Rengaraj Deivendran, Kim Duk Kyung, Zheng Ying Hui, Lee Sang In, Kim Heebal, Han Jae Yong
Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea.
Biol Reprod. 2008 Sep;79(3):413-20. doi: 10.1095/biolreprod.108.067959. Epub 2008 Apr 30.
Tissue-specific novel transcripts expressed during sexual development were examined by RT-PCR, quantitative RT-PCR (qRT-PCR), and in situ hybridization to provide data for chicken genomics. Public databases for transcript data have been constructed with known and unknown sequences of various tissues from different animals. However, the expression patterns and functions of the transcripts are less known. From the The Institute for Genomics Research Gallus gallus library, we examined 291 tentative consensus (TC) sequences that assembled 100% with transcripts by RT-PCR during male and female sexual development from Embryonic Day 6 to 25 wk of age. We found 85 TC sequences that were specific to testicular development; of these, 43 TC sequences were exclusively upregulated in 25-wk-old testis. Another 52 TC sequences were not specific to one tissue, but occurred in the testis and ovary at different developmental ages. Twelve testis-specific TC sequences upregulated in 25-wk-old testis were randomly selected and further examined with qRT-PCR. For precise localization, these 12 testis-specific TC sequences were examined by in situ hybridization with 25-wk-old adult testis. Six TC sequences were strongly expressed in secondary spermatocytes and haploid spermatids until spermatozoa release. Another six TC sequences were differentially expressed in the adluminal compartment of seminiferous tubules. Among the testis-specific TC sequences, TC120901 is a known gene, phospholipase C, zeta (PLCZ1). Our data provide potential insight into gene expression and genomic information on novel transcripts that are important to avian reproduction.
通过逆转录聚合酶链反应(RT-PCR)、定量逆转录聚合酶链反应(qRT-PCR)和原位杂交技术,对性发育过程中表达的组织特异性新转录本进行了检测,以提供鸡基因组学的数据。已构建了包含不同动物各种组织已知和未知序列的转录本数据公共数据库。然而,这些转录本的表达模式和功能尚不清楚。从基因组学研究所的原鸡文库中,我们检测了291个初步一致性(TC)序列,这些序列在胚胎第6天到25周龄的雄性和雌性性发育过程中通过RT-PCR与转录本100%组装。我们发现85个TC序列对睾丸发育具有特异性;其中,43个TC序列仅在25周龄睾丸中上调。另外52个TC序列并非特定于一个组织,但在不同发育阶段的睾丸和卵巢中均有出现。随机选择了12个在25周龄睾丸中上调的睾丸特异性TC序列,并通过qRT-PCR进一步检测。为了精确定位,用25周龄成年睾丸进行原位杂交检测这12个睾丸特异性TC序列。6个TC序列在次级精母细胞和单倍体精子细胞中强烈表达,直至精子释放。另外6个TC序列在生精小管的近腔室中差异表达。在睾丸特异性TC序列中,TC120901是一个已知基因,即磷脂酶Cζ(PLCZ1)。我们的数据为对鸟类繁殖重要的新转录本的基因表达和基因组信息提供了潜在的见解。