大葉蝴蝶蘭開花時間相關基因PbGI之選殖及分析與主動誘變蝴蝶蘭之特性分析

Molecular cloning and analysis of flowering-time gene PbGI from Phalaenopsis bellina and characterization of activation-tagged Phalaenopsis

陳霈慈(Pei-Tsu Chen)

指導教授 ： 蔡新聲 ； 陳靖棻

朝陽科技大學/理工學院/生物技術研究所/碩士(2009年)

摘要

光和溫度影響蝴蝶蘭開花時間，然而有關其分子調控途徑的研究甚少。目前對於光週期調控開花的部分，僅在阿拉伯芥有較深入的研究，故本論文以夏天開花之大葉蝴蝶蘭為材料，初步針對其光週期調控開花的上游基因GIGANTEA（GI）進行選殖與分析。利用基因同源性設計引子進行5’及3’RACE後，成功選殖到PbGI cDNA，全長3,480 bp，可轉譯成1,159個胺基酸。經序列分析後，得知PbGI在序列長度、親源演化分析及胺基酸組成上，皆與OsGI較為相似。利用多重序列排比分析得知PbGI與其它物種之GI之間有大於69%的保守性，並且與AtGI和OsGI相同有5個預測的穿膜區域，故推測為一穿膜蛋白質。集群與對應分析結果顯示，PbGI的RSCU值與AtGI最為相似。經基因套數分析後得知PbGI為單一複本數，與HvGI和OsGI相同。而基因表現量分析的結果則顯示PbGI與AtGI和OsGI同樣受光與生理時鐘調控且常態表現，且在花朵、氣根與葉片中亦能偵測到其表現量，其中以花瓣和成熟葉片表現量為較高。綜合前述，可推測PbGI應為GI同源基因。另外，利用主動誘變技術研究蝴蝶蘭開花時間的部分，從30株轉殖株觀察到1株開花時間延遲且外表形態改變的蝴蝶蘭，經由分析有可能為pTAG-8單一插入或是其他原因所致，其所影響之因素值得未來進行深入探討。

關鍵字

蝴蝶蘭 ； 開花基因

並列摘要

Although light and temperature affect the flowering time in Phalaenopsis, the molecular mechanism of flowering in such floral crop remain unclear. Possible flowering-time pathways are mainly studied in Arabidopsis. To have a better understanding on the photoperiodic flowering of Phalaeanopsis, this studies started with a circadian controlled gene GIGANTEA(GI) in Phalaenopsis bellina. According to the gene homology and 5’ and 3’ RACE, the full-length of PbGI (3,480 bp) has been cloned and bioinformatically analyzed. The deduced amino acid sequences, composition and phylogenic analysis indicated that PbGI is more closed to rice GI (OsGI) and shared more than 69% of similarity with other GI homologues. PbGI is predicted to have five transmembrane regions like other GI homologues, meanwhile the cluster and correspondence analysis of its RSCU values are more similar to AtGI’s. By Southern blots and real-time PCR analysis, it was found that PbGI, a single copy gene like HvGI and OsGI, is controlled by light conditions and can express constantly in almost all tissues, especially in mature leaves and petals. Therefore, PbGI must be a GI homolog. On the other hand, an activation-tagged strategy was used to study the flowering time in Phalaenopsis, and one of the 30 transgenic Phalenopsis was observed with a delayed flowering phenotype. Such change might caused by a single insertion of pTAG-8 or other factors that remained to be studied.

並列關鍵字

Phalaenopsis ； GIGANTEA

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