文心蘭老化相關基因OSAG243的釣取與分析

Abstract

文心蘭外銷可分為空運與海運，但由於現階段設備不足，切花空運品質不易維持，且空運成本較高，因此目前還是以海運為主。海運需時較久，赴日銷售時的瓶插壽命不長，嚴重影響到文心蘭切花的品質，因此如何延緩文心蘭切花老化為目前文心蘭切花外銷的重要研究方向。本實驗室曾利用抑制性雜合扣除法 (Suppression Subtractive Hybridization)、蛋白質二維電泳、Differential Display等方式，釣取出一些文心蘭老化相關基因OSAGs (Oncidium Senescence-Associated Genes)，大約200~300bps的片段，並利用半定量RT-PCR進行進一步的篩選。以Real-time PCR的方法再做一次確認，確定OSAG243為老化相關基因。利用RACE釣取OSAG243 cDNA全長，並將全長經由NCBI blast，比對到了阿拉伯芥的一個C3HC4-type RING finger protein (At243)。 將OSAG243大量表現於阿拉伯芥，轉植株和野生型的葉型及花型並無顯著差異。而At243RNAi轉殖株及at243 T-DNA插入突變株(T-DNA insertion line)雖然在葉型及花型上也與野生型沒有差異，但卻發現有提早開花的現象，大約在8片簇生葉時就會開花，而野生型阿拉伯芥要在11~12片艚生葉時才會開花。以時間上來說，At243RNAi轉殖株及at243 T-DNA插入突變株比野生型提早6天開花。檢測FLC、FT、SOC1、GL1等和開花有關的基因，發現FLC在at243 T-DNA插入突變株中的表現量有減少，而FT及SOC1則增加，GL1的表現量和野生型沒有差異，表示提早開花的現象可能與FLC被抑制表現有關。FLC的表現會因為組蛋白H3-K4-trimethylation的程度變高而增加，且前人研究指出，At243可能是BRE1 orthologue，BRE1和組蛋白H2B ubiquitination有關，並進一步促使組蛋白H3 methylation。西方墨點分析的結果發現，At243RNAi轉殖株及at243突變株中組蛋白H3-K4-trimethylation的程度比野生型少，表示抑制At243表現會使得組蛋白H3-K4-trimethylation的程度變少，而這可能就造成At243RNAi轉殖株及at243突變株中FLC的表現被抑制，因而使植株提早開花。播種後40天的at243 T-DNA插入突變株葉綠素含量也比野生型少，表示at243 T-DNA插入突變株可能有提早老化的現象。 總的來說，大量表現OSAG243於阿拉伯芥並不會影響阿拉伯芥的生理功能，但抑制阿拉伯芥內生At243的表現可能會減少組蛋白H3-K4-trimethylation的程度並抑制FLC的表現，最終影響開花時間。

The cutting-flower of Onicidium Gower Ramsey is the most important export flowers in Taiwan. However, the vase life of the onicidium cutting-flower is not long enough for better economical value. Therefore, how to delay the senescence of onicidium cutting-flower is the prior way to study. Our laboratory used several methods, including suppression subtractive hybridization, differential display, and protein 2D-PAGE, to isolate the senescence-associated genes (SAGs). OSAG243 is one of them, and results from real-time PCR revealed that OSAG243 is a senescence-associated gene. The full length cDNA of OSAG243 was isolated using Rapid Amplification of cDNA Ends (RACE). After OSAG243 was compared to database in NCBI, it showed that OSAG243 is a C3HC4-type RING finger protein. In order to understand the functions of OSAG243 in plant, Arabidopsis expressing OSAG243 driven by 35S promoter or AGL5 promoter was constructed. In terms of phenotype and dark-induced senescence, no significant difference between wild-type and transgenic Arabidopsis was observed. Surprisingly, the flowering time of At243RNAi and Arabidopsis with T-DNA insertion in At243 gene is seven days earlier than that of wild-type Arabidopsis. Semi-quantitative RT-PCR indicated that depression of FLC caused the early flowering phenotype. The chlorophyll content of 40-day-old At243 T-DNA insertion line was lower than that of wild-type Arabidopsis. It indicated that At243 T-DNA insertion line might be senescent earliler than wild-type Arabidopsis. Westren blot results indicated that knocking out At243 in Arabidopsis caused lower level of methylation in histone H3, and it would down-regulate the expression of FLC. Conclusively, overexpression of OSAG243, a RING finger protein, may not affect the physiological functions of Arabidopsis. However, knocking out the At243 in Arabidopsis may decrease methylation level of histone H3 and down-regulate the expression of FLC, and finally influence the timing of flowering.