選殖豬母源性胚胎拉鏈狀白胺酸蛋白質激酶之調控區域及其特性分析

Abstract

早期豬胚胎發育過程中，以桑椹胚往囊胚的轉換為最重要的時期，不同基因在此時期也有不同的表現。利用 cDNA 微陣列及定量 PCR 在此時期驗證了十三個差異表現的候選基因，其中母源性胚胎拉鏈狀白胺酸蛋白質激酶 (maternal embryonic leucine zipper kinase, MELK) 在豬基因組中仍未被報導。了解這些基因的調控機制可用以改善豬胚的發育。本研究主要目的為選殖候選基因調控序列並驗證其活性，並且探討 MELK 基因受何種因子調控。 在豬、人、鼠及牛不同物種進行序列比對來預測出調控序列位置並選殖出 MELK (453 bp)、ataxia-telangiectasia mutated protein (1067 bp)、F-box protein 32 (1133 bp) 及 beta-2-microglobulin (556 bp)，並藉由報導基因於體外驗證其活性。藉由序列長度連續截切找出 MELK 啟動子上重要的調控區域，並使用線上軟體 (Genomatix) 預測所有轉錄因子結合位並找出在 -129 bp 至 -56 bp 為重要的基本轉錄區域及 -348 bp 至 -272 bp 為具正向調控之區域。為了釐清這些轉錄因子結合位對於轉錄活性的影響，我們用點突變方式構築四種不同區段轉錄因子突變之質體，此結果顯示點突變在重疊的 estrogen response element、steroidogenic factor binding element 及 RXR heterodimer binding site 上；MEF 3 binding site 與 heat shock factor binding site 上或 TATA-box 都顯著地降低啟動子的表現活性。然而，這群轉錄因子結合位仍待後續研究如：染色質免疫沉澱 (chromatin immunoprecipitation, CHIP) 或膠體電泳位移分析 (electrophoretic mobility shift assay, EMSA) 證實之。 總括而言，我們選殖了多個候選基因之啟動子序列並確認其活性，亦完成這群候選基因啟動子構築的螢光表現載體，未來可應用於胚胎的顯微注射研究其在活體的表現情形。在最後我們分析了 MELK 啟動子序列並於調控區域內找到數個具活性的轉錄因子結合位，推測其可正向調控 MELK 下游基因的表現。

Transition from morula to blastocyst is a critical step in early embryonic development of the pigs. This process coincides with the change in gene expression pattern. Thirteen differentially expressed candidate genes during this period were previously identified by cDNA microarray and quantitative PCR, among which the gene of maternal embryonic leucine zipper kinase (MELK) remains novel in the porcine genome. A better understanding of the regulatory mechanism of the gene may provide tools to improve the successful development of porcine embryos. The purpose of this study is to clone and verify the promoter regions of these candidate genes, specifically focusing on MELK which was previously shown in other species to regulate cell proliferation and cell-cycle progression. Sequence alignment among porcine, human, murine and bovine to predict the promoter regions of MELK (453 bp), ataxia-telangiectasia mutated protein (1067 bp), F-box protein 32 (1133 bp) and beta-2-microglobulin (556 bp), and reporter analysis in vitro demonstrated their activities. Serial deletion analysis was applied to specifically investigate the regulatory activity of MELK promoter. The on-line prediction software (Genomatix) predicted several putative transcription factor binding elements present in this region with -129 bp to -56 bp harboring the basal transcription activity and also some other positive regulatory elements located from -348 bp to -272 bp. To elucidate their roles in transcriptional regulation, we created four constructs with point-mutation in different sets of potential transcription factor binding elements. Our results showed that mutations on the overlapping region of the estrogen response element, steroidogenic factor binding element and RXR heterodimer binding site, that of MEF3 binding site and heat shock factor binding site, or TATA box, significantly decreased the promoter activity. However, the cognate protein factors binding to these elements remains to be proven by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) in subsequent experiments. In conclusion, we cloned and verified the promoters of several candidate genes. In addition, we completed their promoter-specific reporter constructs to be used for embryo microinjection and gene ontology study. We also characterized the promoter region of MELK gene and showed that there were potential transcription factors likely to positively regulate the MELK gene expression.