Abstract
熱逆境對生物體是很嚴重的的問題，將生物細胞暴露在高溫下會增加一群蛋白質的生成，我們稱之為熱休克蛋白(heat shock proteins, HSPs)。熱休克蛋白可依其分子量區分為不同的族群；某些HSP100和存在原核生物中的caseinolytic protease (Clp)的調節單位蛋白很像，因此這一類的蛋白被稱為HSP100/Clp蛋白家族。這個蛋白家族的成員有一特性，他們彼此之間的序列有很高的相似度，尤其是在他們的胺基酸序列上有高保守性的核酸結合區域。有些Clp蛋白會和Clp蛋白酵素的核心單元ClpP結合，用以調控蛋白質分解的作用。本實驗室在先前的研究中，以Pisum sativum的ClpC cDNA作探針，從阿拉伯芥的cDNA基因庫中篩選出ClpA-like的cDNA，定序列後並命名為AtclpA。但是根據在基因資料庫的序列比對，我們發現這個cDNA應該還缺少5'端的一段序列，因此參考最近註冊的阿拉伯芥ClpC cDNA (AF022909)設計引子利用逆轉錄法和聚合酵素鏈鎖反應(RT-PCR)希望從mRNA的模版經由放大作用得到AtclpA所缺少的5'端序列片段嵌入pGEM-T Easy載體並命名為pGEM-T1.3。將pGEM-T1.3經pflMI和NotI限制酵素作用過的NotI-PflMI DNA片段嵌入經同樣處理的pAtclpA中以構築完整的cDNA並將此cDNA命名為AtclpC。完整的AtclpC cDNA有3095個鹼基對，並包含5'端106個及3'端205個鹼基對的未轉譯區域，其編碼區可以演譯成927個胺基酸的蛋白質。接著利用活體外的轉錄和轉譯系統，欲得知AtclpC是否可以轉譯成完整的蛋白質。因為並未看到我們預期的片段，所以關於此cDNA的正確性仍須進一步的確認。另外探討此AtclpC的mRNA在4℃低溫，37℃高溫，及脫水逆境處理兩小時之後的表現量，發現似乎低溫及高溫逆境會減少其mRNA的表現量，但脫水處理則只有少量影響。這些關於 AtclpC的探討希望能提供更進一步研究其機制或功能的依據。Heat stress is a critical problem to various organisms, and theexposure of cells to an increased temperature enhances thesynthesis of several proteins called heat shock proteins(HSPs). Heat shock proteins are classified into severalsubgroups according their molecular weight. Some 100 kDa heatshock proteins (HSP100) are discovered to be the homologue ofthe regulatory sub- unit of the caseinolytic protease (Clp)and are grouped into the HSP100/Clp family. The Clp familymembers share highly conserved sequences, especially thenucleotide binding domains (NBDs). In our previous work, aClpA-like cDNAwas isolated from Arabidopsis cDNA library usingthe Pisum sativum clp cDNA (Pspclp) as a probe. This ClpA-like cDNA was subcloned into pBluescript II KS+ and namedAtclpA. But this cDNA was found that it was truncated from the5'-region according to the results from similarity search indatabase. In order to construct the full-length cDNA, RT-PCRwas applied to amplify the 5' region fragment according to thesequence of ClpC cDNA (AF022909). The RT-PCRamplified fragmentwas cloned into pGEM-T Easy vector and designated pGEM-T1.3TheNotI-PflMI fragment of pGEM-T1.3 including the 5'-region wasligated to the NotI and PflMI digested pATClpA to construct afull-length cDNA and named AtclpC. AtclpC cDNA is 3095 bp inlength, including 106 bp 5'-untranslated region and 205 bp3'-untranslated region. The coding region can be deduced intoa polypeptide of 927 amino acids. No product was detected whenthe AtclpC was subject to in vitro transcription andtranslation reactions, fur- ther examinations on thecorrectness of the AtclpC need to be done. The ex- pressionsof mRNA of the AtclpC were investigated by treating theArabidopsis plant in cold stress (4□C), heat stress (37□C),and dehydration (60% re- lative humidity). The resultsshowed that the AtclpC mRNA level seemed de- cline after twohr treatment with cold or heat, but only a little reductionupon dehydration stress. These results provide informations forfurther study on the expression, structure and function ofthe AtclpC.