A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.

鳄形类（Crocodilians）是演化上独具特色的爬行动物，与蜥蜴亲缘关系较远，且被认为是鸟类的近亲类群。相较于鸟类与哺乳类，学界针对鳄形类免疫球蛋白轻链（Ig light chain）的研究仍较为匮乏。本研究依托扬子鳄（Alligator sinensis）基因组细菌人工染色体（BAC）文库与公开基因组数据，解析了该物种免疫球蛋白轻链基因座（IgL gene loci）的基因组组织结构。扬子鳄存在两种IgL同种型——λ型与κ型，这与卡罗林安乐蜥（Anolis carolinensis）的情况一致。Igλ基因座包含6个Cλ基因，每个Cλ基因上游均带有1个Jλ基因；同时在(Jλ-Cλ)n序列上游存在86个具有潜在功能的Vλ基因。Igκ基因座则仅含1个Cκ基因、6个Jκ基因以及62个功能性Vκ基因。所有VL基因可被划分为总计31个基因家族，其中Vλ家族19个，Vκ家族12个。通过比对哺乳类、鸟类、蜥蜴及蛙类的轻链基因染色体定位，本研究数据进一步证实扬子鳄确实存在两种IgL同种型：Igλ与Igκ。通过对克隆获得的Igλ/κ互补DNA（cDNA）进行分析，本研究发现表达型Vλ与Vκ基因存在显著的家族使用偏好性。对重组VJ接头区域的分析显示，在表达型λ与κ轻链序列中均存在N核苷酸与P核苷酸。对V基因的系统发育分析显示，哺乳类、鸟类、爬行类及爪蟾（Xenopus）共享部分V基因家族，提示这些保守的V基因家族为直系同源基因，并在IgL的演化历程中得以保留。本研究数据显示，扬子鳄的IgL基因库具有极高的多样性与复杂性，该结果为脊椎动物免疫球蛋白基因的演化研究提供了新的视角。