Abstract
當生物體面臨逆境 (stress) 時，大多數者會產生生理、形態或者是生化上的變化，使其適應或抵抗這種惡劣的環境。以溫度為例，若生物遭受高於正常生長溫度10℃左右之逆境時，體內原本可順利合成的蛋白質會被抑制，同時卻會誘導另一群蛋白質的生成，這種現象稱為熱休克反應 (heatshock response)，這群蛋白質也被稱為熱休克蛋白 (heat shockproteins)；其主要功能為保護蛋白質免受逆境造成傷害。熱休克蛋白之分子量從十幾kDa到上百kDa，依不同生物體之蛋白類型亦有所不同，分子量約在70 kDa的HSP70，其整個hsp70基因家族相似度極高，根據其表現受高溫影響之不同，可區分為兩大類：一為hsp70，需高溫誘導才表現者，另一則為hsc70，無需高溫誘導，於正常生長環境下就會表現。本實驗室先前從蕃茄之基因庫中篩選出具有hsc70基因，定名為Lehsc70-3；先將Lehsc70-3啟動子不同區域構築於氯黴素乙醯轉移酵素(chloramphenicolacetyltransferase，CAT)之前，希望藉由PEG transformation方式將之送入大豆原生質體中，分析啟動子之不同區域對於基因表現的影響。在Lehsc70-3啟動子中具有兩個GARE序列，所以針對同一品系之蕃茄，探討在不同濃度的吉貝素處理下，此基因表現是否有所影響，結果發現Lehsc70之mRNA與吉貝素濃度呈現劑量效應 (dosage effect)，不過當吉貝素濃度超過10-5 M，Lehsc70-3mRNA表現量卻不如預期的多。除此之外，利用數計式電導度計快速檢測植物組織於不同溶液中經過熱處理後的損傷程度。論文中並使用anti-HSP70單株抗體來偵測蕃茄體內HSP/HSC70家族蛋白質之表現量，以便日後運用此抗體來探討蕃茄HSP/HSC70之生理作用，期望進一步了解整個Lehsc70基因之調控機制。Most organisms share a common molecular response in theirphysiological, morphological or biochemical changes when theyface the environmental stress. Under high temperature or otherforms of stress, all protein synthesis of organism is reducedexcept for a group of proteins called heat shock proteins(HSPs). The phenomenon is called heat shock response. HSPs seemto have a main function to protect other proteins from stress.HSPs range from 16 to 110 kDa, with the protein profile varyingamong organisms. Although the sequences of hsp70 family areevolutionarily highly conserved, hsp70 family is divided intotwo classes according to the expression in response to heatinduction. HSP70s are strictly heat-inducible, but HSC70s (heatshock cognates) are expressed in the absence of heat shock.TheLehsc70-3 is a member of hsc70 genes previously isolated from atomato genomic library. To analyze the Lehsc70-3 promoter,recombinant plasmids containing various regions of Lehsc70-3promoter and the chloramphenicol acetyltransferase (CAT)reporter gene were constructed. We tried to introduce therecombinant plasmids into soybean cells by PEG transformationfor examining the influence of the various regions in Lehsc70-3promoter with the aid of CAT reporter gene. And Lehsc70-3promoter has two GAREs (gibberellin A3 response element). Tostudy the significance of GAREs, tomato leaves were sprayed withdifferent concentrations of GA3 prior to RNA extraction. TheLehsc70-3 mRNA level increased in tomato leaves by the GA3treatment in a manner of dosage effect in GA3of 10-6 M and 10-5M. However, GA3 at a concentration above 10-5 M did not maintaina dosage effect on the expression of Lehsc70 mRNA. The degree ofheat injury was monitored by measuring electrolyte leakage fromcells exposed to 37℃ for 2 hours in different bathing solutionusing a conductivity meter.A monoclonal (mouse) anti-heat shockprotein 70 antibody was also used to detect the expression ofthe tomato HSP/HSC70 in this study in order to furtherunderstand the possible function of HSC70 and the regulatorymechanism of Lehsc70 genes in tomato. A monoclonal (mouse) anti-heat shock protein 70 antibody was also used to detect theexpression of the tomato HSP/HSC70 in this study in order tofurther understand the possible function of HSC70 and theregulatory mechanism of Lehsc70 genes in tomato.