Cloning And Analysis Of Megalobrama Amblycephala Retinol Binding Protein Related Genes

Megalobrama amblycephala commonly known as wuchang fish, Cypriniformes, Cyprinidae, Megalobrama, is only distributed in the middle and lower reaches of Yangtze river region, such as Liangzi Lake, Yuni Lake and Poyang Lake. It is an important economic species of freshwater fish due to its rapid growth, strong disease resistance, optimum temperature range, less illness, easily catching, good tastes and rich nutrition. In recent years, more and more researches about M. amblycephala have been carried out. This research mainly includes cloning of retinol-binding protein gene, analyze the related gene expression in different developmental stages and different organizations of M. amblycephala. The main results are as follows:1. Cloning and analysis of M. amblycephala rbp4Genomic DNA (4915 bp) and full-length cDNA (815 bp) sequences of M. amblycephala rbp4 were obtained. Further comparative analysis between genomic DNA and cDNA sequences showed that the gene contained five exons and four introns, and its splice sites conformed to the GT/AG rule. The full-length cDNA consisted of 34-bp 5’-untranslated region,202-bp 3’-UTR including a poly-A signal sequence, and 579-bp open reading frame (ORF) encoding 192 amino acid residues. By comparing rbp4 amino acids sequences from the different species, it was found that M. Amblycephala showed higher homology with Cyprinus carpio (80%), Clarias batrachus(93%), Danio rerio (88%), Oncorhynchus mykiss (82%), Tukifugo rubripes (71%), while much lower homology with Chelonia my das (64%), Felis catus (59%), Bos taurus (58%) and Homo sapiens (58%).2. Temporal and spatial expression patterns of M. amblycephala rbpQuantitative RT-PCR were performed, and the results of temporal and tissue expression pattrns of rbplb, rbpla, rbp4 genes showed that there had differences in three kinds of rbp gene expression both in different tissues and developing periods. Rbplb had high expression in kidney and spleen, but was extremely low or not expressed in intestine and eyes; Rbp2a and rbp4 were both highly expressed in liver, rbp2a was expressed at low level in intestine but not in the eye; Rbp4 was expressed at low level in the eye but not in intestine. In addition, the expression of the same gene in different development periods also had differences:the rbplb had a continuing expression in early embryonic development of M. amblycephala, and continued to rise after hatching stage; the expression of rbp2a and rbp4 were extremely low or not expressed in the early embryonic development.3. Cloning and sequence analysis of the rbp4 promoterThermal asymmetric interlaced (TAIL) PCR as an efficient and useful method was used to isolate the promoter sequence of the rbp4 from M. amblycephala. Rbp4 SNPs located in the promoter were detected in three different geographic population samples, and two SNP sites are found,-385A>G and -329-C> T. After CRS-PCR (created restriction site PCR) products were amplified and digested, the gene and genotype frequency was calculated in three geographic populations.4. Effect of 9-cis-retinoic acid on promoter activityTwo recombinant vectors carrying two haplotypes,385G-329T and 385A-329C, were constructed and transfected into cells. Luciferase activity analysis showed that 9-cis retinoic acid (9-cis-RA) increased the promoter activity of both pGL3-385A-329C and pGL3-385G-329T. And, luciferase activity increased gradually and peaked at 1 μM of 9-cis-RA in cells transfected with pGL3-385G-329T, while reached the highest level at 100 nM in cells carrying the pGL3-385A-329C.