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

Rajamuddin, Muh. Alias L.; Ali, Alimuddin; Widyastuti, Utut; Faizal, Irvan

doi:10.22146/ijbiotech.9304

Cited by 4 publications

(4 citation statements)

References 12 publications

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“…ahare?? has also been reported for the GFP marker gene (Rajamuddin et al 2014) and the H-carrageenase gene (Rajamuddin 2016). The CaMV 35s promoters have also been successfully used to express transgenes in other species of red algae, such as the GUS reporter gene in Porpbyra yexoensis (Cheney et al 2001) and the Lac2 reporter gene in Gradarid gracilis (Huddy et al 20 12).…”

Section: Quantification Of Goc Gene Expression Inmentioning

confidence: 70%

Overexpression of Gα Gene Increases Growth and Hyposaline Tolerance in Kappaphycus alvarezii Transgenic Plantlets

Sulistiani

2021

BIOTROPIA

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G proteins are membrane proteins that play roles in signal transduction in living organisms. They consist of a, p and y subunits. The G protein a subunit (Ga) plays a role in plant resistance toward biotic and abiotic environmental stresses. Transgenic plantlets of Kappaph_yczis aluare+i carrying the Ga gene (derived from soybean) have been successfully obtained through Agrobacterium tnmefaaciens-mediated transformation. The present study aimed to: 1. compare the growth of non-transgenic and transgenic plantlets of K. alvareei in vitro using Provasoli enriched seawater (PES) medium with normal salinity and hyposalinity and 2. analyze the expression level of the Ga gene in transgenic plantlets using quantitative Polymerase Chain Reaction (qPCR). The results showed that all transgenic plantlets (six clones) had significantly higher daily growth rate (DGR, %/d) than that of nontransgenic under the condition of normal salinity (30 ppt) and hyposalinity (15 and 20 ppt) for 5 weeks of observation. At 15 ppt, transgenic plantlets were more tolerant than non-transgenic ones, as most thalli of transgenic plantlets remained brown in color, whereas most thalli of non-transgenic plantlets were bleached. The results of the qPCR analysis showed that the expression of the Ga gene in transgenic plantlets increased by 6.43 -8.03 times compared with that of non-transgenic plantlets. The result of Pearson correlation analysis showed that relative expression of Ga gene had a strong correlation, both with DGRs in normal salinity and hyposalinity of transgenic plantlets (correlation coefficient > 0.7). The correlation was linearly positive, where increased expression of the Ga gene was strongly associated with an increase in DGRs.

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Section: Quantification Of Goc Gene Expression Inmentioning

confidence: 70%

Overexpression of Gα Gene Increases Growth and Hyposaline Tolerance in Kappaphycus alvarezii Transgenic Plantlets

Sulistiani

2021

BIOTROPIA

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“…Perbaikan mutu genetik pada rumput laut telah dirintis sejak tahun 2010 melalui kerja sama penelitian antara Balai Penelitian dan Pengembangan Budidaya Air Payau (BPPBAP), Maros dengan Pusat Antar Universitas, Institut Pertanian Bogor (PAU-IPB). Informasi yang diperoleh antara lain lain introduksi gen GFP di bawah regulasi promoter sehingga menghasilkan transgenik rumput laut sampai tahap filamen (Rajamuddin et al, 2014), kloning gen penyandi karaginan, merakit vektor ekspresi, serta introduksi gen karaginan rumput laut K. alvarezii (Rajamuddin et al, 2016), serta transformasi gen lain untuk meningkatkan ketahanan terhadap cekaman lingkungan antara lain gen PaCS (Daud et al, 2013;Suryati et al, 2014), gen lisozim (Handayani et al, 2014), gen methalothionin type-II (Mamt2) (Fajriah et al, 2015;Suryati et al, 2015).…”

Section: Pendahuluanunclassified

REGENERASI DAN PERBANYAKAN RUMPUT LAUT Kappaphycus alvarezii HASIL TRANSFORMASI GEN SUPEROKSIDA DISMUTASE (MaSOD)

Suryati

Triana

Widiastuti

et al. 2017

Jurnal Riset Akuakultur

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Transformasi gen superoxide dismutase (MaSOD) pada rumput laut Kappaphycus alvarezii menggunakan Agrobacterium tumefacient telah dilakukan secara in vitro. Transformasi gen MaSOD ke dalam genom rumput laut diharapkan dapat mengurangi cekaman oksidatif terutama yang disebabkan oleh perubahan suhu, salinitas, dan cemaran logam di perairan. Penelitian ini bertujuan untuk regenerasi rumput laut hasil introduksi gen MaSOD dan non-transgenik pada labu kultur. Regenerasi dan perbanyakan rumput laut hasil transformasi gen MaSOD dilakukan di laboratorium pada labu kultur yang diletakkan dalam “culture chamber” yang dilengkapi dengan aerasi menggunakan media kultur yang diperkaya dengan pupuk PES, Grund, Conwy, dan SSW sebagai kontrol, salinitas 20, 25, 30, 35, dan 40 g/L, pH 4, 5, 6, 7, dan 8. Intensitas cahaya antara 500-2.000 lux dengan fotoperiode terang dan gelap 8:16; 12:12; dan 16:8. Untuk merangsang pertumbuhan eksplan dilakukan pemeliharaan dengan penambahan hormon tumbuh IAA dan BAP dengan perbandingan 1:1, 1:2, dan 2:1. Penelitian dilakukan secara bertahap. Evaluasi transgenik dilakukan menggunakan teknik PCR. Hasil penelitian memperlihatkan bahwa sintasan yang paling tinggi diperoleh menggunakan media PES (94%), salinitas 30 g/L (90%), pH 7 (96%), intensitas cahaya pada 1.500 lux (80%), fotoperiode 12:12 (84%), komposisi ZPT dengan campuran IAA dan BAP dengan perbandingan 2:1. Hasil analisis PCR memperlihatkan K. alvarezii transgenik putatif mengandung transgen MaSOD sebanyak 78% dari hasil transformasi.Superoxide dismutase transformation (MaSOD) gene of seaweed Kappaphycus alvarezii mediated by Agrobacterium tumefacient has been successfully done in vitro. MaSOD genes introduced into the seaweed genome is expected to reduce oxidative stress caused by environmental conditions such as changes in temperature, salinity and metal contamination of the water. This study aimed to regenerate both the MaSOD transformed seaweed and non-transgenic in a culture flask. Regeneration and multiplication of those seaweed were conducted in a laboratory flask cultures placed in a “culture chamber” which was aerated and enriched with fertilizers PES, Grund, Conwy, and SSW as a control, salinity 20, 25, 30, 35, and 40 g/L, pH: 4, 5, 6, 7, and 8. the light intensity between 500-2000 lux, with light and dark photoperiod 8:16; 12:12; and 16:8. To stimulate the growth of explants the addition of growth hormone IAA and BAP with ratios of 1:1, 1:2 and 2:1 were performed. This study was a multiple-step of process, by which transgenic explants was identified by PCR method. The results showed that the highest survival rate was obtained using media PES (94%), salinity 30 g / L (90%), pH= 7 (96%), the intensity of light at 1500 lux (80%), photoperiod= 12: 12 (84%), and ratio of IAA and BAP 2: 1. The results of PCR analysis showed the putative K. alvarezii transgenic MaSOD was 78% of explants.

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“…Aplikasi transgenesis pada rumput laut (alga) memperlihatkan potensi besar di masa mendatang (Walker et al, 2005;Hallmann, 2007;Potvin & Zhang, 2010). Beberapa penelitian transgenik pada alga yang telah berhasil dilakukan, antara lain: pengujian ekspresi gen β-glucuronidase (GUS) pada Laminaria japonica (Li et al, 2009), gen LacZ pada Gracilaria (Huddy et al, 2012), gen Sitrat sintase pada K. alvarezii (Daud et al, 2013), gen green fluorescent protein (GFP) pada K. alvarezii (Rajamuddin et al, 2014), gen Lizosim pada K. alvarezii (Handayani et al, 2014), dan gen Metallothionein tipe II pada K. alvarezii (Fajriah et al, 2014).…”

Section: Pendahuluanunclassified

“…Proses transfer gen untuk pembuatan K. alvarezii transgenik dapat dilakukan menggunakan metode elektroporasi (Rajamuddin et al, 2014), dan metode perantara A. tumefaciens (Daud et al, 2013;Fajriah et al, 2014;Handayani et al, 2014).…”

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BINARY VECTOR CONSTRUCTION OF KAPPA(κ)-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION

Rajamuddin

Ali

Widyastuti

et al. 2016

J. Ilmu dan Teknologi Kelautan Tropis

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Increasing of kappa (κ)-carrageenan content in Kappaphycus alvarezii seaweed is potentially be achieved by applying transgenesis technology. This study was performed to obtain a construction of κ-Carrageenase gene and Agrobacterium tumefaciens to carry those construction genes. The κ-Carrageenase (κ-Car) gene was involved in κ-carrageenan biosynthesis. The κ-Car gene sequence was ligated between the 35S CaMV promoter and tNos terminator sequences to generate pMSH/κ-Car expression vector. Transformation of pMSH/κ-Car plasmid to Escherichia coli was performed by heat-shock method, and to Agrobacterium tumefaciens by tri-parental mating method. The results showed that several colonies of E. coli and A. tumefaciens grew in the selective culture mediums containing antibiotic. PCR analysis using primers 35S-Forward and tNos-Reverse with DNA template from those bacterial colonies resulted DNA fragment of about 2,000 bp, the same as the total length of 35S CaMV promoter, κ-Car gene and tNos terminator sequences. Therefore, the construction of pMSH/κ-Car gene was succeeded and a colony of A. tumefaciens transformant carrying pMSH/κ-Car plasmid was successfully produced. Keywords: Agrobacterium tumefaciens, kappa(κ)-Carrageenase gene, transgenesis, vector

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

Cited by 4 publications

References 12 publications

Overexpression of Gα Gene Increases Growth and Hyposaline Tolerance in Kappaphycus alvarezii Transgenic Plantlets

Overexpression of Gα Gene Increases Growth and Hyposaline Tolerance in Kappaphycus alvarezii Transgenic Plantlets

REGENERASI DAN PERBANYAKAN RUMPUT LAUT Kappaphycus alvarezii HASIL TRANSFORMASI GEN SUPEROKSIDA DISMUTASE (MaSOD)

BINARY VECTOR CONSTRUCTION OF KAPPA(κ)-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION

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