| Both morphological and molecular evidence show that amphioxus is the basal chordate and is a crucial organism for understanding the chordate evolution including the origin of the immune system, morphology and development. With the rapid development of comparative immunology, as well as the completion of the Branchiostoma floridae genome sequencing, the attention was focused on amphioxus immune increasingly. Therefore, this study on the basis of Amphioxus EST database, used molecular biology methods to study AmphilKK gene after LPS stimulation mechanism, while analyzed the AmphilKK gene evolution. The main results of the thesis are as follow:(1) Taking advantage of the IKK conserved domain and EST library of amphioxus (Branchiostoma floridae), we searched, amplified and validated a627bp cDNA fragment from amphioxus. Based on the627bp fragment, a480bp and a2543bp fragment were amplified by5’-RACE and3’-RACE respectively. These three fragments (i.e.627bp,480bp and2543bp fragment) were assembled by overlapping to be a3087bp nucleotide sequence representing the complete AmphilKK cDNA. The complete AmphiIKK cDNA was further verified by end to end PCR. The complete AmphilKK cDNA consisted of an open reading frame (ORF) of2334nucleotides, a5’-UTR of164nucleotides and a3’-UTR of589nucleotides with a consensus polyadenylation signal (AATAAA). The ORF encodes a putative protein with777amino acids with a molecular weight of89.3kDa and a theoretical PI of6.26, which shares high similarity with a portion of the genes encoding the IKK family of serine/threonine kinases. This cDNA sequence and deduced protein sequence have been submitted to GenBank (Genbank ID:JQ653961).(2) Analysis of the AmphilKK protein sequence revealed that it had a conserved serine/threonine kinase domain, in which there was a serine/threonine kinase active site and a conserved MAPKK activation loop. Like human IKKa and IKKβ, a potential helix-loop-helix motif, a leucine zipper-like amphipathic a-helix, and a C-terminal NEMO-binding domain were also identified in the amino acid sequence of AmphiIKK. Alignment of the predicted amino acid sequence of AmphiIKK with IKKa and IKKβ sequences from other known IKKs using the MatGAT program revealed that AmphiIKK had the highest identity (43.3%) to Pinctada fucata IKK and shared43.8-64.0%similarity and23.6-43.3%identity to the other known IKK homolog. An unrooted phylogenetic tree was constructed using Bootstrap Neighbor-Joining method of MEGA5.0software. As shown in Fig.2, a clear clade division is observed between invertebrate and vertebrate proteins. Clade Ⅰ included the invertebrate, and may be further classified into two subclades:subclade-Ⅰ, Millipede; subclade-Ⅱ, Mollusk and Chordate. Clade Ⅱ is all vertebrates and can be further classified into two subclades:subclade-Ⅰ, IKKas and subclade-Ⅱ, IKKPs. The AmphiIKK located between millipedes and vertebrates. The high bootstrap values supported the precision of topology.(3) In order to examine whether AmphiIKK was ubiquitously expressed, quantitative RT-PCR was employed to quantify the expression of AmphilKK in different tissues including muscle, gill, intestine, notochord, hepatic cecum and gonad. The result showed that the AmphiIKK gene was constitutively expressed in all tissues tested. It was worth to noting that the expression level of AmphilKK gene was abundant in notochord, moderate in muscles, gills, intestine and hepatic cecum, and low in gonad. To investigate the potential biological role of AmphilKK in amphioxus’s innate immunity, the mRNA level of AmphilKK was measured at different time point after LPS stimulation by quantitative RT-PCR. During the first4hours after stimulation with LPS, we observed about equal fold expression compared to PBS control. After6h stimulation, the expression of the AmphilKK gene was up-regulated and this trend lasted until8h, but almost no difference at the other time point. |
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