在阿拉伯芥表現嗜熱菌Meiothermus taiwanesis MtDnaJ及MtDnaK對非生物逆境反應之分析

Abstract

氣候變遷導致非生物逆境嚴重影響作物的生長及產量。其中為提升作物的逆境耐受性，在轉殖基礎應用上有用基因之發掘及利用益形重要。目前極端環境下生長之生物，如耐高溫或鹽鹵的溫泉菌其分子伴蛋白系統相關基因的應用即為研究熱點。分子伴蛋白（molecular chaperone）系統由DnaJ (Hsp40)-DnaK (Hsp70)-GrpE (NEF)構成，主要功能可避免變性蛋白凝集沉澱，協助展開多肽鏈回復正常摺疊及功能構形，進而維持細胞內蛋白質恆定。過去已有將原核或真核熱休克蛋白各自大量表現於大腸桿菌或植物以增進其對不同非生物性逆境耐性之先例。本研究主要將臺灣温泉亞嗜熱菌(Meiothermus taiwanesis)的MtDnaJ及MtDnaK以CaMV 35S啟動子或T7啟動子分別大量表現於阿拉伯芥或大腸桿菌中以分析MtDnaJ及MtDnaK是否可提升阿拉伯芥及大腸桿菌對熱逆境及其他不同非生物逆境耐受性。首先，將溫泉菌MtDnaJ及MtDnaK與其他生物之熱休克蛋白進行演化樹親緣分析及蛋白質序列比對，發現不同物種的熱休克蛋白確實有高度保守性，可能具有相似之生理性狀。其次，進行不同逆境下阿拉伯芥轉殖株的外表型耐受性評估。結果發現絕大部分品系在熱休克處理下普遍都具後天獲得熱耐性（acquired thermotolerance）。而在鹽處理下，雜交轉殖株不論是幼苗發芽勢、根長生長勢或是成株試驗存活率都較佳。其中一個大量表現MtDnaJ及MtDnaK蛋白的雜交品系H8表現尤佳，由此推測大量表現雙重熱休克蛋白MtDnaJ及MtDnaK的雜交品系可能因為表達較完整的分子伴蛋白系統功能，所以有較佳耐性表現。另外以大腸桿菌系統測試，發現在一般生長情況下，大量表現MtDnaJ及MtDnaK基因表達融合蛋白的品系有較佳生長趨勢。綜合以上推論，大量表現原核臺灣亞嗜熱菌熱休克蛋白MtDnaJ或MtDnaK，尤其將兩者共同表現於阿拉伯芥可有效提升植株或細菌對非生物逆境如高溫、鹽害及滲透壓逆境的耐受性。

Climate change results in severely abiotic stresses and affects crop growth, development and yields. For the improvement of crop stress tolerance, the stress-tolerant gene mining and applications for transgenic biotechnology application become more important. The extreme environment organism, such as thermotolerance or halophytic hot spring bacteria has been used for its molecular chaperone system related genes to enhance crop stress resistance. The Hsp70 molecular chaperone system is composed of DnaJ (Hsp40)-DnaK (Hsp70)-GrpE (NEF), and the main function is to prevent denatured protein aggregation and precipitation, help the unfold polypeptides refolding and gain the functional composition, and furthermore maintain the protein homeostasis in cells. Previous studies showed that prokaryotes or eukaryotes overexpressing chaperone-related genes could enhance their abiotic stresses tolerance. Thus, in this study we overexpression of Meiothermus taiwanesis MtDnaJ and MtDnaK driven by CaMV 35S promoter or T7 promoter, respectively in Arabidopsis thaliana or Erscherichia coli to test if this strategy could confer the abiotic stresses tolerance in Arabidopsis and E. coli. First, the phylogenic analysis and polypeptides alignment of MtDnaJ/MtDnaK and other organisms showed that indeed these proteins are highly conserved in proteins of various species and may share similar physiological functions. Second, phynotypic analyses of transgenic Arabidopsis under multiple stresses showed that almost all transgenic lines have acquired thermotolerance. On the other hand, under the salt stress, the double cross transformants with MtDnaJ/MtDnaK expressed better phenotype no matter in seed viability, root length, or adult survival rate, especially the MtDnaJ- and MtDnaK- overexpression transformant, H8. This may be due to the establishment of a more complete Hsp70 chaperone system that leads to better gene expression and to confer stress tolerance. In E. coli system, the better growth curve was observed with the overexpression of MtDnaJ and MtDnak fusion gene under normal condition. Combined all the results, the overexpression of prokaryotic Meiothermus taiwanesis MtDnaJ or MtDnaK, especially co-expression of both genes by PCR-fusion, could confer plants with the tolerance of high temperature, salt stress and osmotic stresses.