臺灣博碩士論文加值系統

面對外界環境刺激及生長發育的改變，生物體可藉由蛋白質磷酸化及去磷酸化的機制，
經由訊息傳導網路影響各種生理作用。為研究於果實後熟過程中參與訊息傳導之蛋白激
特性，故採用聚合連鎖反應法進行基因選殖。自所獲得之香蕉後熟相關蛋白激cDNA選
殖系中，選取一cDNA選殖系pBPK3進行定序分析，得其全長為1172bp，解碼266個胺基酸，
推演之分子量為29814 Da，等電點8.13。經電腦比對，此基因與阿拉伯芥、老鼠及人類之
dual specific mitogen-activated protein kinase kinase (MAPKK) 基因具較高之相似
性。相對於BPK3基因之表現產物為1.35 kb及2.5 kb，於後熟香蕉果實中表現最強，並隨
果 實後熟程度增加，其mRNA蓄積量呈現明顯增加而後期微降的趨勢。為瞭解於香蕉後熟
過程表現之蛋白激基因，其基因結構特性與可能扮演之生理角色，故混合四個cDNAs (p
BPK3、pBPK6、pBPK9與pBPK10) 為探針，進行基因組庫之篩選。利用溶斑雜交法於一百五
十萬個溶斑形成單位中，共獲得33個相對於此4個cDNAs之基因組選殖系。相對於pBPK3 cD
NA之基因組選殖系lBPSK70，所得序列長度為3644bp，具有一個外顯子而不具內隱子。此
基因 含一長度為1047bp之蛋白質開放解碼框架，共解碼348個胺基酸，推演之分子量為38
329Da，等電點為9.41。另外兩個自香蕉中選殖出的蛋白激基因BPK6及PK5，分別與真核
生物 中的receptor protein kinase/Raf以及ribosomal protein kinase具較高的相似性
，且 此二基因於後熟的果實中均有表現。這些與真核生物MAPK訊息傳導成員 (component
s of MAPK cascade)類似基因之分離，顯示在後熟的果實中，分子訊息之傳導可能利用與
動物 或酵母菌系統類似之訊息傳導途徑。

Reversible protein phosphorylation mechanism is thought to play a key role in
diverse biological signal transduction systems in animal, yeast, and plant. To
identify protein kinases which functions during fruit ripening, we have isola
ted several cDNA clones from Musa spp. using PCR and degenerate oligonucleotid
es designed based on the conserved regions of the catalytic domain of protein
kinases. One of the cDNA clones (pBPK3) was completely sequenced and character
ized.The cDNA clone is 1172bp in length and encodes a polypeptide of 266 amino
acids with a calculated molecular mass of 29,814 Da. The putative BPK3 protei
n shows extensive homology to MAPKK genes from Arabidopsis, yeast and vertebra
tes. Major (1.35kb) and minor (2.5kb) transcripts were detected in bract, anth
er, and most abundantly in ripened banana fruit. The mRNA level of BPK3 increa
sed markedly at stage 2 fruit, remained nearly constant during ripening stages
and slightly decreased at stage 7 fruit. To investigate the structure of pro
tein kinase genes, a genomic library of Musa spp. was screened using four cDNA
s (pBPK3、pBPK6、pBPK9 and pBPK10) as probes. Thirty-three positive clones wer
e obtained by screening 1.5x106 plaques under high-stringency hybridization co
ndition. One of the genomic clone lBPSK70 was characterized and revealed the p
resence of only one exon. The gene is 3644 bp long, and the encoded protein co
ntains an open reading frame of 348 amino acids with calculated molecular mass
of 38,329 Da and predicted pI of 9.41 . Sequence comparison revealed that pBP
K3 cDNA was derived from BPSK70 gene. The two protein kinase genes fragments (
BPK6 and PK5) were also sequenced, and the predicted polypeptides show closely
related to receptor-like serine/threonine kinase / or Raf and ribosomal prote
in kinase, respectively. Northern analysis revealed that the two genes express
ed in ripening banana fruit.The existence of these protein kinase genes has in
dicated that the signaling pathway during fruit ripening may resemble eukaryot
ic MAPK signaling cascades.