苦瓜植物生長素及乙烯受體基因之選殖與分析

Cloning and Analysis of Auxin and Ethylene Receptor Genes from Bitter Gourd (Momordica charantia)

李念穎(Nien-Ying Lee)

指導教授 ： 杜宜殷 ； 黃鵬林

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10517

摘要

為瞭解植物生長素及乙烯對苦瓜生長發育之調控機制並加以應用，本論文選殖苦瓜植物生長素受體cDNA、基因及苦瓜乙烯受體cDNA並進行分析。
苦瓜植物生長素cDNA受體是以阿拉伯芥TIR1基因片段為探針，篩選苦瓜cDNA庫得到選殖系pMcAR97，其內含有長1938 bp的苦瓜TIR1 cDNA，可轉譯496個胺基酸，但缺少5’端轉譯區294個核苷酸。進一步以pMcAR97 cDNA為探針篩選苦瓜基因組庫，選取基因組選殖系λMcAR7，經定序分析6693 bp，知苦瓜TIR1基因McTIR1具3個顯子及2個隱子，編碼594個胺基酸，其上具有F-box及白胺酸保守序列，預測之分子量為66.8 kDa，等電點為6.27，與阿拉伯芥及棉花TIR1之胺基酸序列同源性分別為78％與77.2％。分析McTIR1解碼區上游的2.65 kb之啟動子區域，其上具有多種反應序列(responsive element)，可能受光、離層酸、茉莉酸甲酯、高溫逆境、乾旱逆境等因子調控。南方氏雜交分析結果顯示McTIR1為低拷貝基因。北方雜交分析結果顯示McTIR1在果實發育初期表現量大於後期；在不同器官的表現則於幼葉表現量最高，雌花次之，根部表現量最低；於較高濃度IAA處理之果實，表現量略高於低濃度處理者；以5 × 10-5 M IAA處理苦瓜不同時數之試驗中，8小時之果實表現量最高；不同種類植物生長素對McTIR1之表現影響無差異。
以阿拉伯芥乙烯受體ETR1基因片段為探針，篩選苦瓜cDNA庫，得到兩種苦瓜乙烯受體cDNA。選殖系pMcER102所含之cDNA長2,800 bp，屬於ETR1型乙烯受體cDNA，對應之基因命名為McETR1。McETR1編碼之蛋白質具有741個胺基酸，推演之分子量為82.73 kDa，等電點為6.79。選殖系pMcER287-4所含之cDNA長2,521 bp，屬於ERS1型乙烯受體cDNA，對應之基因命名為McERS1。McERS1編碼之蛋白質具有637個胺基酸，推演之分子量為71.06 kDa，等電點為6.37。McETR1及McERS1之胺基酸序列N端具有3個疏水性保守區，推測為跨膜組區，近C端之組胺酸激酶組區上則有乙烯受體五個序列保守區H、N、G、F、及G，但McETR1具有磷酸根分子接受區，McERS1則不具該區。分別比對各物種之ETR1型與ERS1型乙烯受體胺基酸序列，顯示McETR1與阿拉伯芥ETR1之同源性為81.8%，與甜瓜和胡瓜之ETR1乙烯受體同源性最高，分別達95.0% 及94.2%；McERS1與阿拉伯芥ERS1之同源性為70%，亦與甜瓜和胡瓜之ERS1型乙烯受體同源性最高，達95.5% 及95.1%；McERS1與雙子葉作物ERS1型乙烯受體之序列同源性較高，且同科作物間的乙烯受體胺基酸序列同源性較大。

關鍵字

植物生長素受體 ； 苦瓜 ； 乙烯受體 ； 訊息傳導 ； TIR１

並列摘要

To understand the regulation mechanism of auxin and ethylene in bitter gourd, the auxin receptor gene and the ethylene receptor cDNA were cloned from bitter gourd for analysis.
The fragment of TIR1 gene from Arabidopsis was used as probe to screen the cDNA library of bitter gourd. The TIR1 cDNA in clone pMcAR97 consists of 1938 bp and encodes 496 amino acids, lacking 98 amino acids in 5’ terminus. The pMcAR97 cDNA was then used to screen the genomic library of bitter gourd. The genomic clone λMcAR7 contains McTIR1 gene with 3 exons and 2 introns. McTIR1 encodes an open reading frame consisting of 594 amino acids with a calculated molecular weight of 66.8 kDa and a predicted pI of 6.27. F-box motif and Leucine-rich repeats are found in the amino acid sequence. The McTIR1 protein exhibits 78.0% and 77.2% identity in amino acid sequences with TIR1 from Arabidopsis thaliana and Gossypium hirsutum, respectively. Many predicted responsive elements related to light, ABA, MeJA, heat stress were found in the sequence of 2.65 kb 5’-flanking region. Southern analysis indicated that McTIR1 is a low-copy gene. Northern analysis indicated that McTIR1 expressed more in the early developmental stage of bitter gourd. The McTIR1 mRNA was most abundant in young leaves and female flowers, and least in roots of bitter gourd. Gene expression of McTIR1 was induced by higher concentration of IAA treatment in the fruit sections. McTIR1 expressed abundant mRNA in fruit sections under 5 × 10 -5 M IAA treatment for 8 hrs. Different kinds of auxin shows no effect to the expression of McTIR1.
The fragment of ETR1 gene from Arabidopsis was used as probe to screen the cDNA library from bitter gourd. The cDNA in clone pMcER102 consists of 2,800 base pairs. The coding protein of pMcER102 cDNA, named McETR1, consists of 741 amino acids with a calculated molecular mass of 82.73 kDa and a predicted isoelectric point of 6.79. Another clone pMcER287-4 also contains an ethylene receptor cDNA, which consists of 2,521 base pairs. The coding protein, named McERS1, consists of 637 amino acids with a calculated molecular mass of 71.06 kDa and a predicted isoelectric point of 6.37. Both of McETR1 and McTIR1 contains three predicted hydrophobic transmembrane -spanning domains in the N-terminal region, the histidine kinase domain near the C terminus, and possesses 5 conserved motifs “H, N, G1, F, G2”. However, McETR1 contains a receiver domain in the C terminus while McERS1 doesn’t. The predicted McETR1 protein exhibits 81.8% identity in amino acid sequence with Arabidopsis ETR1, and exhibits 95.0% and 94.1% identity with ETR1 sequences from Cucumis melo and C. sativus, respectively. The predicted McERS1 protein exhibits 70.5% identity in amino acid sequence with Arabidopsis ERS1, and exhibits 95.5% and 95.1% identity with ERS1 sequences from Cucumis melo and C. sativus, respectively. Ethylene receptors from plants among the same family or among monocotyledons share higher amino acid sequence identity to each other. The predicted McERS1 protein shares higher amino acid sequence identity with ERS1 ethylene receptors from dicotyledons than from monocotyledons.

並列關鍵字

Auxin receptor ； Bitter gourd ； Ethylene receptor ； signal transduction ； TIR1

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