半結球萵苣缺磷情況下之生理反應與基因表現

Abstract

磷為植物必需巨量營養元素之一，不僅是遺傳物質與細胞結構的重要組成，也是多項生理生化反應的重要因子。前人研究指出萵苣 (Lactuca sativa L.) 的磷使用效率因種類與栽培種而異，因此本研究探討半結球萵苣對不同磷處理的生理反應。此外，利用微陣列 (microarray) 與定量反轉錄PCR (quantitative reverse transcription PCR, qRT-PCR) 技術分析半結球萵苣磷處理下之基因表現。 半結球萵苣栽培於不同光強度下表現不同生長速度，其缺磷反應速度也不一樣。苗期在高或低光強度下缺磷3天即顯著降低地上部與地下部的無機磷濃度，地上部鮮重於高光強度下缺磷3天顯著下降，低光強度下則缺磷5天才顯著下降。生長期在高或低光強度下缺磷5天顯著降低地上部、地下部無機磷濃度與地上部鮮重，含水量在缺磷10天後顯著下降，過氧化氫在缺磷10天後顯著上升，葉綠素與類胡蘿蔔素在高光下缺磷5天顯著上升，低光下則10天才上升。採收期在高或低光強度下缺磷5天顯著降低地上部與地下部無機磷濃度，但即使缺磷10天對地上部鮮重、含水量、葉綠素、類胡蘿蔔素、過氧化氫與硝酸鹽等濃度皆無顯著影響。 本研究利用微陣列分析找到半結球萵苣於缺磷逆境下表現上升的基因Lsa031437.1，此基因功能可能參與植物體內氧化還原反應，其對缺磷處理的表現情形更進一步透過定量反轉錄PCR證實。之後將Lsa031437.1啟動子序列結合GUS報導基因之構築利用農桿菌滲入法短暫表現於萵苣葉片。GUS染色結果發現植株生長於磷肥充足與缺磷狀況其葉片GUS表現量相似。此結果說明Lsa031437.1 promoter:GUS構築結合農桿菌滲入法可能不適用於監測萵苣含磷狀態。

Phosphorus (P), one of the essential macronutrients of plant, is not only an important component of genetic materials and cellular structure, but also a key factor involved in many physiological and biochemical reactions. Previous studies showed that phosphorus use efficiencies of lettuce (Lactuca sativa L.) varied depending on morphological types and cultivars. Therefore, this research investigated the physiological responses of semi-heading lettuce under various phosphorus treatments. In addition, gene expression analysis was conducted using microarray and quantitative reverse transcription PCR (qRT-PCR) techniques in semi-heading lettuce under various phosphorus treatments. Semi-heading lettuce plants cultured under different light intensities displayed different growth rates, and the phosphorus starvation responses were also different. During seedling stage, shoot and root phosphate concentrations were significantly decreased after Pi starvation for 3 days under either high or low light intensities. The shoot fresh weight was significantly decreased after Pi starvation for 3 days under high light intensity condition, and was significantly decreased after Pi starvation for 5 days under low light intensity condition. During growing stage under either high or low light intensities, the shoot fresh weight, shoot and root Pi concentrations were significantly decreased after Pi starvation for 5 days, the water content was significantly decreased after Pi starvation for 10 days, and the hydrogen peroxide concentration was significantly increased after Pi starvation for 10 days. The chlorophyll and carotenoids concentrations were significantly increased after Pi starvation for 5 days under high light intensity condition, but increased after Pi starvation for 10 days under low light intensity condition. During harvesting stage under either high or low light intensities, shoot and root Pi concentrations were significantly decreased after Pi starvation for 5 days. However, during this stage, even after Pi starvation for 10 days, there were no significant difference between Pi treatments in shoot fresh weight, water content, chlorophyll concentration, carotenoids concentration, hydrogen peroxide concentration and nitrate concentration. In this research, the Pi starvation induced gene- Lsa031437.1 was identified in semi-heading lettuce via microarray analysis. The function of Lsa031437.1 may involves in the reaction of oxidation-reduction in plants. Expression patterns of Lsa031437.1 in responses to Pi starvation treatments were further confirmed by qRT-PCR techniques. Then, the Lsa031437.1 promoter fusion with β-glucuronidase (GUS) construct was transiently expressed in lettuce leaf via agroinfiltration. GUS staining results showed that the expression levels of GUS signal in leaves were similar when plants grown under Pi sufficient and starvation conditions. This result indicated that Lsa031437.1 promoter:GUS construct in combination with agroinfiltration technique may be not suitable for monitoring phosphorus status in lettuce.