Wahyudi, Yusuf Arif (2018) Respon Molekular Sekuens Nukleotida Reseptor β-aktin dan HSP (Heat Shock Protein) Pada Benih Kakap Putih (Lates calcalifer) Terinfeksi Viral Nervous Necrosis. Magister thesis, Universitas Brawijaya.
Abstract
Penyakit viral nervous necrosis (VNN) atau viral encephalopathy and retinopathy (VER) masih menjadi permasalahan utama bagi perikanan budidaya laut, payau, dan tawar di dunia (Costa and Thompson., 2016). Invasi VNN/VER menyebabkan aktifnya mekanisme pertahanan tubuh ikan (immune respons). Respon imun menyebabkan terjadinya ekspresi gen untuk menghasilkan beberapa gen imun sebagai protein antiviral sehingga dapat menyebabkan ekspresi gen aktin sitoskeleton, mempengaruhi stabilisasi, dan biologi inang, termasuk sintesis protein, seperti HSP (heat shock protein) dalam membantu lipat dan degradasi protein pada saat diubah atau terdenaturasi (Hsp70), juga dalam protein folding, degradasi, transduksi sinyal, regulasi siklus sel, adaptasi seluler jangka panjang (Hsp90). Penelitian ini bertujuan untuk menentukan pengaruh serangan VNN terhadap respon reseptor β-aktin dan HSP organ target benih kakap putih (Lates calcalifer) melalui analisa RT-PCR, nested RT-PCR, dan Sekuensing sehingga dapat menggambarkan virulensi secara kualitatif dan kuantitatif, serta melakukan analisa ekspresi sekuens gen reseptor β-aktin dan HSP dengan membandingkan data pada GenBank. Penelitian dilakukan di laboratorium Balai KIPM Surabaya I selama 3 bulan, Januari – Maret 2018. Metode penelitian yang akan digunakan adalah deskriptif kualitatif. Deteksi menggunakan metode RT-PCR, nested RT- PCR, dan Sequensing. Analisa data sekuensing menggunakan program Mega ver.6 (software) dan dibandingkan dengan sekuens data GenBank. Hasil penelitian yaitu invasi virus RNA VNN pada sampel benih kakap putih (Lates calcalifer) terdeteksi sebanyak 6 (enam) sampel positif dengan menggunakan metode nested RT-PCR pada band 294 bp. Invasi virus RNA VNN pada sampel benih kakap putih (Lates calcalifer) menyebabkan ekspresi gen reseptor β-aktin dan HSP, terdeteksi pada sampel (+) VNN menggunakan metode RT-PCR pada masing-masing band yaitu : β-aktin = 150 bp; Hsp40 = 964 bp; Hsp60 = 652 bp; Hsp90α = 219 bp; Hsp70 = 562 bp; Hsc70 = 148 bp; dan Hsp90β = 229 bp. Ekspresi gen reseptor β-aktin dan Hsc70 juga terdeteksi pada sampel negatif (-) VNN dengan menggunakan metode deteksi RT-PCR pada β-aktin = 150 bp dan Hsc70 = 148 bp. Secara semikuantitatif menggunakan analisa software imageJ, ekspresi gen reseptor β-aktin dan HSP menghasilkan kecenderungan meningkat pada sampel positif (+) VNN dibandingkan sampel negatif (-) VNN. Analisa nukleotida hasil sekuensing pada virus RNA VNN menunjukkan kekerabatan 99% dengan isolate RGNNV dan pola sekuens nukleotida yang terbentuk kearah pola substitusi transisi. Analisa nukleotida hasil xi sekuensing gen reseptor β-aktin dan HSP menunjukkan terjadinya peningkatan pola substitusi kearah substitusi transisi dan pembentukan asam amino hidrofilik/polar, serta adanya kekerabatan atau homologi pola nukleotida hasil ekspresi β-aktin dan HSP pada ikan lainnya data Genbank. Penelitian lanjutan mengeksplorasi penggunaan respon reseptor HSP dan β-aktin sebagai penanda biologis (biomarker) pada berbagai kasus infeksi lainnya, selain infeksi virus RNA VNN. Khususnya penelitian untuk mengeksplorasi secara biomolekuler fungsi pathogenomik dan proteomik sebagai respon antiviral. Penelitian lanjutan untuk membandingkan ekspresi HSP dan β-aktin pada infeksi virus RNA VNN di setiap fase perkembangan ikan, mulai dari benih, remaja, dan dewasa sehinggga diperoleh data yang lebih spesifik dan mendetail.
English Abstract
Nervous necrosis virus (NNV) or viral encephalopathy and retinopathy (VER) disease is still a major problem for marine, brackish, and freshwater aquaculture in the world (Costa and Thompson, 2016). NNV or VER invasion leads to an active immune response mechanism. Immune responses lead to gene expression to produce several immune genes as antiviral proteins that may induce expression of actin cytoskeleton genes, affect stabilization, and host biology, including protein synthesis, such as HSP (heat shock protein) in helping fold and degradation of proteins at the time of change or denatured (Hsp70), also in protein folding, degradation, signal transduction, cell cycle regulation, long- term cellular adaptation (Hsp90). This study aims to determine the effect of NNV invasion on the response of β-actin and HSP receptors of Asian seabass (Lates calcalifer) through RT-PCR, nested RT-PCR, and sequencing analysis. The research was conducted at Fish Quarantine and Inspection Of Surabaya I laboratory for January - March 2018. The research method that will be used is descriptive qualitative. Detection using RT-PCR method, nested RT-PCR, and Sequensing. Analysis of sequencing data using the Mega ver.6 program and compared with the GenBank data sequence. The result of the research is the invasion of NNV on the sample of Asian Seabass (Lates calcalifer) was detected by 6 (six) positive samples by using nested RT-PCR method at 294 bp band. The invasion of the NNV up regulation of expression the β-actin and HSP receptor genes, detected in the positive NNV sample using the RT-PCR method in each band : β-actin = 150 bp; Hsp40 = 964 bp; Hsp60 = 652 bp; Hsp90α = 219 bp; Hsp70 = 562 bp; Hsc70 = 148 bp; and Hsp90β = 229 bp. Expression of the β-actin and Hsc70 receptor genes was also detected in the negative NNV sample by using the RT-PCR detection method. Semiquantitatively using imageJ software analysis, the expression of the β-actin and HSP-receptor genes results in an increasing trend in the positive NNV sample compared to the negative NNV sample. Nucleotide sequencing analysis on NNV showed 99% association with RGNNV isolate and nucleotide sequence pattern formed towards transition substitution pattern. Nucleotide analysis resulted from sequence of β-actin and HSP receptor genes showed an increase of substitution pattern toward transition substitution and hydrophilic / polar amino acid formation, as well as the kinship or homology of nucleotide pattern of β-actin expression and HSP on other fish of Genbank data. Further research to explored the use of HSP and β-actin receptor responses as biological markers (biomarkers) in many other infectious cases, in addition to NNV infection. Especially research to explore the biomolecular pathogenomic and proteomic functions as an antiviral response. Further studies to compare the expression of HSP and β-actin genes in NNV infection in every phase of fish development : fry, juvenile, and adults to obtained more specific and detailed data.
Other obstract
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| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | TES/639.3/WAH/r/2018/041806699 |
| Uncontrolled Keywords: | Nukleotida ReseptorB-Actin, Heat Shock Protein (HSP) |
| Subjects: | 600 Technology (Applied sciences) > 639 Hunting, fishing & conservation > 639.3 Culture of cold-blooded vertebrates |
| Divisions: | S2/S3 > Magister Budidaya Perairan, Fakultas Perikanan dan Ilmu Kelautan |
| Depositing User: | Endang Susworini |
| Date Deposited: | 19 Jul 2022 03:08 |
| Last Modified: | 19 Jul 2022 03:08 |
| URI: | http://repository.ub.ac.id/id/eprint/192266 |
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