苦瓜CTR1同源基因之選殖與表現分析

Cloning and Expression Analysis of Constitutive Triple Response 1 Homologous Genes in Bitter Gourd

蘇世珩(Shih-Heng Su)

指導教授 ： 杜宜殷

共同指導教授 ： 黃鵬林(Pung-Ling Huang)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10321

摘要

為取得苦瓜乙烯訊息傳遞CTR1同源基因及其啟動子，選取苦瓜基因組選殖系λMCTR11及λMCTR16進行定序分析，結果顯示λMCTR16包含苦瓜McCTR1基因之解碼區（coding region），λMCTR11則含有McCTR1啟動子區域長3,656 bp。苦瓜McCTR1具有16個顯子（exon）及15個隱子（Intron），解碼846個胺基酸，屬於Serine/Threonine激酶。利用南方氏雜交分析及限制酶圖譜比對，推測該基因為單一拷貝。根據McCTR1啟動子序列分析結果，可推知苦瓜McCTR1表達會受到乙烯（ethylene）、植物生長素（auxin）、激勃素 (gibberellin)、茉莉酸 (jasmonic acid)、離層酸 (abscisic acid)、水楊酸(salicylic acid)、光、病原菌以及創傷等調控。
經以反轉錄聚合酶連鎖反應（RT-PCR）基因表現分析，得知McCTR1於莖與葉表達量最大，果實與雌花表達量較少。在授粉後不同時間的果實中，此基因於果實發育初期表達量高，授粉後第12天，果實已達生理成熟，因此基因表達量最低，第12天至第21天果實進入後熟階段，乙烯生成量增加，誘導McCTR1表達量增加，至第24天果實完全後熟且黃化腐爛，因此基因表達量亦開始下降。另外，外施乙烯亦會誘導McCTR1表現，以外施10μL/L誘導1小時基因即大量表達，之後隨著處理時間增加，基因表達量又開始下降；以不同濃度的乙烯測試，0.1μL/L誘導最大量的基因表現。不同種類的生長素則以 2,4-dichlorophenoxyacetic acid (2,4-D)以及indole-3-acetic acid (IAA)可誘導McCTR1表現，其中以10-4 M的IAA誘導之McCTR1 mRNA量最大。進一步以離層酸及cycloheximide處理，都會誘導McCTR1表達，而以水楊酸及激勃素則會抑制該基因的表達。
啟動子活性分析結果顯示，菸草轉殖株的GUS活性於根尖與根毛表達量大，葉與莖交接處，也可以偵測到GUS表達。過量表達McCTR1之T1轉殖株共6株，其中三株之外表型出現植株小且葉簇生的現象，另三株之外表型則與野生型無顯著不同。

關鍵字

三相反應 ； 苦瓜 ； 訊息傳遞 ； 乙烯

並列摘要

In order to obtain the promoter and coding sequence of CTR1 homologous gene, two genomic clones λMCTR11 andλMCTR16 isolated from bitter gourd were sequenced and characterized. According to the result of sequence analysis, McCTR1 includes 16 exons and 15 introns. The open reading frame of McCTR1 encodes a 846-amino acid serine/ threonine protein kinase with a molecular mass of 90 kD. McCTR1 belongs to a single or low copy gene in bitter gourd genome based on Southern analysis and restriction mapping. Conserved elements responsive to ethylene, auxin, gibberellin (GA), abscisic acid (ABA), salicylic acid (SA), Jasmonic acid, light, elicitor, and wounding were found in the promoter region.
RT-PCR analysis indicated that McCTR1 mRNA expressed in all organs especially in the stem and leaves and early developmental stage of fruit. The gene expression of McCTR1 was induced by exogenous ethylene especially with 0.1μL/L for 24 hours and 10μL/L for 1 hour. Accumulation of McCTR1 mRNA in bitter gourd fruit was induced after treatment with 10-4 M Indoleacetic acid (IAA) for 30 minutes. Not only IAA, expression of McCTR1 gene was also induced by 2,4-dichloro- phenoxyacetic acid (2,4-D), ABA, SA, and cycloheximide (CHX) but reduced by GA.
On the other hand, tobacco plants transformed with McCTR1::GUS expressed GUS in root tip, root hair, and the junction of leaf and stem. There are 6 T1 Arabidopsis transformed with CaMV35S::McCTR1. The phenotypes of 3 putative transformants are similar to mutant ctr1 and the other three are similar to wild type Arabidopsis.

並列關鍵字

ethylene ； signal transduction ； bitter gourd ； triple respose

參考文獻

Bleecker, A. B. 1999. Ethylene perception and signaling: an evolutionary perspective, Trends Plant Sci. 4: 269-274.

Cao, J. F., P. Linstead, F. Berger, J. Kieber, and L. Dolan. 1999. Differential ethylene sensitivity of epidermal cells is involved in the establishment of cell pattern in the Arabidopsis root. Physiol. Plant. 106:311-317.

Chang, C., and R. Stadler. 2001. Ethylene hormone receptor action in Arabidopsis. BioEssays 23:619-627.

Chang, C., S. F. Kwok, A. B. Bleecker, and E. M. Meyerowitz. 1993. Arabidopsis ethylene – response gene ETR1: Similarity of product to two-component regulators. Science 262:539-544.

Chao, Q., M. Rothenberg, R. Solano, G. Roman, W. Terzaghi, and J. R. Ecker. 1997. Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell 89: 1133-1144.