Comparison of Variation Growth Hormone (gh) of Gene Sequence in the Salmo Trutta Caspius , Atlantic Salmon and Salmo Trutta

Comparison of variation Growth hormone (GH) of gene sequence in the Salmo trutta caspius , Atlantic salmon and Salmo trutta

¹*Abolhasan Rezaei, ²Sheyda Akhshabi

1*: Department of Genetics-School of basic science, Islamic Azad University Tonekabon Branch,Iran.

2: Young Researchers Club, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran.

Corresponding Author: Abolhasan Rezaei

a.rezaei@tonekaboniau.ac.ir

Mailing Address: Islamic Azad University Tonekabon Branch-Iran. P.O. Box 4864161187, Tonekabon, Iran.

Abstract

The Salmo trutta caspius are used for studies of the Growth hormone (GH) gene, this gene have two major functions, 1: The studies for genetics polymorphism in salmo trutta caspius with other salmonids. 2: The GH gene cause of increasing metabolism and growth body system in fish, especially salmonids. In this research, we found the full length of the growth hormone gene of salmo trutta caspius. In related to, first, we isolated DNA genomics from fin tissue for studies about PCR techniques, then were designed pair of primers from some salmons of GH genes accessed in the Gene Bank. These primers amplified almost, fragments of 810, 310 and 920 bp. The fragments after purification on the gel electrophoresis were sent to the sequencing. The fragment sequences were compared between salmo trutta caspius, Atlantic salmon and salmo trutta by the GH gene. The results are shown that there is a high homology between Atlantic salmon (97%), and low homology between salmo trutta (23%).

Introduction:

The family Salmonidae comprises 11 genera and includes salmon, trout, charr, freshwater whitefishes, ciscos and graylings (Nelson, et al., 2006). The Salmons are important for aquaculture and considerable of economics. Furthermore Salmonids are most of the main food in Asia, especially Iran, in Europe contain most of the countries and America and Canada(Davidson et.al., 2010), Salmo trutta caspius species are living in the Caspian Sea. This species is very rarely in the world. Because salmo trutta caspius is very sensitive to pollution in the water. The adult female salmons also for spawning should be travel to fresh water. So, is very existence study and researches on the salmo trutta caspius. In related to we examined about polymorphism of the salmo trutta caspius and other salmonids for getting result amount of the relationships between salmo trutta caspius and other salmons. In the Europe, the Middle East, western Asia, and parts of North Africa. From north to south, its range extends from northern Norway and north-eastern Russia, to the Atlas Mountains of North Africa. Other trout species have been also investigated. Some major scientific questions can be explored using salmonid genomes. The a lot of genes can be investigated to salmons such as mitochondrial DNA (Karlson et. al., 2009), microsatellites (Williamson et al., 2002) and growth hormone genes.(Gross et al., 1995 and 1996. Rayynanen et al.,2004. Agelon et al., 1988). In related to, we examined growth hormone (GH) gene for detection polymorphism and common ancestor in Salmons.

The GH gene of the Atlantic salmon (Salmo salar) and Salmo trutta was selected to be the reference sequence for all salmonids on the basis of its importance for the aquaculture industry and because so much previous work has been carried out at the GH genes on this species.

The GH gene in solminds are two types, type 1 and 2, resulting from their polyploid ancestry (Agellon et al., 1988; Rentier-Delrue et al., 1989.. Forbes et al. _1994) found sequence of the full length length of GH gene in Salmo trutta caspius (Accession number, JN241634.1).(Rezaei et al., 2011 a,b)

We aligned GH gene in Salmo trutta caspius with Atlantic salmon and Salmo trutta, by the NCBI Network system program. Results are shown there is a high homology with Atlantic salmon (97%), but there is a lower homology with Salmo trutta intron C, intron D, GH1 and intron d GH2 . However we cannot get exactly result from Salmo trutta, because there was not full length of GH gene of Salmo trutta in the Gene Bank.

The objectives of this study were to study polymorphism within Salmo trutta caspius populations and amount of relationships with Atlantic salmon and Salmo trutta, moreover, the capability transforming GH gene in salmons for our future planning.

Materials and Methods

The Salmo trutta caspius used the experiment originated from two rivers in Iran (river of Dohezar and river of Sardabroud in north of Iran). These fishes had been 3 years age old. They were anesthetized with MS2220 and extracted of blood from caudal vein. Blood samples (2-5 ml) were removed from the fish via caudal puncture (G.18 needle) using a heparinized syringe (as an anticoagulant for blood sampling) after using MS222 (1:10,000) as an anesthetic to minimize stress.

Process of designing primers: For designing primers we had to use some sequences from Gene Bank, NCBI Network system. In related to, we thought this sequences has been high homology with Salmo trutta caspius, because Atlantic salmons and also other bony fishes have same shape and morphology with salmo trutta caspius, therefore we had risk and was synthesized primers. Also, these sequences were around 4700 nucleotides, so, for getting high performance, we designed primers some part of the full length of GH gene of Atlantic salmon by the NCBI Network program and DNAMAN genetics program. These primers amplified 330, 850, 910 bp. from first to end of the GH gene of Salmo trutta caspius.

The PCR programs: The PCR reaction used 10 microgram PCR reactions contained: 1 µl template DNA, 2 µl forward primer (100ng/µl), 2 µl reverse primer (100 ng/µl), 2 µl dNTP mix (2.5mM each), 5 µl 10X ChromTaq Assay buffer, 0.5 µl ChromTaq enzyme (3U/µl), Water 37.5 µl, in a total volume, 50 µl. 94o of 5 min, 35 cycles of 94oC 30 Sec., 55oC 30 Sec., and 72oC 1 min. Two to ten µl of each PCR reaction were run on 1 % agarose gels in TAE buffer containing ethidium bromide. One µl 500bp, DNA ladder (Gibco-BRL) was used as a size standard. Then the PCR products after purification by the Chromous kit purification were sent to the Choromous Geni Company-India for doing sequence.

Results

Analysis of the polygenetics of GH gene in the salmo trutta caspius

The primers were designed to four segments of the GH gene, because full length of the GH gene in the Atlantic salmon has been around 4700 nucleotides. The fragments contain of the exons and

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