in the same tissues and presented in identical sets of cells (Figure 3). However, as observed from injection experiments with 2 heterogenous promoter constructs, GFP generally appears about 10 hours earlier than RFP. The first detection of GFP in skin and muscle was around 5 hpf and 22 hpf, respectively; in comparison, the appearance of RFP in the 2 tissues was 16 hpf and 30 hpf, respectively. The timing of GFP and RFP appearance in embryos injected with different combinations of DNA constructs is summarized in Figure 4. Although RFP was detected later than GFP, the intensity of its fluorescence increased steadily during development. By 72 hpf, only RFP fluorescence was detected in skin cells in embryos injected with pCK-EGFP/pCKRFP (Figure 3, C and D). Similarly, under the MLC promoter, RFP fluorescence became predominant after 5 dpf and GFP fluorescence was greatly overshadowed, even under the optimal excitation light for GFP (Figure 3, G and ...
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... 2 promoters used in the present study have been previously characterized in transient transgenic zebrafish embryos (Ju et al., 1999) or by direct injection of naked DNA into adult muscles (Xu et al., 1999). When pCK-EGFP was injected into zebrafish embryos, GFP was specifically expressed in the surface skin cells in early embryos ( Figure 1, A and B), consistent with our earlier observation (Ju et al., 1999). In embryos injected with pMLC-EGFP, GFP expression was specifically detected in trunk skeletal muscles (Figure 1, C and D). These expression patterns have been confirmed in stable transgenic zebrafish lines by expression of GFP under the 2 promoters (our unpublished observations). Therefore, the 2 promoters used in the present study were highly tissue-specific. To determine whether the 2 fluorescent proteins driven by different tissue-specific promoters can be expressed correctly in targeted tissues, pMLC-EGFP/pCK-RFP and pCK- EGFP/pMLC-RFP were respectively coinjected into zebrafish embryos. As shown in Figure 2, both fluorescent proteins were presented faithfully in the expressing tissues according to the respective promoters. In embryos injected with pMLC-EGFP/pCK-RFP, GFP and RFP were correctly expressed in muscle and skin cells, respectively (Figure 2, A and B). RFP was first detected in skin cells at 16 hpf, while GFP was first expressed in muscle fibers of somites at 22 hpf. It is interesting to note that strong expression of RFP can be detected as orange color under the excitation light for GFP (Figure 2, A); however, no GFP fluorescence can be observed under the excitation light for RFP (B). In embryos injected with pCK-EGFP/pMLC-RFP (Figure 2, C – F), GFP was first detected at the superficial layer of injected embryos at 6 hpf (shield stage), and the expression continued in the outmost layer of skin cells during embryogenesis. This is in contrast to the earliest detection of RFP expression at 16 hpf in embryos injected with pMLC-EGFP/pCK-RFP. In muscle cells, RFP was first observed at 30 hpf, also later than the earliest detection of GFP (22 hpf) under the same MLC promoter. Thus, GFP is detected earlier than RFP in the same tissue under the same promoter. Unexpectedly in the embryos injected with pCK-EGFP/ pMLC-RFP, GFP was also observed in muscle fibers at the same stage as RFP detection. It is apparent that all GFP- expressing muscle fibers also express RFP (Figure 2, C). This phenomenon is likely due to the formation of a het- eroconcatemer of the 2 injected plasmid DNAs, and thus the muscle-specific elements from pMLC-RFP affected the expression of gfp , as previously reported by Muller et al. (1997). During embryogenesis, RFP accumulation increased steadily in muscle fibers. In comparison, GFP fluorescence in muscle fibers became relatively weak and seemed to dis- appear after 72 hpf (Figure 2, E). Despite the observation of “ ectopic ” expression of GFP in muscles of the embryos injected with pCK-EGFP/pMLC- RFP, we never observed the expression of RFP in the muscles of the embryos injected with the reciprocal combination, pCK-RFP/pMLC-EGFP. Neither was “ ectopic ” expression of GFP or RFP observed in skin cells from embryos injected with either combination, pCK-EGFP/pMLC- RFP or pCK-RFP/pMLC-EGFP. To investigate whether there is any interference between the 2 fluorescent proteins if they are expressed in identical cells, coinjection of pCK-EGFP/pCK-RFP or pMLC-EGFP/ pMLC-RFP was carried out. In both cases, GFP and RFP were correctly expressed in the same tissues and presented in identical sets of cells (Figure 3). However, as observed from injection experiments with 2 heterogenous promoter constructs, GFP generally appears about 10 hours earlier than RFP. The first detection of GFP in skin and muscle was around 5 hpf and 22 hpf, respectively; in comparison, the appearance of RFP in the 2 tissues was 16 hpf and 30 hpf, respectively. The timing of GFP and RFP appearance in embryos injected with different combinations of DNA constructs is summarized in Figure 4. Although RFP was detected later than GFP, the intensity of its fluorescence increased steadily during development. By 72 hpf, only RFP fluorescence was detected in skin cells in embryos injected with pCK-EGFP/pCKRFP (Figure 3, C and D). Similarly, under the MLC promoter, RFP fluorescence became predominant after 5 dpf and GFP fluorescence was greatly overshadowed, even under the optimal excitation light for GFP (Figure 3, G and ...
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... 2 promoters used in the present study have been previously characterized in transient transgenic zebrafish embryos (Ju et al., 1999) or by direct injection of naked DNA into adult muscles (Xu et al., 1999). When pCK-EGFP was injected into zebrafish embryos, GFP was specifically expressed in the surface skin cells in early embryos ( Figure 1, A and B), consistent with our earlier observation (Ju et al., 1999). In embryos injected with pMLC-EGFP, GFP expression was specifically detected in trunk skeletal muscles (Figure 1, C and D). These expression patterns have been confirmed in stable transgenic zebrafish lines by expression of GFP under the 2 promoters (our unpublished observations). Therefore, the 2 promoters used in the present study were highly tissue-specific. To determine whether the 2 fluorescent proteins driven by different tissue-specific promoters can be expressed correctly in targeted tissues, pMLC-EGFP/pCK-RFP and pCK- EGFP/pMLC-RFP were respectively coinjected into zebrafish embryos. As shown in Figure 2, both fluorescent proteins were presented faithfully in the expressing tissues according to the respective promoters. In embryos injected with pMLC-EGFP/pCK-RFP, GFP and RFP were correctly expressed in muscle and skin cells, respectively (Figure 2, A and B). RFP was first detected in skin cells at 16 hpf, while GFP was first expressed in muscle fibers of somites at 22 hpf. It is interesting to note that strong expression of RFP can be detected as orange color under the excitation light for GFP (Figure 2, A); however, no GFP fluorescence can be observed under the excitation light for RFP (B). In embryos injected with pCK-EGFP/pMLC-RFP (Figure 2, C – F), GFP was first detected at the superficial layer of injected embryos at 6 hpf (shield stage), and the expression continued in the outmost layer of skin cells during embryogenesis. This is in contrast to the earliest detection of RFP expression at 16 hpf in embryos injected with pMLC-EGFP/pCK-RFP. In muscle cells, RFP was first observed at 30 hpf, also later than the earliest detection of GFP (22 hpf) under the same MLC promoter. Thus, GFP is detected earlier than RFP in the same tissue under the same promoter. Unexpectedly in the embryos injected with pCK-EGFP/ pMLC-RFP, GFP was also observed in muscle fibers at the same stage as RFP detection. It is apparent that all GFP- expressing muscle fibers also express RFP (Figure 2, C). This phenomenon is likely due to the formation of a het- eroconcatemer of the 2 injected plasmid DNAs, and thus the muscle-specific elements from pMLC-RFP affected the expression of gfp , as previously reported by Muller et al. (1997). During embryogenesis, RFP accumulation increased steadily in muscle fibers. In comparison, GFP fluorescence in muscle fibers became relatively weak and seemed to dis- appear after 72 hpf (Figure 2, E). Despite the observation of “ ectopic ” expression of GFP in muscles of the embryos injected with pCK-EGFP/pMLC- RFP, we never observed the expression of RFP in the muscles of the embryos injected with the reciprocal combination, pCK-RFP/pMLC-EGFP. Neither was “ ectopic ” expression of GFP or RFP observed in skin cells from embryos injected with either combination, pCK-EGFP/pMLC- RFP or pCK-RFP/pMLC-EGFP. To investigate whether there is any interference between the 2 fluorescent proteins if they are expressed in identical cells, coinjection of pCK-EGFP/pCK-RFP or pMLC-EGFP/ pMLC-RFP was carried out. In both cases, GFP and RFP were correctly expressed in the same tissues and presented in identical sets of cells (Figure 3). However, as observed from injection experiments with 2 heterogenous promoter constructs, GFP generally appears about 10 hours earlier than RFP. The first detection of GFP in skin and muscle was around 5 hpf and 22 hpf, respectively; in comparison, the appearance of RFP in the 2 tissues was 16 hpf and 30 hpf, respectively. The timing of GFP and RFP appearance in embryos injected with different combinations of DNA constructs is summarized in Figure 4. Although RFP was detected later than GFP, the intensity of its fluorescence increased steadily during development. By 72 hpf, only RFP fluorescence was detected in skin cells in embryos injected with pCK-EGFP/pCKRFP (Figure 3, C and D). Similarly, under the MLC promoter, RFP fluorescence became predominant after 5 dpf and GFP fluorescence was greatly overshadowed, even under the optimal excitation light for GFP (Figure 3, G and ...
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... 2 promoters used in the present study have been previously characterized in transient transgenic zebrafish embryos (Ju et al., 1999) or by direct injection of naked DNA into adult muscles (Xu et al., 1999). When pCK-EGFP was injected into zebrafish embryos, GFP was specifically expressed in the surface skin cells in early embryos ( Figure 1, A and B), consistent with our earlier observation (Ju et al., 1999). In embryos injected with pMLC-EGFP, GFP expression was specifically detected in trunk skeletal muscles (Figure 1, C and D). These expression patterns have been confirmed in stable transgenic zebrafish lines by expression of GFP under the 2 promoters (our unpublished observations). Therefore, the 2 promoters used in the present study were highly tissue-specific. To determine whether the 2 fluorescent proteins driven by different tissue-specific promoters can be expressed correctly in targeted tissues, pMLC-EGFP/pCK-RFP and pCK- EGFP/pMLC-RFP were respectively coinjected into zebrafish embryos. As shown in Figure 2, both fluorescent proteins were presented faithfully in the expressing tissues according to the respective promoters. In embryos injected with pMLC-EGFP/pCK-RFP, GFP and RFP were correctly expressed in muscle and skin cells, respectively (Figure 2, A and B). RFP was first detected in skin cells at 16 hpf, while GFP was first expressed in muscle fibers of somites at 22 hpf. It is interesting to note that strong expression of RFP can be detected as orange color under the excitation light for GFP (Figure 2, A); however, no GFP fluorescence can be observed under the excitation light for RFP (B). In embryos injected with pCK-EGFP/pMLC-RFP (Figure 2, C – F), GFP was first detected at the superficial layer of injected embryos at 6 hpf (shield stage), and the expression continued in the outmost layer of skin cells during embryogenesis. This is in contrast to the earliest detection of RFP expression at 16 hpf in embryos injected with pMLC-EGFP/pCK-RFP. In muscle cells, RFP was first observed at 30 hpf, also later than the earliest detection of GFP (22 hpf) under the same MLC promoter. Thus, GFP is detected earlier than RFP in the same tissue under the same promoter. Unexpectedly in the embryos injected with pCK-EGFP/ pMLC-RFP, GFP was also observed in muscle fibers at the same stage as RFP detection. It is apparent that all GFP- expressing muscle fibers also express RFP (Figure 2, C). This phenomenon is likely due to the formation of a het- eroconcatemer of the 2 injected plasmid DNAs, and thus the muscle-specific elements from pMLC-RFP affected the expression of gfp , as previously reported by Muller et al. (1997). During embryogenesis, RFP accumulation increased steadily in muscle fibers. In comparison, GFP fluorescence in muscle fibers became relatively weak and seemed to dis- appear after 72 hpf (Figure 2, E). Despite the observation of “ ectopic ” expression of GFP in muscles of the embryos injected with pCK-EGFP/pMLC- RFP, we never observed the expression of RFP in the muscles of the embryos injected with the reciprocal combination, pCK-RFP/pMLC-EGFP. Neither was “ ectopic ” expression of GFP or RFP observed in skin cells from embryos injected with either combination, pCK-EGFP/pMLC- RFP or pCK-RFP/pMLC-EGFP. To investigate whether there is any interference between the 2 fluorescent proteins if they are expressed in identical cells, coinjection of pCK-EGFP/pCK-RFP or pMLC-EGFP/ pMLC-RFP was carried out. In both cases, GFP and RFP were correctly expressed in the same tissues and presented in identical sets of cells (Figure 3). However, as observed from injection experiments with 2 heterogenous promoter constructs, GFP generally appears about 10 hours earlier than RFP. The first detection of GFP in skin and muscle was around 5 hpf and 22 hpf, respectively; in comparison, the appearance of RFP in the 2 tissues was 16 hpf and 30 hpf, respectively. The timing of GFP and RFP appearance in embryos injected with different combinations of DNA constructs is summarized in Figure 4. Although RFP was detected later than GFP, the intensity of its fluorescence increased steadily during development. By 72 hpf, only RFP fluorescence was detected in skin cells in embryos injected with pCK-EGFP/pCKRFP (Figure 3, C and D). Similarly, under the MLC promoter, RFP fluorescence became predominant after 5 dpf and GFP fluorescence was greatly overshadowed, even under the optimal excitation light for GFP (Figure 3, G and ...
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... 2 promoters used in the present study have been previously characterized in transient transgenic zebrafish embryos (Ju et al., 1999) or by direct injection of naked DNA into adult muscles (Xu et al., 1999). When pCK-EGFP was injected into zebrafish embryos, GFP was specifically expressed in the surface skin cells in early embryos ( Figure 1, A and B), consistent with our earlier observation (Ju et al., 1999). In embryos injected with pMLC-EGFP, GFP expression was specifically detected in trunk skeletal muscles (Figure 1, C and D). These expression patterns have been confirmed in stable transgenic zebrafish lines by expression of GFP under the 2 promoters (our unpublished observations). Therefore, the 2 promoters used in the present study were highly tissue-specific. To determine whether the 2 fluorescent proteins driven by different tissue-specific promoters can be expressed correctly in targeted tissues, pMLC-EGFP/pCK-RFP and pCK- EGFP/pMLC-RFP were respectively coinjected into zebrafish embryos. As shown in Figure 2, both fluorescent proteins were presented faithfully in the expressing tissues according to the respective promoters. In embryos injected with pMLC-EGFP/pCK-RFP, GFP and RFP were correctly expressed in muscle and skin cells, respectively (Figure 2, A and B). RFP was first detected in skin cells at 16 hpf, while GFP was first expressed in muscle fibers of somites at 22 hpf. It is interesting to note that strong expression of RFP can be detected as orange color under the excitation light for GFP (Figure 2, A); however, no GFP fluorescence can be observed under the excitation light for RFP (B). In embryos injected with pCK-EGFP/pMLC-RFP (Figure 2, C – F), GFP was first detected at the superficial layer of injected embryos at 6 hpf (shield stage), and the expression continued in the outmost layer of skin cells during embryogenesis. This is in contrast to the earliest detection of RFP expression at 16 hpf in embryos injected with pMLC-EGFP/pCK-RFP. In muscle cells, RFP was first observed at 30 hpf, also later than the earliest detection of GFP (22 hpf) under the same MLC promoter. Thus, GFP is detected earlier than RFP in the same tissue under the same promoter. Unexpectedly in the embryos injected with pCK-EGFP/ pMLC-RFP, GFP was also observed in muscle fibers at the same stage as RFP detection. It is apparent that all GFP- expressing muscle fibers also express RFP (Figure 2, C). This phenomenon is likely due to the formation of a het- eroconcatemer of the 2 injected plasmid DNAs, and thus the muscle-specific elements from pMLC-RFP affected the expression of gfp , as previously reported by Muller et al. (1997). During embryogenesis, RFP accumulation increased steadily in muscle fibers. In comparison, GFP fluorescence in muscle fibers became relatively weak and seemed to dis- appear after 72 hpf (Figure 2, E). Despite the observation of “ ectopic ” expression of GFP in muscles of the embryos injected with pCK-EGFP/pMLC- RFP, we never observed the expression of RFP in the muscles of the embryos injected with the reciprocal combination, pCK-RFP/pMLC-EGFP. Neither was “ ectopic ” expression of GFP or RFP observed in skin cells from embryos injected with either combination, pCK-EGFP/pMLC- RFP or pCK-RFP/pMLC-EGFP. To investigate whether there is any interference between the 2 fluorescent proteins if they are expressed in identical cells, coinjection of pCK-EGFP/pCK-RFP or pMLC-EGFP/ pMLC-RFP was carried out. In both cases, GFP and RFP were correctly expressed in the same tissues and presented in identical sets of cells (Figure 3). However, as observed from injection experiments with 2 heterogenous promoter constructs, GFP generally appears about 10 hours earlier than RFP. The first detection of GFP in skin and muscle was around 5 hpf and 22 hpf, respectively; in comparison, the appearance of RFP in the 2 tissues was 16 hpf and 30 hpf, respectively. The timing of GFP and RFP appearance in embryos injected with different combinations of DNA constructs is summarized in Figure 4. Although RFP was detected later than GFP, the intensity of its fluorescence increased steadily during development. By 72 hpf, only RFP fluorescence was detected in skin cells in embryos injected with pCK-EGFP/pCKRFP (Figure 3, C and D). Similarly, under the MLC promoter, RFP fluorescence became predominant after 5 dpf and GFP fluorescence was greatly overshadowed, even under the optimal excitation light for GFP (Figure 3, G and ...
Citations
... GUS activity was measured using a Synergy HT microplate reader (BioTek, USA). For subcellular localization and BiFC assays, Confocal laser scanning microscopy was used to detect fluorescence at excitation wavelengths of 546 nm for RFP and 488 nm for GFP, as described previously [43]. Alternatively, transfected protoplasts were harvested for RNA extraction and subsequent gene expression analysis. ...
... Another practice application of this novel protein variant is in ornamental aquaculture. Fluorescent fish is a reality in this science (Wan et al. 2002;Choe et al. 2021) and are available commercially. However, a fish able to produce dual fluorescence is extra attractive for ornamental pisciculture. ...
The marine environment is a rich reservoir of diverse biological entities, many of which possess unique properties that are of immense value to biotechnological applications. One such example is the red fluorescent protein derived from the coral Discosoma sp. This protein, encoded by the DsRed gene, has been the subject of extensive research due to its potential applications in various fields. In the study, a variant of the red fluorescent protein was generated through random mutagenesis using the DsRed2 gene as a template. The process employed error-prone PCR (epPCR) to introduce random mutations, leading to the isolation of twelve gene variants. Among these, one variant stood out due to its unique spectral properties, exhibiting dual fluorescence emission at both 480 nm (green) and 550 nm (red). This novel variant was expressed in both Escherichia coli and zebrafish (Danio rerio) muscle, confirming the dual fluorescence emission in both model systems. One of the immediate applications of this novel protein variant is in ornamental aquaculture. The dual fluorescence can serve as a unique marker or trait, enhancing the aesthetic appeal of aquatic species in ornamental settings.
... Since the 1980s, gene constructs containing a promoter, a protein-encoding sequence, and other elements have been introduced into an organism's genome (Du, et al., 1992;Zhu, 1993;Zhu et al., 1985). Fluorescent transgenic fish were the first transgenic animal product marketed to the public (Wan, et al., 2002). The fast-growing AquaBounty Atlantic salmon, which is now marketed, is the only transgenic fish intended for use as food (Hallerman, et al., 2023). ...
... Advancing the ornamental fish industry through genetic engineering Application of transgenic technology for genetic improvement of cultured fishes had been mooted in the early 1990s (Fletcher and Davies 1991), after the successful generation of transgenic goldfish (Zhu et al. 1985). Since the first report on the transgenic zebrafish (Glofish) (Wan et al. 2002), several transgenic lines of ornamental fish have been produced; medaka (Zeng et al. 2005;Cho et al. 2013Cho et al. , 2014 and tetra fish (Pan et al. 2008;Leggatt and Devlin 2020). As in Glofish, the fluorescent genes namely green fluorescent protein (gfp) (Wan et al. 2002;Zeng et al. 2005;Leggatt and Devlin 2020), yellow fluorescent protein (yfp), cyan fluorescent protein (cfp) (Cho et al. 2014) and red fluorescent protein (rfp) (Wan et al. 2002;Pan et al. 2008) have been successfully transferred to produce attractive luminous fish. ...
... Since the first report on the transgenic zebrafish (Glofish) (Wan et al. 2002), several transgenic lines of ornamental fish have been produced; medaka (Zeng et al. 2005;Cho et al. 2013Cho et al. , 2014 and tetra fish (Pan et al. 2008;Leggatt and Devlin 2020). As in Glofish, the fluorescent genes namely green fluorescent protein (gfp) (Wan et al. 2002;Zeng et al. 2005;Leggatt and Devlin 2020), yellow fluorescent protein (yfp), cyan fluorescent protein (cfp) (Cho et al. 2014) and red fluorescent protein (rfp) (Wan et al. 2002;Pan et al. 2008) have been successfully transferred to produce attractive luminous fish. The skeletal muscle gene promoters regulate the fluorescent genes to express various beautifully glowing colours under different light emission (Cho et al. 2014). ...
... Since the first report on the transgenic zebrafish (Glofish) (Wan et al. 2002), several transgenic lines of ornamental fish have been produced; medaka (Zeng et al. 2005;Cho et al. 2013Cho et al. , 2014 and tetra fish (Pan et al. 2008;Leggatt and Devlin 2020). As in Glofish, the fluorescent genes namely green fluorescent protein (gfp) (Wan et al. 2002;Zeng et al. 2005;Leggatt and Devlin 2020), yellow fluorescent protein (yfp), cyan fluorescent protein (cfp) (Cho et al. 2014) and red fluorescent protein (rfp) (Wan et al. 2002;Pan et al. 2008) have been successfully transferred to produce attractive luminous fish. The skeletal muscle gene promoters regulate the fluorescent genes to express various beautifully glowing colours under different light emission (Cho et al. 2014). ...
The ornamental fish industry has continued to flourish since eighteenth century with increased fascination by enthusiasts in the striking body colours and patterns displayed in the fishes, a beneficial outcome of rigorous selective programmes. The expression of these pigmented colours is the result of the differentiation and orientation of specialised chromatophores located within the dermal layer. The different types of chromatophores found in many ornamental fish species, are the basis of the unique colour hues and patterns. This review discusses the current approaches for enhancing the body pigmentation and pattern of ornamental fishes. Two factors are considered to be the main drivers of body colour regulation: feed additives (pigments) and rearing environment setup, i.e. tank colour and light. Potential candidate pigment genes to manipulate the ornamental fish body pigmentation and pattern have been elucidated through mapping of putative regulatory pathways, buoyed by the rapid development of next generation sequencing technologies. The effects of feed additives, tank background colour and light on various ornamental fish species, and regulatory pathways of involved genes offer valuable insights for enhanced variety production prior to genetic engineering and are herein discussed. It is hoped that the systematic analysis of the current knowledge in this review would be a boon for the ornamental fish community to step up efforts to boost the ornamental fish breeding industry.
... For some species like nile tilapia and midas cichlid, although red strains are available, but the color is not strong and uniform (Henning et al. 2010;Segev-Hadar et al. 2021). In this case, transgenic technology possess a great power to introduce new and fluorescent colors (Wan et al. 2002;Gong et al. 2003). A successful example is GloFish, which are genetically engineered first in zebrafish with highly expressed green (GFP), red (RFP), and yellow (YFP) fluorescent proteins under the control of a strong muscle-specific gene promoter (Gong et al. 2003). ...
Albinism is the most common color variation described in fish and is a fascinating trait of some ornamental fish species. Albino mutants can be generated by knocking out core genes affecting melanin synthesis like slc45a2 in several fish species. However, genetic mutation remains challenging for species with unknown genome information. In this study, we generated albino mutants in two selected ornamental fish species, royal farlowella (Sturisoma panamense), and redhead cichlid (Vieja melanura). For this purpose, we carried out phylogenetic analyses of fish slc45a2 sequences and identified a highly conserved region among different fish species. A pair of degenerate primers spanning this region was designed and used to amplify a conserved slc45a2 fragment of 340 bp from the two fish species. Based on the amplified sequences, a target site in the 6th exon was used for designing guide RNA and this targeted site was first verified by the CRISPR/Cas9 system in the zebrafish (Danio rerio) model for the effectiveness. Then, specific guide RNAs were designed for the two ornamental fish species and tested. Most of the injected larvae completely lost black pigment over the whole body and eyes. DNA sequencing confirmed a high degree of mutation at the targeted site. Overall, we described a fast and efficient method to generate albino phenotype in fish species by targeting the conserved 6th exon of slc45a2 gene for genome editing via CRISPR/Cas9 and this approach could be a new genetic tool to generate desirable albino ornamental fish.
... For the fusion constructs, the same sequence, encoding the 3xGly linker peptide, was used to connect the RAI genes with mRFP (Table 2) This is not unexpected, as normally the second reading frame on the bicistronic mRNA is translated to a lesser extend compared to the first one, ranging in most cases between 20 -50 % (Mizuguchi et al., 2000). Moreover, in comparison to eGFP, mRFP seems to have a lower detection sensitivity while bearing a higher stability at the same time (Wan et al., 2002). This is reflected in the plasmid functionality tests, since the observed red fluorescence was often weak and very difficult to detect, which is accordingly not directly attributable to a lower expression level. ...
... This is due to the mosaic expression as a result of the injection process and to the comparably low intensity of mRFP fluorescence (Wan et al., 2002). ...
The molecular mechanisms that control the development of paired extremities are broadly conserved among vertebrate species. The paired fins of fish - pectoral and pelvic fins - are homologous to the fore- and hindlimbs of land vertebrates. Consequently, a fundamental knowledge about signalling processes in zebrafish paired fin development might help to understand limb patterning and congenital limb defects in humans. All-trans-retinoic acid (RA) is a key factor in many developmental processes including limb development. The current model for forelimb development was predominantly determined from studies in mice (Cunningham et al., 2013; Mic et al., 2002, 2004; Sandell et al., 2007; Zhao et al., 2009), chicken (Nishimoto et al., 2015) and zebrafish (Begemann et al., 2001; Gibert et al., 2006; Grandel & Brand, 2011; Grandel et al., 2002). It suggests an antagonism between RA and fibroblast growth factors (FGFs) along the anteroposterior axis, which mediates the correct positioning of the limb field and establishes a permissive environment for the induction of limb budding (Cunningham et al., 2013; Zhao et al., 2009). Moreover, RA cooperatively interacts with β-catenin signalling and Hox genes to control Tbx5 expression during forelimb development in chicks (Nishimoto et al., 2015). Examinations in zebrafish agree with the requirement of RA for pectoral fin induction (Gibert et al., 2006). For hindlimb development, however, the roles of RA are still controversial. The idea of a similar role for RA in fore- and hindlimb development (Nishimoto et al., 2015) contrasts with the opinion that RA is dispensable for hindlimb development (Zhao et al., 2009). In the zebrafish model, comparable studies investigating the role of RA on pelvic fin development are missing, which is why this thesis focused on this particular question. Gene expression analysis on zebrafish larvae revealed the presence of Rdh10a, Aldh1a2, Cyp26b1 and Cyp26c1 transcripts during the early stages of pelvic fin bud formation. The expression pattern of these genes, which are involved in RA synthesis and metabolism, indicated the establishment of an anteroposterior RA gradient in the early pelvic fin bud. Later, activity of RA signalling associated genes was detected along the forming fin rays. Based on heat-shock treatments of transgenic Hsp70l:Cyp26a1 zebrafish larvae, overexpression of Cyp26a1 and thus a reduction of the RA level was achieved during pelvic fin formation. From the obtained results an important role of RA in the development of pelvic fins during early stages of fin bud formation was concluded. A complete inhibition of the formation of endo- and exoskeletal pelvic fin structures could be achieved if the heat-shock treatment was started before the first signs of a morphological fin bud appeared. After the onset of fin bud formation, Cyp26a1 overexpression resulted in the reduction of the overall length of the pelvic girdle accompanied by the lack of diverse skeletal elements, mostly the posterior process and the radials. These results indicate a putative role of RA in the pelvic fin initiation process, which seems to occur during a limited time frame. Moreover, they suggest a role of RA in pelvic girdle patterning and chondrogenesis. Additionally, a participation in fin ray formation and growth is likely. However, since the entire organism is affected in these experiments, unspecific effects cannot be ruled out. Therefore, the main focus of this work was to establish the binary Gal4-UAS system with the aim to manipulate RA signalling in a spatially and temporally controlled manner. On the one hand, driver lines provide the expression of either a hormone- or light-inducible Gal4 variant under the control of tissue-specific enhancers. Here, three Gal4 variations - ERT2-Gal4-VP16, KalTA4-ERT2 and GAVPO (Akerberg et al., 2014; Distel et al., 2009; Gerety et al., 2013; Kajita et al., 2014; Wang et al., 2012) - were investigated and considered suitable for the use in zebrafish. Tissue-specifity was achieved by selecting enhancers of the genes Prrx1a, Prrx1b and Pitx1, which are active specifically in pectoral and/or pelvic fins (Chan et al., 2010; Hernández-Vega & Minguillón, 2011). On the other hand, effector lines express genes encoding either a dominant-negative retinoic acid receptor (dnRarα2a) (Stafford et al., 2006) or the RA metabolizing enzyme Cyp26a1 under the control of five repetitive (5x) or four non-repetitive (4xnr) upstream activating sequences (UAS) (Akitake et al., 2011; Goll et al., 2009). Driver and effector constructs are equipped with minimal Tol2 cis sequences mediating transgene integration into the genome by Tol2 transposase activity. Moreover, different marker genes facilitate the identification of single or multiple transgenic zebrafish. As a proof-of-principle, the activation of dnRarα2a expression in F3 embryos of 5xUAS:dnRarα2a-IRES-eGFP zebrafish by injection of KalTA4-ERT2-GI mRNA, followed by induction with 4-hydroxy-tamoxifen (4-OHT) was demonstrated. Altogether, the basis for a valuable genetic tool was created, that combines several advantages: a simple and practical application, a simplified screening process, the visualisation of transgene activity and the optimization for the zebrafish model organism.
... One of the pioneering attempts was that using muscle-specific gene, mylz2, and a skinspecific gene, keratin 8 promoters for GFP and development of a transgenic zebrafish showing green fluorescence bright enough to be observed under daylight. Subsequent attempts also yielded fruitful results with red and yellow fluorescent proteins [71][72][73][74] . This became the most successful commercial application of transgenic technology, marketed under trade name of 'Glo Fish' after sufficient media stir regarding ANDi (the first transgenic primate), and Alba (the transgenic GFP rabbit) 75 . ...
One of the greatest challenges in the ornamental fish industry is to replicate accurate natural colour of fishes in captivity. Numerous attempts to preserve colour in captivity have been ineffective in reducing its fading, making it an important determinant in the selection of ornamental fish species for trade in terms of saturation, brightness and hue. Colour development of ornamental fishes has been widely studied, yielding curious insights about evolutionary genetics and having a discerning role, either as deceptive or attractive (aposematic) signals in mating as well as in camouflaging (Delphic) patterns during predator-prey interactions. This article discusses colour enhancement strategies with reference to nutritional interventions through carotenoid-rich feed ingredients, genetic manipulation or injection of colour in subcutaneous layers of the skin. An insight into the mechanism of pigmentation shows that motility and pigment dispersion of chromatophores are the two drivers by which fishes control integumentary colour variation. Research on colour development and its enhancement has witnessed novel techniques to support the ornamental fish industry. Therefore, this article also sheds light to answer questions on various issues pertaining to environmental and physiological effects on colouration. It attempts to provide insight on potential research areas, with caution on ethical and legal issues to ensure sustainability, so as to restrict risks of unwanted inheritance of colour patterns. It also highlights the problems of identity crisis among conspecifics thereby bringing a 'rainbow revolution' to the ornamental industry.
... A principios del siglo XXI, se desarrollaron a nivel de laboratorio los primeros peces ornamentales transgénicos. La técnica para la obtención de estos OVM consistió en la introducción de genes que producen proteínas fluorescentes de colores verde (GFP, Green Fluorescent Protein), rojo (RFP, Red Fluorescent Protein), entre otros, extraídos primero de la medusa abisal Aequorea victoria (Murbach y Shearer, 1902) y luego de la anémona de mar [(Anemonia manjano (Carlgren,1900)] y otros organismos marinos (Gong et al., 2001;Wan et al., 2002;Udvadia & Linney, 2003). ...
In 2006, the first transboundary movement of fluorescent zebrafish to the Peruvian territory was identified. Little is known about the possible environmental impact due to the uncontrolled release of these transgenic fluorescent fish. As well as, the origins and gene flow of these hydrobiological organisms. The fluorescence genes GFP
(Green Fluorescent Protein) and RFP (Red Fluorescent Protein) in transgenic Zebrafish introduced into the Peruvian territory and Zebrafish from Colombia, Chile and Taiwan as positive controls were analyzed by PCR. All nucleotide sequences corresponded to the red fluorescent protein (drFP583) of the marine anemone Discosoma
sp. with a size of 505pb (RFP) and its green variant (GFP) with a size of 570pb. Likewise, the phylogenetic analysis evidenced an Asian origin of the transboundary movement of these fish along a South American route via Colombia, mainly to Peruvian territory.
... A principios del siglo XXI, se desarrollaron a nivel de laboratorio los primeros peces ornamentales transgénicos. La técnica para la obtención de estos OVM consistió en la introducción de genes que producen proteínas fluorescentes de colores verde (GFP, Green Fluorescent Protein), rojo (RFP, Red Fluorescent Protein), entre otros, extraídos primero de la medusa abisal Aequorea victoria (Murbach y Shearer, 1902) y luego de la anémona de mar [(Anemonia manjano (Carlgren,1900)] y otros organismos marinos (Gong et al., 2001;Wan et al., 2002;Udvadia & Linney, 2003). ...
In 2006, the first transboundary movement of fluorescent
zebrafish to the Peruvian territory was identified. Little is
known about the possible environmental impact due to
the uncontrolled release of these transgenic fluorescent
fish. As well as, the origins and gene flow of these
hydrobiological organisms. The fluorescence genes GFP
(Green Fluorescent Protein) and RFP (Red Fluorescent
Protein) in transgenic Zebrafish introduced into the
Peruvian territory and Zebrafish from Colombia, Chile
and Taiwan as positive controls were analyzed by PCR. All
nucleotide sequences corresponded to the red fluorescent
protein (drFP583) of the marine anemone Discosoma
sp. with a size of 505pb (RFP) and its green variant
(GFP) with a size of 570pb. Likewise, the phylogenetic
analysis evidenced an Asian origin of the transboundary
movement of these fish along a South American route
via Colombia, mainly to Peruvian territory.
... BiFC and intracellular localization were visualized by fluorescence microscopy. GFP was observed with the filter BP450-490 (blue light) and RFP was observed with the filter BP546 (yellow light) (Wan et al., 2002). ...
The MBW complex, consisting of MYB, basic helix-loop-helix (bHLH)and WD40 proteins, regulates multiple traits in plants, such as anthocyanin and proanthocyanidin biosynthesis and cell fate determination. The complex has been widely identified in dicot plants, whereas few studies are concentrated on monocot plants which are of crucial importance to decipher its functional diversities among angiosperms during evolution. In present study, a WD40 gene from Freesia hybrida, designated as FhTTG1, was cloned and functionally characterized. Real-time PCR analysis indicated that it was expressed synchronously with the accumulation of both proanthocyanidins and anthocyanins in Freesia flowers. Transient protoplast transfection and biomolecular fluorescence complementation (BiFC)assays demonstrated that FhTTG1 could interact with FhbHLH proteins (FhTT8L and FhGL3L)to constitute the MBW complex. Moreover, the transportation of FhTTG1 to nucleus was found to rely on FhbHLH factors. Outstandingly, FhTTG1 could highly activate the anthocyanin or proanthocyanidin biosynthesis related gene promoters when co-transfected with MYB and bHLH partners, implying that FhTTG1 functioned as a member of MBW complex to control the anthocyanin or proanthocyanidin biosynthesis in Freesia hybrida. Further ectopic expression assays in Arabidopsis ttg1-1 showed the defective phenotypes of ttg1-1 were partially restored. Molecular biological assays validated FhTTG1 might interact with the endogenous bHLH factors to up-regulate genes responsible for anthocyanin and proanthocyanidin biosynthesis and trichome formation, indicating that FhTTG1 might perform exchangeable roles with AtTTG1. These results will not only contribute to the characterization of FhTTG1 in Freesia but also shed light on the establishment of flavonoid regulatory system in monocot plants, especially in Freesia hybrida.
