3种鲤科鱼β珠蛋白基因的比较研究

COMPARISION OF β GLOBIN GENE IN THREE FISHES OF CYPRINOID

根据 β珠蛋白的N末端和C末端氨基酸序列的保守性设计 2 0bp长的简并引物 ,用RT PCR方法扩增并克隆了鲤、草鱼、鲫的 β珠蛋白基因cDNA。结果显示克隆 3种鱼的 β珠蛋白基因cDNA全长为 4 4 1bp。对它们所编码的氨基酸序列的比较可以看出虽然它们都属于鲤科但氨基酸的组成却有较大的差异。根据所克隆的cDNA分别设计特异引物用PCR方法克隆了 3种鱼的基因组DNA全长 ;从起始密码子到终止密码子的长度分别为鲤 6 6 7bp、草鱼6 2 9bp、鲫 6 6 7bp。 3种鱼中均含有 3个外显子和 2个内含子且内含子的插入位置相同。内含子的碱基序列差异很大。鲫与鲤内含子 1和 2的长度完全相同 ,而与草鱼的内含子长度则相差较大。

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 the most 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 β chain. Primer A is 5′ GT (GTAC)GA(ATGC)TGGAC(ATGC)GA(CT)(CG)(AC)(ATGC)GA3′;Primer B is 5′TG(AG)TA(CT)T(CG)(CT)CT(ATGC)CA(ATGC)A(AG)(ATGC)GC 3′.Using RT PCR method, β globin 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 β globin 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 the β globin gene at the DNA level, genomic DNA was amplified using specific primers based on cDNA sequence. The result shows that the fragments of Cyprinus carpio; Ctenopharyngodon idellus; Carassius auratus β globin 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 and 67 77%, 71.07%, 63.89% A+T respectively. The splice junctions of both introns conform to the GT/AG rule, containing GT at their 5′ donor junctions and AG at their 3′ 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 a changing 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.