番茄果實專一性過氧化酶基因之分子特性

Molecular characterization of fruit-specific class III peroxidase genes in tomato

王啓正(Chii-Jeng Wang)

指導教授 ： 葉開溫

國立臺灣大學/生命科學院/植物科學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.02247

摘要

為了研究番茄果實專一性第三群過氧化酶基因（以下簡稱過氧化酶基因）之特性與功能，本研究從番茄未熟果選殖出來4個過氧化酶基因：LePrx09、LePrx17、 LePrx35及 LePrxA，其中LePrx35 及LePrxA為新的番茄過氧化酶基因。蛋白質的二維結構分析顯示此4個過氧化酶基因具有第三群過氧化酶的特性，也就是具有B、D、F的保守區域，這些區域為與heme結構及2個鈣離子交互反應的保守氨基酸序列、穩定分子結構的鹽橋及4個雙硫鍵，因此確認其為第三群過氧化酶基因無誤。系統分子演化分析24個或118個茄科作物過氧化酶蛋白序列可分成3群，LePrx09、LePrx17、LePrx35及LePrxA分別與受傷、病害及木質素合成相關的過氧化酶在同一群中，因此推斷此4個基因功能可能與受傷、病害及木質素合成有關。經RNA表現部位分析顯示，LePrx35及LePrxA僅在未熟果中表達，LePrx17 及 LePrx09 則在未熟果及綠熟果中表達，且LePrx09則主要在綠色果實中的中果皮表達，其他3個基因則主要在果肉組織中表達，利用基因槍傳送LePrx09啟動子驅動GUS基因的啟動子活性分析也支持此一結果。cis-element分析顯示LePrx09、LePrx17、LePrx35及LePrxA之啟動子區域至少含有1個負責因糖抑制（sugar- repressive）的調控片段，還有許多與受傷，病原及荷爾蒙反應有關的cis-elements，此外，LePrx09，LePrx17及 LePrxA的啟動子有果實專一性的cis-elements：TGTTACA或TCCAAAA。使用番茄未熟果進行各種荷爾蒙、受傷處理、病原菌感染及過氧化氫處理， LePrx09之表達量可被ethephon、茉莉酸、水楊酸、受傷處理、早疫病原菌感染及過氧化氫所誘導而增加，LePrx17表達量可被茉莉酸處理及早疫病原菌感染所誘導增加。利用基因轉殖大量表達LePrx09，可使番茄的轉殖株葉片對過氧化氫具有抗性，大量表達其他3個過氧化酶基因之轉殖植株葉片對過氧化氫之抗性則無顯著增加，推論LePrx09參與過氧化氫的訊息傳導徑路並調整果實的生長及抗逆境能力．因此，未來若能找到LePrx09的啟動子區域及其上游基因的序列在抗逆境種原的關聯性，將可作為番茄抗逆境或抗病性育種的分子標誌。

關鍵字

番茄 ； 第三群過氧化酶基因 ； 果實專一性

並列摘要

To research the characters and function of fruit-specific class III peroxidase gene of tomato, expression of four tomato class III peroxidase (LePrx) genes, LePrx09, LePrx17, LePrx35 and LePrxA, was isolated and charactered from immature tomato fruits, and the function in the regulation of fruit growth was studied. LePrx35 and LePrxA were identified as novel peroxidase genes in tomato. Analysis of amino acid sequences revealed that these genes are belonged to class III peroxidases group and contain B, D and F conserved domains, which bind heme groups, two calcium atoms, and form a stable molecular structure by a buried salt bridge motif and four disulphide bridges. Phylogenetic analysis for 24 and 118 full-length peroxidase proteins of Solanaceae crop species show three groups constructed and some wounding, pathogen and lignin synthesis related peroxidases were grouped with the four LePrxs. The temporal expression patterns at various fruit growth stages revealed that LePrx35 and LePrxA were expressed only in immature green fruits, whereas LePrx17 and LePrx09 were expressed in both immature and mature green fruits. Cis-element analysis revealed that all the upstream of the four LePrxs have at least sugar repressive element and other wounding, pathogen and hormones related cis-elements. The promoter of LePrx09, LePrx17 and LePrxA has fruit-specific cis-elements, TGTTACA or TCCAAAA. Moreover, tissue-specific expression profiles indicated that only LePrx09 was mainly expressed in the mesocarp but not the inner tissue of immature fruits. Histochemical localization of GUS activity analysis also revealed the mesocarp-expression pattern. The effects of hormone treatments and stresses on the four genes were examined; only the expression levels of LePrx17 and LePrx09 were altered. Transcription of LePrx17 was up-regulated by jasmonic acid (JA) and pathogen infection, and expression of LePrx09 was induced by ethephon, salicylic acid (SA) and JA, in particular, as well as wounding, pathogen infection and H2O2 stress. Tomato plants over-expressing LePrx09 displaying enhanced resistance to H2O2 stress, suggesting that LePrx09 may participate in the H2O2 signaling pathway to regulate fruit growth and disease resistance in tomato fruits. Thus, if the sequences of LePrx09 promoter region and its upstream genes were involved in the stresses resistant tomato entries, it would be as a useful marker for stresses resistance breeding.

並列關鍵字

tomato ； class III peroxidase genes ； fruit-specific

參考文獻

6. References

Amaya I, Botella MA, de la Calle M, Medina MI, Heredia A, Bressan RA, Hasegawa PM, Quesada MA, Valpuesta V (1999) Improved germination under osmotic stress of tobacco plants overexpressing a cell wall peroxidase. FEBS Letters 457: 80-84

Andrews J, Adams SR, Burton KS, Edmondson RN (2002a) Partial purification of tomato fruit peroxidase and its effect on the mechanical properties of tomato fruit skin. Journal of Experimental Botany 53: 2393-2399

Andrews J, Adams SR, Burton KS, Evered CE (2002b) Subcellular localization of peroxidase in tomato fruit skin and the possible implications for the regulation of fruit growth. Journal of Experimental Botany 53: 2185-2191

Andrews J, Malone M, Thompson DS, Ho LC, Burton KS (2000) Peroxidase isozyme patterns in the skin of maturing tomato fruit. Plant, Cell & Environment 23: 415-422