Studies on Transferring Heat Shock Protein Cognate 70 Gene into Cabbage (Brassica oleracea L. var. capitata L.)

Abstract

自然環境中，高溫是植物常遭受的逆境之一，為防禦此種傷害，植物發展出一套耐受高溫的方法，即熱休克反應(heat shock response)。此反應會促使植物細胞在短暫時間內停止大部份原來正常合成的蛋白質，而新合成熱休克蛋白(heat shock proteins;HSPs)，用以調整生理、生化反應，來抵抗高溫可能帶來對植物致命性的傷害。除了高溫等逆境誘導表現外，在平時不受逆境誘導即有表現的熱休克蛋白稱為heat shock proteins cognate(HSC)。HSC的存在，顯示除了逆境時，在一般生理情況下，熱休克蛋白仍具有重要功能。 隨著生物技術的進步，轉殖抗逆境基因至作物以提高其逆境忍受力，是提高作物產量有效的途徑。植物在溫度逆境下會產生熱休克蛋白以保護自身細胞渡過逆境，若能加強其抗逆境基因表現，可能會提高作物抗逆境的能力。因此，本試驗嘗試利用農桿菌轉移番茄hsc70-2基因到甘藍(新峰、高峰)植株內，探討在CaMV 35S或rbcS啟動子之驅勤下，大量表現hsc70-2基因，對增加甘藍耐熱之可行性。試驗同時包括在番茄hsc70-2基因5'端與CaMV 35S啟動子間，黏接上大豆之rbcS transit peptide sequence，比較hsc70-2在轉殖甘藍之葉綠體與細胞質對耐熱性的差異。本研究亦藉由轉殖反義之番茄hsc70-2基因，探討hsc70-2在耐熱性中所扮演的角色。 轉移六種構築載體之hsc70-2基因的轉殖甘藍，以南方墨點及北方墨點分析之結果頰示，轉移之hsc70-2基因已轉移到轉殖植株，並且表現hsc70-2基因。以40°C處理轉殖攜帶CaMV 35S啟動子的hsc70-2基因之轉殖甘藍二天，或以電導度所測得之50℃熱殺傷時間都顯示轉殖植林較對照組有顯著的增加耐熱性。The heat shock response is a stress phenomenon in many organisms including plants. This response, induced by an elevation of the ambient temperature, is characterized at the molecular level by de novo synthesis of heat shock proteins (HSPs). Heat shock protein cognate 70 (HSC70), expressed both in normal and stress conditions, is a prominent chaperone found in the cytosol of virtually all eukaryotic cells. In this study, we attempted to improve the ability of heat tolerance of cruciferous vegetables by transferring tomato hsc70-2 gene into cabbage. Tomato hsc70-2 gene was constructed into plant transformation vectors driven by CaMV 35S or Rubisco small subunit (rbcS) promoter with or without soybean transit peptide, and introducted into hypocotyl or cotyledon of cabbage and Chinese cabbage via Agrobacterium-mediated transformation. To reveal the role that HSC70 plays in plants, antisense tomato hsc70-2 gene was also constructed into plant transformation vectors driven by CaMV 35S or rbcS promoter, and introduced into cabbage. The results Southern and Northern hybridization analysis indicated that sense or antisense tomato hsc70-2 genes were present in the genome of transformed plants, and expressed in most of transformed plants. Increase in heat tolerance were found in transgenic cabbages transformed with tomato hsc70-2 gene driven by CaMV promoter as evaluated by the appearances after two days of 40℃ treatment of heat killing time at 50℃