COMPARISION OF B GLOBIN GENE IN THREE FISHES OF CYPRINOID

Fish has many specific characteristics adapted for a variety of physiological needs. Oxygen content in water is not only one of the most important restrictive factors but also one of themost important conditions for the growth of fish. The sensitivity to oxygen is determined by the amino acid sequence of the globin gene. So it is important to try to elucidate the globin gene structure. A pair of consensus degenerate primes was designed basing on N-terminal and C-terminal conservative amino acid of B chain. Primer A is 5cGT (GTAC)GA(ATGC)TGGAC(ATGC) GA(C T) (CG) (AC) (ATGC) GA3c; Primer B is 5cTG(AG) T(CT)T(CG) (CT) CT(ATGC) CA(ATGC) A(AG) (ATGC) GC 3c. Using RT-PCR method, Bglobin cDNA was amplified and cloned from total RNA that was extracted from blood of three fishes that are widely distribution in China (Cyprinus carpio;Ctenopharyngodon idellus; Carassius auratus). The results of the positive clones show the coding sequence is 441 nucleotides in leagth in C. carpio, C. idellus, C. auratus (accession number in GeneBank is AF528161, AF528160, AF528159). The A+ T residues of Bglobin coding sequence of C. idellus, C. carpio, C. auratus is 44. 52%, 44. 29%, and 45. 45% respectively. In order to characterize the structure of theBglobin gene at the DNA level, genomic DNAwas amplified using speci- f ic primers based on cDNA sequence. The result shows that the fragments of Cyprinus carpio; Ctenopharyngodon idellus; Carassius auratusBglobin gene from initiation codon to termination codon is 667bp, 629bp, 667bp respectively. The genomic DNA has 3 extrons and 2 introns which insertion positions are the first splitting codon 30 and the second separating codons 105 and 106. Both introns are extremely rich in A+ T residues. In C. carpio, C. auratus and C. idellus intron 1 is 112bp, 102bp,102bp in length and 65. 59%, 65. 69%, 62. 83% A+ T respectively, whereas intron 2 is 121bp, 121bp, 73bp long and67.77%, 71. 07%, 63. 89% A+ T respectively. The splicejunctions of both introns conform to the GT/AG rule, containing GT at their 5cdonor junctions and AG at their 3c acceptor sites. Globin is species-specific. Globins from different species will have different amino acid sequence and different affinity for oxygen. The three species in this paper belong to cyprinoid. They are closely related. But they differ in their sensitivity to low oxygen content in water, which is linked to the need for dealing with achanging environment or different habitats. So, to study the relationship among the structure of globin gene, affinity for oxygen and hemoglobin is practical significance to the aquatic products industry.