蝴蝶蘭MYB基因之選殖及其啟動子活性分析

Cloning and Promoter Activity Analysis of MYB Genes from Phalaenopsis

許堯斐(Yao-Fei Hsu)

指導教授 ： 黃鵬林

共同指導教授 ： 杜宜殷(Yi-Yin Do)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10529

摘要

MYB (myeloblastosis) 基因之轉譯蛋白為一種轉錄因子 (transcription factor)，高等植物主要以R2R3-MYB群為主，與苯基丙烷代謝路徑 (phenylpropanoid metabolism pathway)、生物性及非生物性逆境調控相關。為了解蝴蝶蘭MYB基因結構及表現調控情形，本研究利用蝴蝶蘭轉錄因子PtMYB3之 cDNA作為探針，篩選蝴蝶蘭基因組庫，獲得PtMYB3完整基因，該基因具有三個顯子與兩個隱子，且隱子邊界序列符合GT-AG規則，基因之三個顯子長度依序為263 bp、672 bp及16 bp；而啟動子序列經比對後，具有離層酸、激勃素、高溫、低溫、光照、茉莉酸及水楊酸等因子之調控序列。啟動子活性分析結果顯示，PtMYB1啟動子可於菸草子葉、葉片和莖頂分生組織表現，於花器中，可於花瓣、柱頭及花藥表現，但未在果實中偵測到啟動子活性，而以MeJA處理菸草PtMYB1pro::GUS轉殖株啟動子活性表現較對照組強，且可誘導根部之啟動子活性表現，而經高溫及黑暗處理則會抑制啟動子活性表現；而PtMYB2啟動子可於阿拉伯芥之葉片、葉片之毛狀體、下胚軸、莖頂分生組織、花萼及果實外皮表現，並且可被IAA、GA、SA、MeJA誘導而增加啟動子活性於葉片之表現量，而經黑暗培養、4℃、創傷及乾旱，可增加PtMYB2啟動子活性表現，以37℃高溫處理後，啟動子活性受到抑制，PtMYB2啟動子於菸草轉殖株之分子驗證已完成，待取得菸草之轉殖純系種子，將進行不同植物生長調節劑及非生物逆境之啟動子誘導分析；而PtMYB3啟動子可於阿拉伯芥之葉片、果實外皮、花萼、柱頭及花藥表現，接著會進行不同植物生長調節劑及非生物逆境之啟動子誘導分析，並完成PtMYB3啟動子於菸草轉殖株之分子驗證。

關鍵字

蝴蝶蘭 ； MYB ； 轉錄因子 ； 基因結構 ； 啟動子活性分析

並列摘要

The MYB (myeloblastosis) transcription factors are present in all eukaryotes and can be classified into three subfamilies depending on the number of characteristic repeats in the MYB domain as referred to as R1, R2, and R3. The R2R3-MYB subgroup mainly exists in higher plants, involving in the regulation of phenylpropanoid metabolism pathway, botic and abiotic stress. To understand gene structures and expression patterns of MYB genes, the cDNA of PtMYB3 was used as probes to screen the genomic library of Phalaenopsis. PtMYB3 gene contains three exons and two introns and the boundaries between exons and introns follow the GT-AG rule. The lengths of three exons are 263 bp, 672 bp and 16 bp, respectively. Based on the analysis of the promoter regions of PtMYB3, some cis-acting elements related to abscisic acid, gibberellic acid, high temperature, low temperature, light, methyl jasmonate and salicylic acid were found. In this research the promoter regions of PtMYB1, PtMYB2, and PtMYB3 were isolated and fused to the uidA (β-glucuronidase) reporter gene to access promoter activity. In the PtMYB1pro::GUS transgenic tobacco, the promoter activity of PtMYB1 was expressed in cotyledon, leaf, apical meristem, and petals of the flower, but no expression of PtMYB1 in fruits was found. The expression of PtMYB1 promoter can be enhanced by methyl jasmonate (MeJA) in roots but supressed by heat shock and dark. The promoter of PtMYB2 was expressed in leaf, trichome, hypocotyls, apical mristem, calyx and peel and can be enhanced by IAA, GA, salicylic acid, and MeJA. The expression of this promoter can be enhanced by dark, 4℃treatment, wounding and drought, but suppressed by 37℃treatment. Gene integrity and copy number for PtMYB2pro::GUS transgenic tobacco plants has been analyzed by Southern hybridization. The promoter of PtMYB3 was expressed in leaf, peel, calyx, pistil, and stamen. Gene integrity and copy number for PtMYB3pro::GUS transgenic tobacco plants will be done. After harvesting the seeds of PtMYB3pro::GUS transgenic tobacco and Arabidopsis, the induction test for different plant regulators and stresses will be conducted.

並列關鍵字

Phalaenopsis ； MYB ； transcription factor ； gene structure ； promoter activity analysis

參考文獻

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