摘要
ants(adenine nucleotide translocators),又称为ATP/ADP载体,利用Genefishing差异筛选技术对普通鲤鱼和德国镜鲤的皮肤转录产物进行筛选,得到了其中一个ant基因的部分片段,利用RT-PCR技术对皮肤中的ants基因进行钓取,得到6种不同的CDS序列。利用CLUSTALW对这6条翻译后的序列进行比对,发现这6条序列编码4种不同的氨基酸序列,表明ants基因至少有6种不同的转录产物(isoforms),但是在蛋白质水平上可能存在4种不同的翻译产物。利用NCBI上的blast对氨基酸序列进行同源性检索发现这4个蛋白产物与斑马鱼ant2最为相似,其相似度分别达到97%,97%,96%,98%。对处于不同分类地位的物种的ants基因与鲤鱼的4个ants表达产物进行进化树分析发现,这4个蛋白产物在93/100再现性水平上明显聚类为ant2,说明皮肤中表达的这些ants基因很可能是ant2。原位杂交结果显示,ant2在受精后4.5 h和6 h不表达,从受精后10 h开始在外包处强烈特异表达,在受精后12 h的胚胎中很弱表达,在受精后24 h的体节中特异强烈表达,在受精后48 h胚胎无表达,但是在着生鳞片的部位特异强烈表达,而在皮肤的其他部位则表达较弱或者不表达。以上的结果表明,ant2基因可能是通过不同的转录产物在精细调控鳞片的发育。
ants （ adenine nucleotide translocator ） , also called ATP/ADP carrier, mediates the exchange of ADP and ATP across the inner mitochondrial membrane, thus playing an essential role in energy metabolism in eukaryotic cells. Genefishing differental display Technique was used to screen different expression products in the transcript level in common carp and germany mirror carp, and obtained a partial sequence of ants gene. To fish for all the expressing ants products in the skin of common carp, RT-PCR was performed, and we obtained 6 different CDS sequences. CLUSTALW was applied to align the translated amino acid sequences and discovered that these 6 CDS can code 4 different amino acid sequences, indicated that ants genes have at least 6 different transcripts, but have 4 different protein products in protein level. Aligned these 4 different amino acid sequences and that of the zebrafish ant2 using blastp on NCBI, found that the percent identities were 97%, 97%, 96%, 98%, respectively. A phylogenetic tree of ants isoforms from common carp and other species located in different taxonomy status was constructed, and showed that these 4 protein products were obviously classified as ant2 with a 93/100 reproducibility, so the ant gene expressed in skin are likely to ant2. The result of Wholemount in situ hybridization revealed that ant2 is intensively expressed specifically in epiboly （ 10 hours after fertilization ） , somite （ 24 hours after fertilization ） and the regions where the scales were developing in skin, but in other regions weak and dispersed expression were detected. According to the above results, we can infer that ant2 likely subtly regulated the development of the scales via different transcripts （ isoforms ） .
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第3期120-128,共9页
Biotechnology Bulletin
基金
中央公益级基本科研业务费项目(2011C015
2010C017)
国家公益性行业(农业)科研专项项目(200903045)
863计划项目(2011AA100401)
"948"项目(2011-G12)
