Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20663
標題: 耐鹽植物冰花鹽誘導基因群之選殖及分析
Screening and analysis of salt-induced cDNAs in halophyte Mesembryanthemum crystallinum
作者: 吳素玫
Wu, Su-Mei
關鍵字: Ice plant;冰花;Salt-tolerant mechanism;Subtractive;耐鹽機制;扣除雜交法
出版社: 植物學系
摘要: 
耐鹽植物Mesembryanthemum crystallinum (common ice plant ; 冰
花)是研究高等植物耐鹽機制的一種模式植物。當培養基中加入200 mM氯
化鈉時,冰花培養細胞可累積特定的逆境蛋白以維持正常生長。本實驗利
用冰花癒傷組織來篩選可被鹽誘導之基因:首先由鹽處理後五天大之癒傷
組織抽取mRNA、反轉錄成cDNA、兩端接上特定的adaptors,將過量之對照
組cDNA與加鹽組cDNA進行扣除雜交,用聚合連鎖反應選擇性擴增扣除雜
交之產物,並clone至pBluescript載體。篩選得54個clones,利用RNA
slot blot鑑定出七個有差別表現之clones。將癒傷組織施以不同濃度的
氯化鈉處理,以RNA slot blot中差別表現比較明顯的三個clones為探針
,並命名為SI1、SI2、SI3,其長度分別約為450、830和800bp。北方墨點
分析結果發現,在施以200或300 mM氯化鈉處理時RNA表現量增加最明顯。
繼以300 mM氯化鈉處理不同天數作time course分析結果發現,RNA表現量
隨培養時間的增長而增加,在加鹽培養第十天時RNA表現量大量增加。這
三個鹽誘導之cDNA clones在植株表現上有組織特異性。cDNA定序後,核
酸序列比對的結果中發現,SI1和SI2與已知基因序列並無很高的相似性
;轉譯後與已知氨基酸序列的一段domain比對結果發現,SI1與果蠅的
putative sodium channel有53% 的相似性;SI2分別與燕麥的MAP kinase
及番茄的wound-induced protein有68% 及100%的相似性;SI3與阿拉伯芥
及蕃茄的ubiquitin conjugating enzyme (UBC) 的DNA序列有61-62% 的
相似性,氨基酸序列有71-72% 的相似性。

Mesembryanthemum crystallinum (common ice plant) is able to
survive in high saline and water-deficit environments. It has
been used as a model plant to study salt-tolerant mechanisms in
higher plants. When cultured ice plant cells were grown in
medium containing 200 mM NaCl, cells were able to maintain
substantial growth rate without any adaptation process. In this
study, cultured ice plant cells were salt-stressed for five
days. Total RNAs were isolated and reverse transcribed into
cDNAs. After ligatihigh saline and water-deficit environments.
It has been used as a model plant to study salt-tolerant
mechanisms in higher plants. Whencultured ice plant cells were
grown in medium containing 200 mM NaCl,cells were able to
maintain substantial growth rate without anyadaptation process.
In this study, cultured ice plant cells weresalt-stressed for
five days. Total RNAs were isolated and reversetranscribed into
cDNAs. After ligation with specific adaptors (forsubsequent PCR
amplification), the cDNAs isolated from salt-grown cells were
hybridized with excess cDNAs isolated from control cells.The
products of subtractive hybridization with both adaptors
attached were selectively amplified by PCR and the rare salt-
induced transcripts were enriched. PCR products were then
cloned into pBluescript vectors and transformed into XL1-Blue
MRF¢ host cells and 54 clones containing insert were isolated
using blue/white color screening. Slot blot analysis showed
three clones (SI1, SI2, SI3) within seven groups of selected
cDNA were differential expressed between control and salt
treatment. Northern blot analyses showed SI1, SI2 and SI3 were
expressed in callus grown in different concentrations of NaCl.
Highest accumulation of these three salt-induced transcripts all
occurred in treatments of 200 or 300 mM NaCl. These three cDNA
clones also showed tissue-specific expression in salt-stressed
plants. DNA sequence analysis of SI1 and SI2 showed no homology
to any sequence in the GenBank. Deduced amino sequence analysis
of SI1 showed 53% homology to putative sodium channel gene of
fruit fly. SI2 showed high homology to oat MAP kinase and
tomato wound-induced protein. SI3 showed 71-72% homology to
certain subfamily of ubiquitin-conjugating enzyme (UBC) in
higher plants.
URI: http://hdl.handle.net/11455/20663
Appears in Collections:生命科學系所

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