耐熱與不耐熱番茄品系雄蕊發育早期具有差異之可溶性蛋白之鑑定

Identification of soluble proteins and corresponding genes from early developing anther that differentiate normal and heat-tolerant tomato lines

呂宏洲(Hung-Chou Lu)

指導教授 ： 林讚標

國立臺灣大學/生命科學院/植物科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00450

摘要

根據前人的研究，花粉母細胞形成發育期或番茄開花前7-15天，對高溫最為敏感並導致高落果率。因此為了鑑定出在高溫逆境下與番茄著果率提昇有關之蛋白質體，我們利用亞蔬中心所提供耐熱番茄 (Solanum lycopersicum, formerly Lycopersicon esculentum) 品系CL5915與不耐熱品系L4783，藉由控制二品系間生長溫度之差異（熱逆境30/25℃與一般生長環境25/20℃），在雄蕊發育早期利用蛋白質體學技術分析可溶性蛋白質，比較並扣除共有之遺傳背景之蛋白質後，將二品系間具有差異的可溶性蛋白質篩選出來，並進一步利用LC-ESI MS/MS進行蛋白質身份鑑定。經由此研究，成功地找出多個具有高度差異性的蛋白質，在功能分析及歸納整理之後，挑選了其中兩個蛋白質glyoxalase I和3beta-hydroxysteriod dehydrogenase，設計degenerative primer，並利用RACE技術將上述2種蛋白質在二種番茄品系中的完整基因序列定出。發現基因序列上的改變可精確地解釋，何以造成蛋白質pI與分子量大小在二品系之間的差異。

關鍵字

耐熱 ； 番茄 ； 雄蕊 ； 二維電泳

並列摘要

According to previous study, developing anther 7-15 days before anthesis in tomatoes was the most sensitive period to high temperature which causes a low fruit set. In order to identify the proteins which are responsible for the better fruit set in heat-tolerant variety than in heat-sensitive one, we used two dimensional electrophoresis systems to separate isolated proteins from developing anther and looked for the proteins up-/down-regulated in heat-tolerant variety. In doing so, two selected cultivars, CL5915, the heat-tolerant, and L4783, the heat-sensitive, were grown at the same time in a phytotron at both 30/25℃ and 25/20℃. The proteins isolated from developing anthers of heat-tolerant variety grown in 30/25℃, were subtracted to remove the common genetic background. The resulting populations of proteins were considered as induced protein positive to heat stress. Using LC-ESI MS/MS, several proteins were identified, including glyoxalase I and 3beta-hydroxysteriod dehydrogenase. Degenerative primers were designed and RACE technique was applied to obtain the complete gene sequences of the two-mentioned proteins from heat-sensitive and heat-tolerant tomatoes. I found substitution of nucleotides of both proteins can explain accurately the changes in isoelectric point and molecular weight of these two proteins.

並列關鍵字

heat-tolerant ； tomato ； anther ； two dimensional electrophoresis

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