青花菜非春化調控開花基因BoFLC3之功能性研究

Functional Characterization of the Non-vernalization Responsive Flowering Gene, BoFLC3, in Broccoli (Brassica oleracea var. italica)

劉佳晴(Chia-Ching Liou)

指導教授 ： 林彥蓉

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902054

摘要

青花菜 (Brassica oleracea var italica) 為全球及臺灣重要蔬菜之一，在臺灣青花菜的生產季節為十一月到隔年四月，為了在半年產季增加青花菜產量，早生青花菜是青花菜育種目標之一。為了輔助早生青花菜育種，了解影響青花菜開花時間之分子機制，有助於以青花菜開花時間之重要基因作為分子標誌來輔助育種。BoFLC3為先前本實驗室針對臺灣非春化型青花菜開花時間進行數量性狀圖譜基因座分析 (QTL mapping) 所發現之開花候選基因之一，先前探勘臺灣青花菜種原間BoFLC3的基因型，不同基因型其開花時間有差異，因此推測此基因為影響臺灣青花菜開花時間之基因。在來自QTL mapping早晚親之BoFLC3兩對偶基因間，其intron I有255 bp之indel多型性，且有三個胺基酸變異，為了證實此基因功能是否與阿拉伯芥AtFLC一樣扮演著開花抑制子，以及為了瞭解三個胺基酸變異是否影響開花時間，將兩對偶基因分別構築在過表現35S promoter後，過表現於wild type Arabidopsis (Col-0)，結果顯示，過表現兩個對偶基因皆延遲阿拉伯芥開花時間，證實BoFLC3與阿拉伯芥FLC功能相似，為抑制開花之基因。另外，兩BoFLC3對偶基因間其啟動子區間有兩段大片段的Indel多型性，一片段為244 bp，另一片段為678 bp，將兩對偶基因啟動子區間結合GUS蛋白，測試兩對偶基因啟動子活性，結果顯示，帶有兩大插入片段之BoFLC3-1啟動子，呈現較低的活性，推測插入片段降低BoFLC3表現，而使BoFLC3抑制開花效果下降，呈現早開花性狀。最後為了進一步瞭解BoFLC3對青花菜開花時間影響程度，產生青花菜boflc3突變株，觀察突變對青花菜開花時間影響，於本研究先建立穩定青花菜轉殖系統，以用於基因編輯技術。此BoFLC3基因功能研究期待將BoFLC3開發成分子標誌，輔助不同開花期的青花菜育種，而進一步得以幫助產季調節。

關鍵字

青花菜 ； 開花時間 ； FLOWERING LOCUS C 基因 (FLC) ； 甘藍 BoFLC3 ； 非春化型

並列摘要

Broccoli (Brassica oleracea var. italica) is one of the important vegetables in the world. Understanding the molecular mechanism of broccoli flowering time can assist the breeding of broccoli with various flowering time for shift production. BoFLC3 has been shown to be associated with the flowering time of non-vernalization type broccolis by the linkage analysis and candidate gene approach. BoFLC3 alleles of late-flowering and early-flowering inbred lines exhibited 3 amino acid substitutions and a 255-bp indel polymorphism in intron I. To validate the function of BoFLC3 conferring flowering time, the full-length genomic DNA of the two BoFLC3 alleles were constructed after 35S promoter and then transformed to an ecotype Arabidopsis, Col-0. The overexpression of both two BoFLC3 alleles postponed flowering initiation, revealed that BoFLC3 played a similar role as AtFLC in inhibition of flowering time and both two BoFLC3 alleles were functional. In addition, promoter assays of two BoFLC3 alleles exhibited 244-bp and 678-bp indel polymorphisms were measured to reveal the importance of these two indels on promoter regions on the expression of BoFLC3. The two insertions in the BoFLC3-1 allele had relative lower promoter activity, implying less repression effect of BoFLC3 on FT associated with earlier flowering than the BoFLC3-2 allele. Furthermore, a stable transformation system of broccoli was established for further characterizing the function of BoFLC3 in broccoli by overexpression and/or genome editing. This study sheds light on the manipulation of BoFLC3 on the breeding of non-vernalization type broccoli with various flowering time to adjust production for food resilience.

並列關鍵字

broccoli ； flowering time ； FLOWERING LOCUS C (FLC) ； BoFLC3 ； non- vernalization

參考文獻

Abou-Elwafa, S. F., Büttner, B., Chia, T., Schulze-Buxloh, G., Hohmann, U., Mutasa-Göttgens, E., & Müller, A. E. (2011). Conservation and divergence of autonomous pathway genes in the flowering regulatory network of Beta vulgaris. J Exp Bot, 62(10), 3359-3374.

Amasino, R. (2004). Vernalization, competence, and the epigenetic memory of winter. Plant Cell, 16(10), 2553.

Ausín, I., Alonso-Blanco, C., Jarillo, J. A., Ruiz-García, L., & Martínez-Zapater, J. M. (2004). Regulation of flowering time by FVE, a retinoblastoma-associated protein. Nat Genet, 36(2), 162-166.

Balasubramanian, S., & Weigel, D. (2006). Temperature Induced Flowering in Arabidopsis thaliana. Plant Signal Behav, 1(5), 227-228.

Berry, S., & Dean, C. (2015). Environmental perception and epigenetic memory: mechanistic insight through FLC. Plant J, 83(1), 133-148.