赤桉類纖維素合成酶基因CslD之選殖

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標題:	赤桉類纖維素合成酶基因CslD之選殖 Isolation of a cellulose synthase-like D gene (CslD) from Eucalyptus camaldulensis
作者:	林秀娥
貢獻者:	嘉南藥理科技大學：生物科技系暨研究所高毓瑩
關鍵字:	赤桉類纖維素合成酶半定量聚合酶連鎖反應即時定量聚合酶連鎖反應半纖維素原位雜交技術 in situ hybridization semi-quantitative PCR real-time PCR RACE cellulose synthase like hemicellulose
日期:	2009
上傳時間:	2011-10-27 14:42:58 (UTC+8)
摘要:	赤桉 (Eucalyptus camaldulensis)，桃金孃科，是一速生樹種，原產地為澳洲，具抗旱、抗寒等特性，且其木材耐腐性強，可供建築及加工用材。於 1986 年首次引入台灣，經由農委會林業試驗所二十年以上之試驗，篩選出數種優良品系。由於其紙漿纖維之品質與產量皆高，因此赤桉被視為一種有潛力的紙漿來源樹種，也因此利用遺傳工程改善紙漿之產量，將成為未來發展趨勢之一。除紙漿生產之外，目前也將桉樹列入生質能源植物候選名單中，主要是因為糧食作物生產燃料將直接衝擊糧食的供應，因此利用高產量纖維質的能源植物，配合新的能源轉換技術生產第二代生質能源，將更符合環保的規範與經濟效益。然而，至今與半纖維素生合成相關之赤桉類纖維素合成酶 (cellulose synthase-like) 尚未有文獻發表。本研究期能選殖出赤桉類纖維素合成酶成員全長基因。因此，本篇論文設計一退化性引子對，並以反轉錄-聚合酶連鎖反應方式，由赤桉嫩葉中選殖出，經過NCBI比對確定為類纖維素合成酶基因成員後，選殖片段為561 bp，其與顫楊 (Populus tremuloides) PtrCslD4核酸相似度為84%。因此命名為EucCslD4。再針對該基因設計專一性引子對，取得類纖維素合成酶全長基因3309 bp，結果指出已選殖出之基因，與顫楊PtrCslD4核酸相似度為86%。本研究採用DIG標記探針進行原位雜交技術 (in situ hybridization)，以了解EucCslD4基因於組織之定位 (localization)。結果發現在CSRII區，EucCslD4於根部組織之根冠位置可觀察到訊號。在第七節間葉片之髓心、未成熟木質部及韌皮部可觀察到訊號，而在第六節間之髓心及十節莖段之未成熟木質部 (immature xylem) 、韌皮部 (phloem) 等部位可觀察到信號。而在CSRI區，EucCslD4於根部組織之根冠位置可觀察到訊號。在第九節間葉片之髓心、未成熟木質部及韌皮部可觀察到訊號，而在第四節間及第六節間之髓心及第八節莖段之未成熟木質部、韌皮部等部位可觀察到信號。再以即時定量聚合酶連鎖反應及半定量聚合酶連鎖反應，該基因於各組織之定量表現，結果顯示EucCslD4在不同組織表現量並不相同，於莖段表現量較高，其次在根部及嫩葉，而在木質部及韌皮部表現量較低。目前已將EucCslD4基因接入雙元載體pBI121中，未來將轉殖進入菸草中，並觀察其型態改變。 Eucalyptus camaldulensis (Murray red gum), Myrtaceae, are trees original in Australia , the drought and cold resistance, and their strong decay resistance of wood for construction and wood processing. It was first introduced into Taiwan in 1896 by TFRI and found with excellent adaptability over 20-year tests. E. camaldulensis is known as a tree offering promise as a potential material for use in paper production, due to the superior quality of its pulp fibers and high pulp yield. To improve the wood quality for pulpwood E. camaldulensis, genetic engineering would be commercially used in the near future. In addition to pulp production, the current will also be included in Eucalyptus biomass energy plants in the list of candidates, mainly because the production of food crops will have a direct impact on fuel supply of food, so the use of high fiber energy production plants, with a new energy conversion technology to produce the second generation of biomass energy, be more in line with the norms of environmental protection and economic benefit. However, little is currently known about the role of Csl genes in E. camaldulensis. Therefore, total RNA was isolated from young leaves of E. camaldulensis and subjected to a two-step RT-PCR using degenerated primers that are specific for the amino acid motif ’CYVQFPQ’ forward primer and ’GWIYGS’ reverse prime, which are highly conserved in all known plant CesAs (Csls) and contain the CSRII region. The clones were then sequenced and analyzed with similarity searches against the nucleotide database in GenBank by BLASTN. Interestingly, sequenced at both ends and shown 84% nucleotide identity to PtrCslD4. To obtain the full-length cDNA of EucCslD4, a RACE PCR technique was adopted. To further confirm that our new cDNA fragments (CSRII fragments, 5′ and 3′ RACE products) were originated from a single, full-length cDNA, PCR primers were designed based on the 5′ and 3′ open reading frame (ORF) sequences. These primers were used to amplify full-length of EucCslD4 cDNAs with 3309 bp in size by RT-PCR. One clone, pCR2.1-TOPO-D4-FL-11, was obtained and showed highly 86% nucleotide identity. Moreover, an in situ hybridization system has been performed to characterize the localization of EucCslD4 in E. camaldulensis. To generate DIG-labeling probes, cDNAs of CSRII region were firstly subcloned into the pGEM-T Easy vector. DNA fragments between the SP6 and T7 promoter sequences in constructs were then amplified by PCR. DIG-labelled sense and antisense RNA probes were prepared using an in vitro transcription kit. This cRNA can be combined with the anti-digoxigenin AP, via of NBT and BCIP has pigment blue-violet color. Signals representing the expression of EucCslD4 could be detected in the immature xylem and phloem of leaves and stems during different developmental stages. Moreover, real-time PCR and semi-quantitative polymerase chain reaction were also conducted to investigate the gene expression of EucCslD4. In our preliminary results showed high expression level in stems and followed by in the roots and leaves, low expression level in xylem and phloem. A binary construct harboring EucCslD4 with pBI121 backbone has been obtained in this study. In the near future, the EucCslD4 would be transferred into tobacco to investigate the role of EucCslD4 in hemicellulose biosynthesis.
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