赤桉纖維素合成酶CesA之基因選殖

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標題:	赤桉纖維素合成酶CesA之基因選殖 Cloning of a cellulose synthase gene (CesA) from Eucalyptus camaldulensis
作者:	陳逸賢 I-Hsien Chen
貢獻者:	高毓瑩嘉南藥理科技大學：生物科技研究所
關鍵字:	雙元載體半定量聚合酶連鎖反應原位雜交赤桉纖維素纖維素合成酶 RACE CesA Eucalyptus camaldulensis cellulose semi-quantitative PCR in situ hybridization binary vector cellulose synthase
日期:	2009
上傳時間:	2009-03-12 11:33:19 (UTC+8)
摘要:	赤桉 (Eucalyptus camaldulensis)，桃金孃科，是一速生樹種，原產地分佈於乾旱及半乾旱地區。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。然而，至今與纖維素生合成相關之基因纖維素合成酶 (cellulose synthase) 尚未在赤桉中被分離出來。本研究與林業試驗所合作，期能選殖出赤桉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纖維素合成酶基因片段1,079 bp，經過NCBI比對確定為纖維素合成酶基因成員後，其與雜交楊 (Populus tremula × Populus tremuloides) PttCesA2核酸相同度為90%。再針對該基因設計專一性引子對，以SMARTTM RACE cDNA amplification kit (Clontech) 取得纖維素合成酶全長基因3,485 bp。且使用CLUSTAL W進行親源分析，結果指出已選殖出之基因，與玫瑰桉已發表六個CesA基因為不同成員，因此命名為EucCesA7，其與雜交楊 (Populus tremula × Populus tremuloides) PttCesA2核酸相同度為80%，此PttCesA2基因為初生細胞壁生成相關基因，其特性可能為調控引張材(tension wood)有關。為了解EucCesA7基因在赤桉中之表達形式，本研究採用DIG標記探針進行原位雜交試驗 (in situ hybridization)，以了解其基因於組織之定位 (localization)。結果發現EucCesA7於葉片、莖段形成層 (cambial zone)、木質部 (xylem) 等部位可偵測到信號，而在第八節莖段可觀察到明顯訊號。再以半定量聚合酶連鎖反應，使用PhotoCap凝膠定量軟體分析該基因於各組織之定量表現，初步結果顯示EucCesA7於木質部表現量較高，嫩葉與莖段的表現量較低；在木質部組織中EucCesA7表現量高於EucCesA4和EucCesA2，在嫩葉以及莖段組織中EucCesA7表現量則低於EucCesA4；高於EucCesA2。綜合以上結果，初步推論EucCesA7與初生細胞壁合成相關。目前已將EucCesA7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 with remarkable growth and wildly distributed in arid and semi-arid climates. 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. The superfamily of cellulose synthase genes (CesA) involves the production of cellulose and has been cloned in many species. However, little is currently known about the role of CesA 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 motifis ’CYVQFPQ’ forward primer and ’GWIYGS’ reverse prime, which are highly conserved in all known plant CesAs and contain the CSRII region. The clones were then sequenced and analyzed with similarity searches against the nucleotide database in GenBank by BLASTN. Interestingly, one 1079 bp putative clone, EucCesA7, was sequenced at both ends and shown 90% nucleotide identity to PttCesA2. To obtain the full-length cDNA of EucCesA7, a RACE PCR technique was adopted by using SMARTTM RACE cDNA amplification kit (Clontech). 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′ untranslated region (UTR) sequences. These primers were used to amplify full-length of EucCesA7 cDNAs with 3485 bp in size by RT-PCR. One clone, pCR2.1TOPO-EucCesA7-FL.18, was obtained and showed highly 80% nucleotide identity. PttCesA2 plays a role in the primary cell wall synthesis, and may involve the gene regulation in the formation of tension wood. Phylogenetic analysis of the CesA sequences was also performed by aligning the sequences with 57 plant CesAs and showed that EuCesA7 belongs to the group that relate to the biosynthesis of primary cell wall. Moreover, an in situ hybridization system has been performed to characterize the localization of EucCesA7 in Eucalyptus. 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 EucCesA7 could be detected in the cambial zone of leaves and stems during different developmental stages. Among all tested tissues, the eighth internode with the strongest signal. Moreover, semi-quantitative polymerase chain reaction, analysis for EucCesA7 gene expression was also conducted. In our preliminary results showed high expression level in xylem and low expression level in leaves when analyzed by PhotoCap gel quantitative software. In comparison with other EucCesA members, EucCesA7 had the highest expression level in xylem followed by EucCesA4 and EucCesA2. In leaf tissue, EucCesA7 has lower expression level than that of EucCesA2 and EucCesA4. A binary construct harboring EucCesA7 with pBI121 backbone has been obtained in this study. In the near future, transgenic tobacco plants carrying EucCesA7 would be obtained to investigate the role of EucCesA7 in cellulose biosynthesis.
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