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標題: 冰花耐鹽相關SKD1基因之分離與表現分析
Isolation and characterization of a salt-induced SKD1-like gene in ice plant
作者: 洪郁惠
Hung, YuHui
關鍵字: Mesembryanthemum crystallinum L;冰花;salt-induced;RACE;SKD1;K+;Na+;ATP binding site;鹽誘導;RACE;SKD1;鉀離子;鈉離子;ATP結合的位置
出版社: 植物學系
冰花(Mesembryanthemum crystallinum L.)是一種可生長於高鈉鹽環境的耐鹽植物,由冰花生長於200 mM NaCl之癒傷組織,利用扣除雜交及聚合連鎖反應選擇性擴增獲得許多鹽誘導的cDNAs,其中一個cDNA命名為SI1 ,其長度約為300 bp,但是其轉錄訊息長度約為1600核酸。欲選殖全長的SI1 clone序列,本實驗設計兩組基因特異引子,以RACE (rapid amplification of cDNA ends) 擴增其5’端的未知序列,結果成功的獲得一個約1000 bp的片段,經DNA定序後,與SI1相接得到1219 bp的序列,推衍其氨基酸序列作比對,結果發現分別與人類及老鼠的一個幫助鉀離子吸收之SKD1 (suppressor of K+ transport growth defect) 蛋白質有69.7 %及70 %的相似性,並與阿拉伯芥的putative suppressor protein有95 %的相似性,將之命名為mcSKD1。此四個SKD1蛋白均有一個ATP結合的位置,且為親水性之蛋白質。南方墨點分析結果顯示,mcSKD1在冰花染色體組中只有一個copy;組織表現分析方面,將癒傷組織施以不同鹽濃度處理,以mcSKD1之cDNA 為探針進行北方墨點分析,結果發現此基因在未加鹽處理時就有表現,提高鹽濃度會使表現量增加。在植株表現分析方面,未加鹽處理時,mcSKD1基因在冰花植株的每個部位皆有表現,其中在根的表現量最低,在花的部位有很高的表現量。隨著鹽處理濃度的提高,此基因在根、莖及葉的表現量亦隨之提高,而且在200 mM NaCl處理後,在新葉的表現量明顯高於老葉。在水耕培養植株根及葉的鹽誘導表現情形變化較土耕不明顯。缺鉀處理冰花癒傷組織五天與十天,此基因的表現量皆比對照組高,代表此基因的表現量會隨著外界的鈉離子、鉀離子的濃度改變而變化,而且在鈉離子累積量最高的組織內,表現量亦最高,推測其功能可能參與根部鉀離子的吸收,以及地上部鈉離子之區隔。

Common ice plant (Mesembryanthemum crystallinum L.) is able to survive in high saline and water-deficit environment. By using substractive hybridization and selective amplification of differentially-expressed cDNAs, several salt-induced cDNA clones have been isolated. In this study, we focused on examining a partial-length cDNA clone SI1. To obtain its full length cDNA sequence, two sets of gene-specific primers were designed according to known SI1 sequence. One 1000 bp fragment was obtained by 5'-RACE (rapid amplification of cDNA ends). The sequence aligned to SI1 and the total length was 1219 bp. Deduced amino acid sequences showed 69.7 % and 70 % similarity to a SKD1 (suppressor of K+ transport growth defect) protein of human (AF038960) and mouse (P46467), respectively, and 95 % similarity to a putative suppressor protein of Arabidopsis thaliana (AC005824). Therefore, we named it as mcSKD1. All SKD1 sequences are hydrophilic and contain one ATP binding site. Southern blot analysis showed that only one copy of the gene existed in haploid genome. Northern blot analyses showed that mcSKD1 was expressed in callus grown in different NaCl concentrations. The highest expression occurred in treatments of 200 or 300 mM NaCl. At whole plant level, mcSKD1 was expressed in all tissues examined and the expression of mcSKD1 increased as the concentration of NaCl in roots, stems and leaves increased. The expression of mcSKD1 was higher in new leaves than old leaves in treatment of 200 mM NaCl. The expression of mcSKD1 was most prominent in 400 mM NaCl- grown roots of hydroponically-grown ice plant. When callus was grown under potassium-deficit medium for five or ten days, the expression of mcSKD1 was increased. The expression of mcSKD1 in related to the ion homeobalance, especially Na+ and K+, are discussed.
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