矛尾鱼Hoxa—11基因克隆及5′端序列分析

Hoxa-11基因调节鱼类鳍和四足动物肢的发育，在脊椎动物进化过程中起着重要的作用，利用人和鼠的Hoxa-11基因保守序列设计了两个兼并引物，通过PCR扩增到了矛尾鱼的Hoxa-11基因，经克隆和DNA序列分析，该片段为2065bp，包括绝大部分外显子Ⅰ，内含子和部分外显子Ⅱ，编码204个氨基酸，其氨基酸序列与人、鼠、鸡、蛙和斑马鱼的同源性分别为66.0%、67.6%、74.4%、72.8%和59.7%。外显子Ⅰ的长度从矛尾鱼到蛙、鸡、鼠和人呈现逐步上升趋势，人比矛尾鱼增长了16%，进一步分析，外显子Ⅰ可分为4个区域；两个高度保守区域，1个中度保守区域和1个可变区域，外显子Ⅰ的长度变化主要是由于可变区域内丙氨酸同聚物以及两侧富含甘氨酸和丝氨酸序列的累积。矛尾鱼只有1个由两个丙氨酸组成的同类物，蛙有1个由5个连续丙氨酸组成的同聚物，而鸡、鼠和人有3个丙氨酸同聚物，其中最大的同聚物由7个连续丙氨酸组成，而且在同聚物两侧出现了富含甘氨酸和丝氨酸序列。这表明可变区域可能与脊椎动物进化和鳍-肢转换过程中新功能的获得有关。同源异型盒所在的外显子Ⅱ区和剪接位点是高度保守的。内含子的长度变化较大，但在其内部也发现了两个高度保守的35bp和16bp的DNA片段，这两个片段在人、鼠、鸡、蛙和矛尾鱼中是完全相同的，这些序列的高度保守性提示其功能上的重要性。

Cloning and Sequence Analysis of 5' Fragment of Hoxa-11 Gene in Latimeria chalumnae

Hoxa-11 gene is essential for the development of fish fins and tetrapod limbs. Based on the published nucleotide sequences of human and mouse Hoxa-11 genes, two degenerate primers were designed. Latimeria Hoxa- 11 gene fragment was amplified by PCR, cloned and sequenced. The acquired Hox gene fragment, which encodes 204 amino acids, is comprised of 2 065bp, including most exon 1, intron and partial exon 2. The homology of latimeria Hoxa-11 protein is 66.0% to human, 67.6% to mouse, 74.4% to chick, 72.8% to frog, and 59.7% to zebrafish, respectively. The exon 2 region including the homeobox and the splice site are highly conserved. However, the exon 1 region has increased in size by 16% from latimeria to human. Sequence analysis further revealed that exon 1 of latimeria Hoxa-11 could be divided into four regions: two highly conserved regions, a moderately conserved region, and a variable region adjacent to the intron. The size variation is primarily caused by the accumulation of alanine repeats and of flanking segments rich in glycine and serine in the variable region. It implies that the variable region might be related to acquisition of new functions in the fin-limb transition and vertebrate evolution. Besides the homeobox, two highly conserved regions in exon 1 and two phylogenetic footprints in the intron were found. The strong sequence conservation suggests an important functional role of these regions.