|
|
|
| Cloning, Molecular Polymorphism and Expression Pattern of β2m Gene in Nile Tilapia (Oreochromis niloticus) |
|
|
|
|
Abstract β2-microglobulin (β2m) is one of the subunits of classⅠmajor histocompatibility complex (MHCⅠ) molecular, which binds non-covalently with the α-chain of the MHC-Ⅰ molecular. Based on the RT-PCR and RACE-PCR, full-length cDNA of β2m was cloned from Nile tilapia (Oreochromis niloticus). In addition, cDNA polymorphism and the mRNA expression pattern were analyzed. The results showed that the two β2m cDNA was 900 and 906 bp in length, respectively, including an open reading frame (ORF) of 351 bp encoding a polypeptide of 116 amino acids. The 5'-UTR and 3'-UTR was 68 nts and 486(492) nts respectively. The two cDNA had different genomic sequence, but they all had 3 exons and 2 introns. The putative protein of the 2 β2m had 92.3% identity. The putative amino acid sequence of β2m had 39.20%~89.8% identity with those of other teleost and mammals. The putative protein of the 2 β2ms exhibited the highest identity with zebra mbuna (Maylandia zebra) (89.80% and 84.7%, respectively). The 2 β2ms had 63.2% and 60.70% identity with catfish (Ictalurus punctatus) respectively. And β2m-2 had the lowest identity with chicken (Gallus gallus) and mouse (Mus musculus)(39.50% and 39.20%, respectively). Predictprotein online analysis found that the 2 β2m all had the typical immunoglobulin(Ig) and major histocompatibility complex protein (MHC) signature YxCxVxH. The phylogenetic tree showed that the β2ms of Nile tilapia clustered with zebra mbuna (Maylandia zebra) first, then clustered with orange-spotted grouper (Epinephelus coioides), large yellow croaker (Pseudosciaena crocea), and seabass (Dicentrarchus labrax), and then clustered with other teleost and mammals. In addition, the rainbow trout (Oncorhynchus mykiss), catfish (Ictalurus punctatus), zebrafish(Brachydanio rerio), and grass carp (Ctenopharyngodon idella) formed a subcluster. The Atlantic cod(Gadus morhua), red seabream(Parus major), and flounder (Paralichthys olivaceus) formed another subcluster. The chicken (Gallus gallus), human (Homo sapiens) and mouse (Mus musculus) formed a separate cluster. Six individuals were used to analysis the cDNA polymorphism. 56 valid sequences were obtained from 60 positive clones (10 positive clones per individuals). And 2~4 cDNA sequences per individuals were obtained. The 56 valid sequences code 6 different cDNA sequences. The 6 cDNA sequences could be divided into 2 different types, and each type had 3 subtype cDNAs. Six cDNA were named as Orni-β2m 0101, Orni-β2m 0102, Orni-β2m 0103, Orni-β2m 0201, Orni-β2m 0202 and Orni-β2m 0203 respectively. To examine the basal expression of β2m, qRT-PCR analysis was carried out in gill, brain, intestine, liver, kidney, stomach, spleen, heart, muscle and skin. To eliminate the individual variation, six fishes were sampled and analyzed separately by qRT-PCR, and their mean values were considered. The results showed that the β2m was detected in all the examined tissues. And the highest expression level of β2m was detected in kidney, spleen and heart, and higher expression in intestine and gill, whereas the lowest expression was detected in muscle and skin. The expressions level of β2m mRNA in the gill, kidney, heart and spleen of Nile tilapia increased first, and then decreased when challenged with Streptococcus agalactiae. In the infected fish gill, kidney, heart and spleen, β2m mRNA expression was increased at 1 day post infection (dpi), and touched the peak at 3 dpi. Then the expression declined at 6 dpi, and at 9 dpi the induction declined to the basal level in kidney and spleen. The results showed that the β2m may play important roles in immune response of Nile tilapia. This observation can contribute to further investigation of immune regulation of nile tilapia，and the results will give us a better understanding for the physiologyical role of β2m in fish.
|
|
Received: 29 February 2016
Published: 06 August 2016
|
|
|
|
|
|
|
