Cloning the Broccoli HMG-I/Y Gene as an Endogenous Reference for Transgene Copy Number Determinations Using Real-time PCR

青花菜高移動性蛋白群 HMG-I/Y 基因之選殖及提供為定量Real-time PCR內生性標準估算轉基因套數之分析評估

林俊宏(Chun-Hung Lin) ； 黃家媛(Jia-Yuan Huang) ； 陳榮芳(Long-Fang O. Chen)

《Botanical Studies》 53卷1期 (2012/01) Pp. 19-30

摘要

植物基因轉殖技術已是作物改良不可或缺之工具，然一般亦知轉基因之嵌入基因組位置與轉基因之套數關係著轉基因之表現與穩定性。TagMan 定量式即時聚合酶連鎖反應（Quantitative real-timePolymerase Chain Reaction, qRT-PCR） 係被發展出具若干優點如測試所需樣品量小、高流通量、高定量範圍及無後PCR 之膠體電泳分析等之快速檢定轉基因套數方法。惟此方法之分析有賴建立一在基因組內低套數且穩定之內生性參考基因為基準。在蕓苔屬（Brassica） 作物中，高移動性蛋白群HMG-I/Y 基因曾被報導利用為qRT-PCR 分析之參考基因，但該基因是否可在青花菜偵測得則有所爭議。本報告因此乃參考已報導之油菜（Brassica napus）及阿拉伯芥HMG-I/Y 基因序列選殖得一全長796 bp 青花菜HMG-I/Y cDNA 序列並加以定性其衍生之胺基酸序列與油菜之相似度達75%。利用已知作物HMG-I/Y基因衍生胺基酸序列在演化樹分析上，其結果與傳統分類分群極為吻合。本研究亦証實HMG-I/Y 基因在青花菜基因組每單套基因組為單套存在，利用研究室之異戊丙烯轉移酶（ipt） 基因及阿拉伯芥胚性開花基因（AtEMF1） 青花菜轉殖材料進行檢測與篩選低套數轉基因，亦獲致該基因利用為內生性標準參考基因以估算轉基因套數之可行性。

關鍵字

定量式聚合酶連鎖反應 ； 轉殖青花菜 ； 阿拉伯芥胚性開花基因 ； 異戊丙烯轉移酶基因

並列摘要

Transgenic technology has become an indispensable tool for crop improvement. However, transgene integration and copy number insertion affect transgenic expression and stability. TaqMan® quantitative real-time PCR (qRT-PCR) was developed as an alternative way to determine transgene copy numbers and has several advantages over other methods: smaller amounts of test materials required, higher throughput capacity, large quantification scope, no post-PCR gel analyses, etc. Nevertheless, the TaqMan qRT-PCR requires an endogenous DNA sequence with confirmed low copy numbers per genome for its internal control. The current reports that use the HMG-I/Y (high-mobile-group protein I/Y) gene as the internal standard for Brassica oleracea are discrepant. Using the HMG-I/Y gene found in rapeseed and Arabidopsis as a reference, we cloned and characterized a full cDNA sequence (796 bp) of the broccoli (Brassica oleracea var. italica) HMG-I/Y gene that shared 75% identity with the deduced amino acid sequences of Brassica napus in our laboratory. We also conducted phylogenetic analyses from the deduced amino acid sequences of the HMG-I/Y gene among known plant species. These corresponded with traditional taxonomy. We demonstrate here that the cloned HMG-I/Y gene, being a single copy in broccoli, can serve as an internal reference gene for estimating copy numbers of transgene among the tested populations of isopentenyl transferase (ipt) and Arabidopsis embryonic flower gene EMF1 (AtEMF-1) transgenic broccoli.

並列關鍵字

Quantitative real time polymerase chain reaction (qRT-PCR) ； AtEMF1 gene ； ipt gene