Cloning And Tissue Expression Analysisof CDNA Encoding FoxA2?FoxC2 And FoxL2 Gene In Chinese Giant Salamander (Andrias Davidianus)

Forkhead-box (winged helix/forkhead) genes generally exist in eukaryotes. According to similarity of the FH domain. Fox gene family is divided into 19 subfamilies from FoxA to FoxS. The conservatism of the structure of FH does not affect the functional diversity of Fox gene, Forkhead-box(Fox) transcription factors through Post-transcriptional modification to produce specific target subset respond to different environmental conditions. Nowadays, the study of fox gene family has been proved to be related to human diseases, and it has become a research hotspot. FoxA2 gene, considered as a switch of genetic control, plays a crucial role in regulating organs like hepar's and pancreas'development and many body metabolic pathways. FoxC2 is an important transcription factor in regulating vasculature like the blood vessel and lymphatic growth and development. Recent studies have shown that FoxC2 plays an important role in the development of several types of cancer through promoting the proliferation of tumor cells and enhancing the metastatic potential of malignant cells. FoxC2 is also known as a major gene linked to lymphedema-distichiasis syndrome. FoxL2, playing a crucial role in female reproduction, especially in differentiation and proliferation of granulosa cells, follicular development and maintenance of ovarian function, can be used as a sensitive marker gene of early ovary development. FoxL2 is known as a major gene linked to blepharophimosis-ptosis-epicanthus inversus syndrome. In this study, FoxA2 cDNA sequence and FoxC2,FoxL2 gene sequences of Chinese giant salamander are cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends(RACE), and their biological characteristics are also analyzed.Analysis of Real-time PCR is performed to examine the expression in various tissues of the Chinese giant salamander. We have obtained the following results:1. Chinese giant salamander FoxA2 cDNA sequence contains 1574bp and encodes 429 amino acids encompassing a FH domain of 91aa. It has been deposited in Genebank with Accession No.KU974963. Gene similarity analysis shows that Chinese giant salamander FoxA2 is highly similar to amphibian but low similar to mammalian at the both nucleic acid and putative amino acid sequence level. The phylogenetic tree shows that the Chinese giant salamander are in a unique evolutionary position, which is gathered together with African clawed frog and tropical clawed frog, neighboured to the bony fish clade.2. Chinese giant salamander FoxC2 gene sequence contains 1841 bp and encodes 474 amino acids encompassing a FH domain of 91 aa. It has been deposited in Genebank with Accession No.KU315724. Gene similarity analysis shows that Chinese giant salamander FoxC2 is highly similar to amphibians but low similar to bony fish and mammalian. The phylogenetic tree shows that spotted gar falls into amphibian clade, neighboured to the Chinese giant salamander group. It indicates that FoxC2 gene evolution of Chinese giant salamander has close relations with bony fish.3. Chinese giant salamander FoxL2gene sequence contains 1296bp and encodes 300 amino acids encompassing a FH domain of 91 aa. FoxL2 sequence has been deposited in Genebank with Accession No.KU310892. Gene similarity analysis shows that Chinese giant salamander FoxL2 is highly similar to amphibian at the both nucleic acid and amino acid sequence level. FoxL2 C-terminal region as transcriptional activation domain still is highly conserved. Biological characteristics analyses show that FoxL2 has many phosphorylation sites which may correlatively associate with transcriptional activation in eukaryotes. The results of phylogenetic analysis show that the Chinese giant salamander form single groups from a big group including amphibians and bony fishes, which exhibits the ancient evolutionary status of Chinese giant salamander FoxL2 gene.4. This paper also makes the expression profile analysis in the Chinese giant salamander's different tissues on the basis of the FoxA2?FoxC2 and FoxL2 gene by Real-time PCR. The results indicate that FoxA2 gene's protein expression quantity is the highest in lungs, followed by that in liver, intestine, ovaries, and almost no expression in testis, spleen, muscle and kidney. However, FoxC2 gene's protein expression quantity is highest in heart and kidney, followed in testis, muscle, lung, ovary and no expressed in the liver, spleen and intestine. FoxL2 gene's protein expression quantity is higher in ovary than in kidney, followed in muscle, testis and lung, and no expression in liver, spleen and intestines.