Cloning And Tissue Expression Analysis Of CDNA Encoding Sox4 Isoforms And Sox7 Gene In Chinese Giant Salamander (Andrias Davidianus)

The Sox (SRY-related HMG box) family of transcription factor is a large gene family that is widely distributed across the animal kingdom and has been subdivided into groups A to K based on their sequence similarity and gene structure. Sox4 belongs to the subgroup C, has key roles in heart outflow tract and valve development, lymphocyte differentiation, neural and glial cell development, endocrine islet formation, osteoblast development. Sox7 belongs to the subgroup F, involved in the regulation of embryonic development and the determination of cell fate. It was shown that Sox7 can induce expression of multiple nodal genes, as well as the expression of genes involved in mesodermal and endodermal differentiation, and is required for Veg-T-induced expression of endodermal markers. The Chinese giant salamander (Andrias davidianus) belongs to one of the most primitive orders of urodele amphibians, the Cryptobranchidae. It is the largest extant amphibian species, and found only in China. In this study, we have successfully cloned the Sox4a, Sox4b, Sox4c and Sox7 cDNA sequence from Andrias davidianus based on the techniques of homology cloning and RACE-RCR, and analyzed the amount of gene expression in different tissues by using fluorescence quantitative PCR technique, the main topics of this paper are as followed:1. Sox4a cDNA seqence was 1153 bp in length, which encoded 224 amino acids and the N-region 59-130aa as highly conservative HMG-box. The putative protein of Sox4a in Andrias davidianus had 63.8%-97.2% identity with other species. We constructed the phylogenetic tree by the amino acids sequences of Sox4a, the result showed that Cyprinus carpio and Acipenser schrenckii split off earlier, Microhyla ornata and Andrias davidianus which belongs to the amphibian in the same branch, while Hypophthalmichthys nobilis and Danio rerio split off at last. This reveals that the evolutionary rate from fish to Andrias davidianus is very fast, and the duplication of these three Sox genes occurred before the divergence of Hypophthalmichthys nobilis and Danio rerio. Sox4b cDNA seqence was 1058 bp in length, which encoded 193 amino acids and the N-region 59-130aa as highly conservative HMG-box. The putative protein of Sox4b in Andrias davidianus had 60.3%-97.1% identity with other species. We constructed the phylogenetic tree by the amino acids sequences of Sox4b, This reveals that Microhyla ornata split off earlier, various fishes then separate, Pelophylax plancyi with Andrias davidianus in the same branch split off at last. Sox4c gene with partial nucleotide fragment was only found in Rana plancyi at present. The putative protein of Sox4c in Andrias davidianus had 81.7% identity with that of Rana plancyi.2. Sox7 cDNA seqence was 847 bp in length, which encoded 188 amino acids and the N-region 44-115aa as highly conservative HMG-box. The putative protein of Sox7 in Andrias davidianus had 64.1%-76.8% identity with other species. We constructed the phylogenetic tree by the amino acids sequences of Sox7, the results showed that Sox7 is an ancient gene which exists in fish, amphibian, reptules and mammal, and Andrias davidianus, Xenopus tropicalis, Xenopus laevis which belong to the amphibian in the same branch, this conform to the course of long-term evolution.3. We obtained 11 members of the Sox gene family in Andrias davidianus which contain Sox2, Sox3, Sox4a, Sox4b, Sox4c, Sox7, Sox9, Soxll, Soxl4a, Soxl4b, Soxl9. Based on the amino acids sequences of these genes, we constructed the phylogenetic tree by Neighbor-joining method to repeat 1000 times in MEGA 5.0, the results showed that Sox4a, Sox4b, Sox4c with Sox11 gathered in the same branch, while Sox9 and Sox7 then separated, the two homologues of Soxl4, Sox2, Sox19 and Sox3 separated finally.4. By fluorescence quantitative PCR analysised the expression of Sox4 and Sox7 in differences tissues of Andrias davidianus. The results indicated that its slight maternal mRNA could be detecte in all tissues, while intestinal maintained a high leve relatively, moderate expression in heart, ovarian, pituitary, and the lowest expression in hepar, brain and kidneys. Sox7 gene was expressed in all tissues, and kidneys maintained a considerable high level, moderate expression in intestinal, heart, brain, ovarian, while the lowest expression in pituitary.