香蕉乙烯訊息傳導MhCTR1基因之表現及啟動子活性分析

Abstract

香蕉為典型的更年型果實，具有重要經濟價值。香蕉果實發育具更年型果實重要特徵，由成熟至後熟階段，乙烯生成量明顯增加，伴隨著呼吸率的上升。為釐清乙烯訊息傳導如何調控香蕉果實後熟，由香蕉 (Hsien Jin Chiao, AAA group)基因組庫篩選分離出阿拉伯芥CTR1 (constitutive triple response 1)同源基因，並命名為MhCTR1。依據基因顯子之組成，MhCTR1與阿拉伯芥AtCTR1、番茄LeCTR1及LeCTR4分屬同群。MhCTR1基因主要表現部位為綠熟果實之果肉及根組織。在果實後熟時期，MhCTR1基因在乙烯生成量增加前有明顯表現，而其主要表達於香蕉果皮開始轉黃之後熟階段3的果肉；MhCTR1基因在後熟果皮則呈持續表現情形。以0.01 μL L-1濃度之乙烯處理香蕉綠熟果實，會增加MhCTR1基因表現。以10 μL L-1濃度處理香蕉綠熟果實，24小時後在果皮及果肉的MhCTR1基因表現均呈下降趨勢。MhCTR1基因在果肉明顯的表現，顯示MhCTR1與果實後熟過程之調控具相關性。 以MhCTR1::GUS阿拉伯芥轉殖株進行MhCTR1基因啟動子活性分析，GUS活性組織化學染色分析結果顯示，MhCTR1基因普遍表現於阿拉伯芥營養組織及生殖組織，在營養組織主要表現於子葉、新葉、葉脈及tricomes；在生殖組織表現於花苞、盛開花的柱頭、花柱、花藥及花瓣。在花朵發育過程，從花苞至盛開花，MhCTR1基因在雌蕊柱頭及雄蕊的花藥及花絲表現逐漸增加；在果實發育過程，MhCTR1基因在長角果的莢片、胎座框、珠柄及花托等部位均有表現；而隨種子發育逐漸成熟，在莢片及胎座框等部位MhCTR1基因表現逐漸下降。以100 μM ABA及7% mannitol處理轉殖株，會抑制MhCTR1基因啟動子的表現。將光照培養3天大的轉殖株移置黑暗中3天，轉殖株呈現典型白化苗型態，而下胚軸表皮及皮層細胞的MhCTR1基因啟動子活性受到抑制，僅表現於中柱。 以CaMV 35S啟動子接上MhCTR1基因cDNA序列，並將表現載體轉殖至阿拉伯芥突變體ctr1，進行互補性試驗分析。CaMV 35S::MhCTR1轉殖系幼苗置於黑暗培養3天，其幼苗呈現的白化苗型態與正常植株相同，而其胚根及下胚軸長度與正常植株相似。分析CaMV 35S::MhCTR1轉殖系4週大植株的葉長及根長，結果顯示其葉長與正常植株長度類似，而其根長則介於正常植株與ctr1突變體之間。利用互補性分析試驗，顯示香蕉MhCTR1基因產物功能與AtCTR1類似。 以MhCTR1基因cDNA序列接上GFP基因序列，並將此重組蛋白基因暫時性表現於阿拉伯芥原生質體。蛋白定位結果顯示，MhCTR1-GFP位於內質網(ER)，與ER標定蛋白表現位置相符。經比對高基氏體及液胞標定蛋白表現型態，顯示MhCTR1-GFP並未表達於高基氏體及液胞。

The banana (Musa spp.) is a typical climacteric fruit of high economic importance. The development of bananas from maturing to ripening is characterized by increased ethylene production accompanied by a respiration burst. To elucidate the signal transduction pathway involved in the ethylene regulation of banana ripening, a gene homologous to Arabidopsis CTR1 (constitutive triple response 1) was isolated from Musa spp. (Hsien Jin Chiao, AAA group) and was designated as MhCTR1. According to the exon composition, MhCTR1, AtCTR1, LeCTR1 and LeCTR4 belong to CTR1 orthologs group I. MhCTR1 is expressed mostly in the mature green pulp and root organs. During fruit development MhCTR1 expression increases just before ethylene production rises. Moreover, MhCTR1 expression was detected mainly in the pulps at ripening stage 3, and correlated with the onset of peel yellowing, while MhCTR1 was constitutively expressed in the peels. MhCTR1 expression could be induced by ethylene treatment (0.01 μL L-1), and MhCTR1 expression decreased in both peel and pulp 24 h after treatment. Overall, changes observed in MhCTR1 expression in the pulp closely related to the regulation of the banana ripening process. The promoter of MhCTR1 gene was fused to GUS reporter gene and introduced into Arabidopsis to produce MhCTR1::GUS transgenic line. Histochemic staining analysis of the transgenic plant revealed that promoter of MhCTR1 gene is active ubiquitously in the period of growth, both in vegetative and reproductive organs. In vegetative tissues, MhCTR1 promoter expressed majorly in cotyledons, young leave, veins and tricomes of mature leave; in reproductive tissues, the promoter activity of MhCTR1 gene is found in floral bud, stamen, pistil, and petal of flower in anthesis. During the flower developmental stage, the promoter activity of MhCTR1 gene is increased gradually in stamen and the stigma of pistil. During the silique development, MhCTR1 promoter expressed obviously in valves, replum and receptacle of immature silique. When the seeds of silique run into mature, the expression of MhCTR1 gene was decreased concomitantly. In the 10-day-old transgenic plant, the expression of MhCTR1 gene was inhibited by treatment of 100 μM ABA and 7% mannitol for 24 hours. When the 3-day-old light-grown seedling was transferred into dark for 3 days, the expression of MhCTR1 gene was abolished in epidermal and cortex cells of hypocotyls and just only expressed in vascular tissue. The complementary test was used to investigate whether or not the banana MhCTR1 gene product functions as ethylene signaling component AtCTR1. CaMV 35S::MhCTR1 construct was introduced into ctr1 mutant to produce the transgenic line. The dark-grown 3-day-old seedling of complementary line restored the inhibited hypocotyl and root length of ctr1 mutant to etiolating phenotype of wild type. MhCTR1 gene restored the mutant to wild type in rosette leave length, and partially recovery the root length from mutant to wild type in 4-week-old adult plant. The construct of MhCTR1 gene tagged with GFP was transiently expressed in protoplast isolated from Arabidopsis leave. In vivo localization analysis revealed that the MhCTR1 was targeted to endoplasmic reticulum (ER) and the expression pattern overlapped with the stained pattern of ER marker protein. In contrast, co-localization of MhCTR1-GFP with RFP-tagged markers for Golgi apparatus and vacuole were not detected in this study.