Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii

Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select a suitable promoter as the ﬁ rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Green ﬂ uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycus alvarezii . GFP gene constructs driven by cytomegalovirus (pCMV-GFP), cauli ﬂ ower mosaic virus (pCaMV-GFP), medaka β -actin (pmBA-GFP) and Japanese ﬂ ounder keratin (pJfKer-GFP) promoters were introduced by electroporation method. Electroporation was performed using a gene pulser (BIORAD) with voltage of 300 V, pulse length of 0.5 ms, pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expression level using a ﬂ uorescent microscope. The results showed that CMV regulated highest number of ﬁ lament callus (34.10%±1.49) expressing GFP at medium to strong ﬂ uorescence levels. CaMV promoter had relatively similar activity with CMV, but lower number of ﬁ lament callus expressing GFP (10.48%±0.25). mBA promoter drove GFP expression at medium level and similar number of ﬁ lament callus (8.85%±2.31) expressing GFP with CaMV, while JfKer promoter had lowest activity by means in number of ﬁ lament callus expressing GFP (4.79%±0.26) and GFP expression level. PCR analysis for transgenic con ﬁ rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressing ﬁ lament callus in the same size (about 0.6 kb) with positive control of plasmid. Thus, CMV and CaMV promoters was an appropriate promoter and foreign gene could be transferred to ﬁ lament callus by electroporation method. Combining this achievement with developing a culture method of ﬁ lament callus to be thallus, stable transgenic breeding in K. alvarezii can be feasible.


Introduction
Kappaphycus alvarezii is one of the major sources of kappa-carrageenan for food, chemical and pharmaceutical industries (McHugh, 2003). This seaweed has been prioritized by the Ministry of Marine Affairs and Fisheries as one of aquaculture commodities targeted to increase on 2014. Increased production of seaweed farming can be achieved through extension and use of high quality seeds in terms of fast growth, resistant to disease and fl uctuation of environmental conditions. Seaweed farming extensifi cation program conducted by the Directorate of Aquaculture successfully increased production of seaweed. However, various problems faced in achieving the production target such as the growth rate being decrease due to disease infection and environmental degradation (Vairappan ,2006;Largo et al., 1997). In addition, disease and environmental factors are also affecting the quality and quantity of carrageenan (Vairappan et al., 2008). On the other hand, the increase in carrageenan consumption worldwide requires more production and high level of bioactive compound in farmed seaweed. Genetic engineering techniques have therefore been expected to improve seed stocks of seaweeds (Hallman, 2007). In fact, transgenic reasearch have been conducted on Laminaria japonica using β-glucuronidase (Li et al., 2009), Kappaphycus alvarezii using LacZ gene (Wang et al., 2010) and Gracilaria changii using LacZ gene as reporter (Huddy et al., 2012;Gan et al., 2003). In Wang et al. study, Kappaphycus alvarezii transgenic was produced by micro-particle bombardment method (Wang et al., 2010).
A p p l i c a t i o n o f g e n e t r a n s f e r (transgenesis) technology in order to increase growth (Devlin et al., 1994, Nam et al., 2001Kobayashi et al., 2007), resistant to disease (Yazawa et al., 2006;Parenrengi, 2010) and adaptation ability to the extreme environmental condition (Wang et al., 1995) have extensively been reported in various finfish and shellfish species. In seaweed, however, research is still limited to development of transgenic method and analysis of promoter activity in Laminaria japonica Aresch and Undaria pinnatifida Suringer (Qin et al., 2005).
The ability of promoter used to control the expression of foreign gene, in this study green fl uorescent protein (GFP) gene was used as reporter, is one of determinant factors affecting the success of transgenesis. Various promoters have been analyzed for their activities in fi sh, such as cytomegalovirus/ CMV (Volckaert, 1994;Arenal et al., 2008;Traxler et al., 1999), β-actin (Yoshizaki, 2001;Alimuddin et al., 2005), heat-shock protein, keratin (Gong et al., 2002 ;Yazawa et al., 2005), myosin light chains (Alimuddin, 2003) and elongation factor-1α (Yasawa et al., 2005;Alimuddin et al., 2007). Promoter activity level is determined by the match between transcription factors (cis regulators) of the promoter with trans regulators in the host (Iyengar et al., 1996;Alimuddin, 2003). Generally, promoters activity originating from the same species or family with the fi sh will be made transgenic are higher than those of originating from different species. In addition, number of promoter which has been isolated is still limited. Thus, it is necessary to evaluate the activity of existing promoters in order to produce seaweed transgenic.
In this study, as an initial step to produce high quality (such as high level of carrageenan content and disease resistant) seed of seaweed using transgenesis technology, four different promoters that drives GFP gene was introduced to obtain a suitable promoter for seaweed. The results of study showed that the two virus derived promoters (CMV and CaMV) and medaka β-actin promoter can regulate high foreign gene expression level in seaweed.

Source of K. alvarezii fi lament callus
K. alvarezii used in this study was derived from the collection of Brackishwater Aquaculture Research Center, Maros, South Sulawesi. Thalli segment, callus and fi lament callus were cultured in the laboratory condition according to method described by Reddy et al. (2003) and Rajamuddin (2010). Thalli segment was culture to obtain callus using solid agar medium containing growth regulators, while callus was culture in semisolid medium to produce fi lament callus. Filament callus were then regenerated in a liquid medium.

Transformation protocol
Each GFP expression vector in concentration of 100 ng/μL was mixed with K. alvarezii callus mass as filament form. Transformation was performed by electroporation method using Gene Pulser II (Biorad, USA) in voltage of 300 V, pulse length 0.5 ms, pulse number 4, and pulse interval 0.1 s at 2 mm cuvette. Those electroporation parameters were obtained from preliminary study that produced high transformation effi ciency and cell viability.

GFP gene expression analysis
GFP expression was analyzed using a fl uorescent microscope (Olympus BH2-RFCA) equipped with reflected light fluorescent attachment (BH2-RFC2). Observation was conducted every hour started at 1 h after electroporation until GFP expression level being stable. Number of cells expressing GFP and their expression level was observed after GFP was stably expressed. GFP expression levels were qualitatively classifi ed as faint, medium and strong fl uorescence.

Transformant regeneration and detection
Transformant carrying GFP was screened and regenerated by culturing the electroporated fi lament callus in the semisolid medium containing kanamisin 100 ppm for 1 month. Detection of transformant was conducted using fl uorescent microscope and PCR method. Genomic DNA was extracted from the highest and lowest GFP expressing transformant using the Puregene DNA Isolation Kit (Gentra, USA). PCR amplifi cation was carried out using a set primer of GFP-F (5'-GGTCGAGCTGGACGG-3') and GFP-R (5'-ACGAACTCCAGCAGG-3'). Amplifi cation of GFP fragment was conducted for 35 cycles of denaturation at 94 o C for 30 s, annealing at 62 o C for 30 s, and extension at 72 o C for 1 min. PCR product was electrophoretically separated using 1% agarose gel.

GFP gene expression
The electroporation parameters used in this study was obtained in our preliminary experiment (unpublished data). As shown in Figures 1A-D, electroporated fi lament callus mass showed green fl uorescence in some of embryos, while no GFP expression in nonelectroporated control was observed (Fig.  4E). This indicated that GFP gene construct was successfully transferred to filament callus of seaweed. Further, percentage of filament callus expressing GFP gene was different between gene construct used ( Table  1, Fig. 4). Highest percentage of filament callus expressing GFP was obtained by using CMV promoter, followed by CaMV, mBA and JfKer (Table 1). GFP gene expression levels driven by CMV, CaMV and mBA were relatively similar while JfKer was lower compared to that of the first three promoters (Fig. 4). The fi rst two promoters is virus origin, while the last two is fi sh origin. Foreign gene expression levels driven by virus origin promoter were similar with mBA promoter of Japanese medaka fi sh origin, but it was lower compared to that of JfKer promoter of Japanese fl ounder fi sh origin. Furthermore, initial time to express GFP after electroporation was fastest in fi lament callus carrying pCMV-GFP and pCaMV-GFP constructs compared with pmBA-GFP, and the later was pJfKer-GFP. Thus, differences in the level and initial time of foreign gene to express are possibly related to difference in recognition of trans-acting protein of the host with cis-acting element of promoter (Iyengar et al., 1996;Alimuddin, 2003).
Hae is hour after electroporation. GFPexpressing cell percentage are given as means ± S.D. Different superscript letters in GFPexpressing cell percentage indicate signifi cant differences (p<0.05) as determined by one-way ANOVA followed by Duncan's test. Rajamuddin et al.

Transformant regeneration
All gene constructs used in this study is contained kanamycin resistance gene so that the transformant can live in the medium containing kanamycin. By adding 100 ppm of kanamycin into the culture medium, the electroporated fi lament callus survived and grew (Fig. 5A-D), while non-electroporated control died after 1 month incubation (Fig. 5E). This indicated that the electroporated fi lament callus carried foreign gene construct. Furthermore, number of fi lament callus expressing GFP after incubation for 1 month (Fig. 5A-D) was higher compared to that of initial condition (Fig. 4), indicating the mitotic division in transgenic cells. Number of GFP-expressing fi lament callus using pCMV-GFP and pCaMV-GFP constructs was relatively similar and higher than that of pmBA-GFP and pJfKer-GFP. This might be correlated to initial number of cells expressing foreign gene before they regenerated. In addition, GFP-expressing cells derived from mitotic division may become a transgenic stable, though this needs a proof by Southern blot analysis.

PCR analysis for transgenic confi rmation
PCR analysis was performed to reconfirm that the green fluorescence in the survived fi lament callus of K. alvarezii in the kanamycin-contained medium is from GFP  transgene expression. As shown in Fig. 6, a DNA band of PCR product from pCMV-GFP (CMV) and pCaMV-GFP (CaMV) expressing filament callus in the same size (about 0.6 kb) with the positive control of pCMV-GFP plasmid (P) was obtained, but no PCR product in the non-electroporated control (C). This indicated that the somatic embryos showing green fl uorescence were transgenic carrying pCMV-GFP and pCaMV-GFP gene constructs. PCR product from pmBA-GFP showed negative of DNA band, altough number of GFP expressing fi lament callus using pCaMV-GFP construct was relatively similar.

Conclusion
Electroporation method could be used to transfer GFP gene into filament callus of Kappaphycus alvarezii. Cytomegalovirus (CMV) and caulifl ower mosaic virus (CaMV) promoters are more suitable for K. alvarezii in controlling GFP gene expression compared with medaka β-actin and Japanese fl ounder keratin promoters.  Figure 6. Result of transgene detection by PCR analysis with DNA template extracted from fi lament callus of Kappaphycus alvarezii Lane CMV, mBA, CaMV and JfKer are PCR product (marked by arrow head) amplifi ed using genomic DNA from pCMV-GFP, pmBA-GFP, CaMV-GFP and pJfKer-GFP expressing fi lament callus, respectively; C= a sample product with genomic DNA from non-transgenic fi lament callus; lane P and N= a sample PCR product with pCMV-GFP plasmid and without DNA template, respectively. M= is 2-log ladder DNA marker (BioLabs, Inc., New England). The amplifi ed fragment is about 0.6 kb in size.
for Research Assessment and Application of Technology (BPPT) Serpong. The pmBA-GFP and pJfKer-GFP constructs were kindly gift of Prof. Dr. Goro Yoshizaki and Prof. Dr. Takashi Aoki, respectively.