Kajian Pola Ekspresi Hrgfp Sebagai Gen Pewarta Pada Embrio Dan Larva Ikan Koi (Cyprinus Carpio Var. Koi)

Luthfiyah, Sa’idah (2017) Kajian Pola Ekspresi Hrgfp Sebagai Gen Pewarta Pada Embrio Dan Larva Ikan Koi (Cyprinus Carpio Var. Koi). Magister thesis, Universitas Brawijaya.

Abstract

Integrasi serta ekspresi DNA transgen yang diintroduksikan dapat dilihat dengan mengetahui pola ekspresi dari hrGFP sebagai gen pewarta pada saat embriogenesis hingga menjadi larva. Pola ekspresi ini lebih dikenal dengan pola ekspresi sementara (transient expression) dan dari ekspresi inilah dapat diketahui DNA tersebut terintegrasi atau tidak. Oleh karena itu, fokus dari penelitian yang akan dilakukan adalah mengetahui pola ekspresi sementara dari hrGFP sebagai gen pewarta dan dari pola ekspresi tersebut dapat dijadikan sebagai sumber informasi apakah penggunaan hrGFP tersebut dapat mengintegrasikan DNA asing kedalam genom ikan koi atau tidak agar pembuatan konstruksi DNA asing yang membawa sifat genetik tertentu dapat mudah diintroduksikan kedalam ikan koi dan didapatkan ikan koi dengan genotip yang unik sesuai dengan yang diinginkan. Metode yang digunakan dalam penelitian ini yakni metode eksperimen. Metode ini digunakan untuk mencari fakta dan hubungan sebab akibat dari suatu objek yang diteliti sehingga didapatkan hasil yang konkrit. Rancangan penelitian yang akan digunakan adalah rancangan acak lengkap. Perlakuan yang dilaksanakan yakni elektroporasi sperma dengan voltage yang berbeda : 10 V, 20 V, dan 30 V dengan 3x ulangan dan dengan konsentrasi DNA 10 ng/μL. Hasil dari elektroporasi nantinya akan dilihat pola ekspresinya pada saat embriogenesis hingga larva sehingga secara kualitatif dapat diketahui kapan ekspresi sementara mulai muncul hingga hilang kembali dan secara kuantitatif dengan melihat nilai intensitas pendaran GFP yang terlihat pada masingmasing perlakuan. Selanjutnya data hasil dari penelitian tersebut akan dianalisa dengan menggunakan analisa sidik ragam dan dilanjutkan hingga uji BNT dan Uji polinomial orthogonal apabila didapatkan hasil sidk ragam yang berbeda nyata. Penelitian ini dilaksanakan di Laboratorium Sentral Ilmu Hayati (LSIH) ; Laboratorium Breeding dan Reproduksi Ikan Fakultas Perikanan dan Ilmu Kelautan Universitas Brawijaya, Malang pada bulan Oktober 2016 – Mei 2017. Berdasarkan hasil penelitian diketahui bahwa perlakuan kuat medan listrik (voltase) yang berbeda memberikan hasil yang berbeda nyata terhadap motilitas, dan viabilitas sperma, namun tidak berbeda nyata pada fertilitas, daya tetas , interaksi hrGFP pada sperma dan interaksi hrGFP pada embrio maupun Larva. hrGFP terlihat berinteraksi dengan sperma pada semua perlakuan kecuali kontrol meskipun dengan tingkat intensitas pendaran yang kecil. Intensitas pendaran tertinggi pada sperma dengan perlakuan A sebesar 38,48. Tertinggi pada fase Organogenesis yakni pada perlakuan A (10V) sebesar 164,452 arbitary. Namun, Pada Larva tertinggi pada perlakuan B (20) sebesar 325,574 arbitary. Pola ekspresi transient pada ikan koi dengan menggunakan hrGFP berbeda dengan spesies lain, Pola ekspresi hrGFP mulai terlihat secara nyata pada fase blastula dan semakin stabil pada Larva. Artinya, hrGFP efektif digunakan sebagai gen pewarta pada kegiatan transgenesis koi.

English Abstract

Integration and foreign DNA expression that are introduced can be seen by knowing the expression pattern of hrGFP as the reporter gene at the time of embryogenesis to become larvae. This expression pattern is better known as the transient expression and from this expression can be known that the DNA is integrated or not. Therefore, the focus of this research is to know the temporary expression pattern of hrGFP as the reporter gene and from that expression pattern can be used as a source of information whether the use of hrGFP can integrate transgen DNA into koi’s genome or not. so making construction of transgenic DNA that carrying certain genetic properties can be easily introduced into koi fish and can get the koi with unique genotypes that are sought. The method that is used in this research is experimental method. This method is used to find facts and causal relationships of an object studied so that obtained concrete results. The research design that is used is complete randomized design. The treatments are sperm electroporation with different voltages: 10 V, 20 V, and 30 V with three times repeated and with 10 ng/μL DNA concentration. The result of electroporation will be seen the expression pattern at the time of embryogenesis until larvae so that qualitatively can be known when the temporary expression began to emerge until lost again and quantitatively by looking at the intensityof luminescence GFP seen in each treatment. Further data of this research will be analyzed by using analysis of variance and continued until BNT test and orthogonal polynomial test if the results obtained a significant variance This study was conducted at the Central Laboratory of Life Sciences (LSIH); Breeding and Reproduction Laboratory of Fisheries and Marine Science Faculty – Brawijaya University, Malang on October 2016 - May 2017.Based on the research, it was known that the different electric field treatment (voltage) gives different results on motility, and sperm viability, but not significantly different on fertility, hatching rate, hrGFP interaction in sperm and hrGFP interaction in embryo and larvae. hrGFP was seen interacted with sperm at all treatments except the control although with a small level of luminescence intensity. The highest luminous intensity was in sperm with treatment A with 38,48 arbitary. Highest at the phase of Organogenesis that was in treatment A (10V) with 164,452 arbitary. However, the highest at larvae was in treatment B (20) with 325,574 arbitary. The pattern of transient expression in koi fish using hrGFP was different from other species, hrGFP expression pattern starts to be noticeable in the blastula phase and more stable in larvae. That is, so based on this research it was known that hrGFP was effectived in expressing GFP as repoter gene.

Item Type:	Thesis (Magister)
Identification Number:	TES/597.483/LUT/k/2017/041705661
Uncontrolled Keywords:	GREEN FLUORESCENT PROTEN, CYPRINUS, FISHES - GENETIES, FISHES - EMBRYOS
Subjects:	500 Natural sciences and mathematics > 597 Cold-blooded vertebrates > 597.4 Miscellaneous superorders of Actinopterygii > 597.48 Cypriniformes > 597.483 Cyprinus carpio (Common carp)
Divisions:	S2/S3 > Magister Budidaya Perairan, Fakultas Perikanan dan Ilmu Kelautan
Depositing User:	Nur Cholis
Date Deposited:	31 Jul 2017 08:46
Last Modified:	13 Dec 2020 09:47
URI:	http://repository.ub.ac.id/id/eprint/895

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