| The immunoglobulin (Ig) isotypes expresssed in different vertebrates are associated with their evolutionary orders:more IgH isotypes emerged in higher jawed vertebrates, showing extensive functional diversification, while the number of IgL isotypes is decreased. From a comparative viewpoint, both Ig isotypes and their expressional mechanisms are dramatically distinct in fish and tetrapods. Being the closest relative of tetrapods, the lungfish appears to be an ideal species to study the Ig gene evolution from fish to tetrapods.In this study, RNA samples of immune associating organs (kidney, intestine, liver and spermary) of the African lungfish, Protopterus annectens, were extracted to construct libraries for transcriptome sequencing. A total of122123Unigenes (575bp in length on average) were obtained. Based on the transcriptome data, three IgMs (IgM1/2/3, sequence identities ranging from52.5%to67.7%), two IgWs (IgW1Land IgW2s, with a sequence identity of65.4%), three IgNs (IgN1/2/3, sequence identities ranging from70.2%to87.6%) and one IgQ were cloned with multiple PCR amplifications. IgN and IgQ are two newly identified IgH isotypes in the lungfish. IgN originates evidently from the IgW in lungfish, although the sequence identities between them are only25.2～29.2%. The phylogenetic tree indicated that IgQ was not closely related with IgW and IgN in the lungfish, but was rather clustered together with IgW/D in other fish. We also showed that in the lungfish,17VH family were used by IgM and IgW2s, and family V and VII were shared by IgM1and IgW2s. However, the remaining V families are not shared by the distinct IgH isotypes, and each V family is only used by a specific IgH isotype. The PCR results showed that VI, VIII were used by IgW1L; VII, IX by IgN1; IX by IgN3. As none of the17VH families were found to be associated with IgN2or IgQ, it is highly likely that there are still other VH families expressed in the lungfish. According to previous studies, all known VH families in vertebrates can be divided into5clans. VH families in clan Ⅰ-Ⅲ belong to tetrapods, members in clan IV belong to teleost and amphibians, and there is no members belonging to birds and mammals in clan V. The phylogenetic tree indicated that11lungfish VH families(Ⅰ, Ⅱ, Ⅴ, Ⅵ, Ⅷ, Ⅹ, ⅩⅡ, ⅩⅢ, XVI, ⅩⅦ) belonged to clan Ⅱ, the others belonging to clan V. These results showed that the lungfish is indeed more closely related with tetrapods than other fish.We also identified7immunoglobulin light chain genes (IgL1～7) in P. annectens for the first time. Screening of the5’RACE library revealed30VL families (LV1～30). The phylogenetic tree of VL classified all IgLs into four clans:σ-cart (IgL1/5/7), σ (IgL6), λ (IgL3/4) and κ (IgL2). Previously, the σ-cart was never found outside the elasmobranch, while the other three IgL isotypes exist in almost all fish. The phylogenetic analysis showed the transitional trait in lungfish VL families. Southern blotting showed the immunoglobulin light chain constant genes existed in single copy in the lungfish genome, while the PCR results indicated that there were germline pre-joined V-J in IgL1and IgL5loci.In summary, in addition to IgM and IgW, this study identified two new IgH isotypes, IgN and IgQ, both of which are specific to the lungfish and evidently originated from IgW. While no IgH genes orthologous to teleost τ/ζ, were found, we also found no evidence supporting the presence of IgY (or IgG and IgE) and IgA (or IgX) encoding genes in this species, strongly suggesting that these IgH isotypes are restricted in tetrapods. Similar to cartilaginous fish, the lungfish also expressed4IgL isotypes. However, further analysis revealed that this species also shared some VL genes with tetrapods, showing a transiting evolution of Ig genes from fish to tetrapods. |
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