苦瓜是台灣最重要的經濟蔬菜之一，但苦瓜的生長和發育受限於淹水以及低溫的影響，不但引起植株的氧化壓力，嚴重時也會影響品質和產量。故農民常以嫁接於絲瓜砧及南瓜砧方式分別增進苦瓜對於淹水及低溫逆境的影響。植物在受到環境逆境影響後容易引起活性氧族(ROS)的反應，植物也發展出一系列防禦機制，其中包括抗氧化酵素解毒系統來清除活性氧族。本研究目的在探討苦瓜、絲瓜及南瓜在逆境下生理參數的變化，及找出對淹水及低溫逆境具有潛力的主要抗氧化酵素並選殖其基因。將苦瓜’月華’、絲瓜’雙依’與南瓜’共榮’之幼苗置於淹水逆境(苦瓜及絲瓜)和低溫10℃逆境(苦瓜及南瓜)下 0~72小時，觀察植株生長、外部形態，測定葉綠素螢光、葉綠素含量、脂質過氧化及電解質滲漏，以及ascorbate peroxidase (APX)、catalase (CAT)、superoxide dismutase (SOD)和glutathione reductase (GR)四種抗氧化酵素的活性。結果顯示，在淹水及低溫逆境下，苦瓜受到的生理傷害包括葉綠素降解，膜通透性增加，脂質過氧化、 Fv / Fm值下降和抗氧化酵素活性增加。絲瓜和苦瓜之間的APX活性在淹水逆境下有顯著差異，絲瓜根部相較於苦瓜根部APX mRNA之表現量，隨淹水時間增加而持續上升，葉部APX mRNA之相對表現量亦同。而在低溫逆境下，苦瓜和南瓜之間的SOD活性顯著不同。目前已經從耐淹水的絲瓜以及耐低溫之南瓜中利用Rapid amplification of cDNA ends (RACE)技術分別找出APX和SOD的全長基因，並將APX及SOD基因轉殖到大腸桿菌中。研究結果將有助於苦瓜栽培，以減少淹水和低溫逆境對作物的傷害及損失，也能在最短的淹水及低溫時間下區分耐逆境與不耐逆境植株，而可做為日後篩選耐淹水及低溫植株(苗期)之生理指標。
Bitter gourd is one of the most important economical vegetables in Taiwan. However, the growth and development of the plants are strongly influenced by flooding and chilling stresses that can be initiated by oxidative stress. Bitter gourds have been grafted onto the sponge gourd and winter squash to cope with flooding and chilling stresses due to its susceptible to both conditions. These scions have developed a series of defense mechanisms and detoxification systems including antioxidants and enzymes to scavenge the reactive oxidative species (ROS). The objectives of our work were to study the changes of physiological parameters in bitter gourd, sponge gourd and winter squash, and identify any antioxidant enzyme under flooding and chilling stresses. The phenotypic traits, physiological parameters (variable fluorescence/maximum fluorescence, electrolyte leakage, malondialdehyde), and antioxidant enzymes (APX, CAT, SOD, and GR) were measured during different time periods of stress.
The results of the study show that the physiological damages include chlorophyll breakdown, membrane permeability increased, and caused lipid peroxidation. The value of Fv/Fm was decreased under flooding and chilling stresses. The APX activity between sponge gourd and bitter gourd was found to be significant difference under flooding stress. APX mRNA transcripts of sponge gourd were relatively higher than bitter gourd based on real-time PCR analysis using actin as internal control.SOD activity between bitter gourd and winter squash was significantly different under chilling stress. Therefore, APX gene from the flooding-tolerant sponge gourd was identified and isolated using RACE technology. Both APX and SOD genes were cloned into E. Coli using transformation methodology. The results may be helpful to those bitter gourd farmers to reduce damages of the plants, and also be informative to screen for suitable scions and for stress tolerant breeding as well.
