香蕉ABC轉運蛋白cDNA之選殖及基因啟動子活性分析

Cloning of cDNA and analysis of gene promoter activity for ABC (ATP-binding cassette) transporter in banana

葉曉君(Hsiao-Jiun Yeh)

指導教授 ： 黃鵬林

共同指導教授 ： 杜宜殷

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10438

摘要

為了探討香蕉ABC轉運蛋白 (ATP binding cassette transporter) 之基因結構及其啟動子表現特性，以香蕉基因組選殖系為探針，利用溶斑雜交法篩選香蕉cDNA庫共ㄧ百五十萬個溶斑形成單位 (plaque forming unit, pfu) ，得到兩類ABC轉運蛋白cDNA，第一類選殖系選取pMaABC-29進行全長定序其序列，總長度為4731 bp，可演繹出全長1452個胺基酸，預測蛋白分子量163 kDa、等電點 (Isoelectronic point) 為8.20；第二類以選殖系pMaABC-74P為代表，cDNA序列長度為4760 bp，為香蕉ABC轉運蛋白Mh-ABC1基因之同源對應cDNA，可轉譯1468個胺基酸，預測分子量為165 kDa、等電點為8.50，比對cDNA及基因之序列，得知此基因在基因解碼區內具23個顯子。兩類香蕉ABC轉運蛋白所演繹之多胜肽皆屬於PDR次家族 (pleiotropic drug resistance subfamily) 之蛋白，胺基酸序列與稻米之PDR蛋白同源性較高。以pMaABC-74P cDNA為探針，進行南方氏雜交分析結果顯示，雜交片段符合基因組選殖系之限制酶圖譜，且此類ABC轉運蛋白基因在香蕉基因組中為單一拷貝或低拷貝基因。以另一cDNA選殖系pMaABC-29 cDNA為探針進行南方氏雜交分析，結果顯示此基因為單一拷貝或低拷貝基因。
利用菸草轉殖株進行啟動子活性分析結果顯示，只有不含預測intron1及5’-UTR之構築pBI121Bid，可在苗期位於下胚軸靠近莖頂分生組織之部位表現GUS活性；且表現隨著苗期發育會逐漸減弱，而其他構築在苗期均無GUS活性之表達，在未開放小花花苞中觀察到，pBI121B、pB2、pBid2轉殖株的花器中，啟動子活性僅專一表達於花藥；pBI121C啟動子活性表現在萼片、柱頭、花柱、子房及花藥等處，而pBI121Bid轉殖株之花器的GUS活性則是表達於花藥與子房中。
Mh-ABC1啟動子序列分析結果，得知在Mh-ABC1啟動子區域中存在數十個可能受到植物荷爾蒙如離層酸 (abscisic acid)、激勃素(gibberellic acid)、茉莉酸 (jasmonic acid)及水楊酸 (salicylic acid, SA) 等所調控之序列，此外亦存在受到環境因子如光線、病原菌 (pathogen)、鹽類 (salt)、乾旱缺水 (drought / dehydration)、低溫 (cold)、病原菌細胞萃取物 (elicitor) 等調控之保守區域。SA處理會促使pBI121Bid轉殖株小苗GUS表達強度增加，50 mM CuSO4處理五種構築之菸草轉殖株小苗，皆可在植株下胚軸與根部交接處偵測到基因表現，Ca2+離子對於pBI121Bid 轉殖株內之GUS活性亦有促進之效果；Al2(SO4)3處理則促進pB2菸草轉殖株小苗全株皆表現GUS活性，同時也能增強pBI121Bid轉殖植株於下胚軸及子葉報導基因之表達。

關鍵字

ABC轉運蛋白

並列摘要

To understand the gene sturcture of banana ABC transporter, plaque hybridization was performed to screen for banana cDNA library. Using gene fragments of Mh-ABC1 as probe., two types of ABC transporter cDNA were obtained. pMaABC-29 cDNA is 4752 bp long encoding, a polypeptide of 1468 amino acids with a isoelectric point of 8.50 whose molecular weight is 165 kDa. The other type of cDNA from clone pMaABC-74P, 4760 bp in length, is
homologous to Mh-ABC1 with 99% homology in nucleotide sequence. Both types of banana ABC transporter belong to PDR (pleiotropic drug resistance) subfamily and share high homology to the PDR proteins in rice. Southern blot
analysis indicated that hybridized fragments using pMaABC-74P as probe match well with the restriction map of Mh-ABC1 gene and this gene belongs to a single- or low-copy gene. According to the Southern analysis using
pMaABC-29 cDNA as probe, the hybridization signals revealed that this gene is also a single- or low-copy gene.

On the other hand,β-glucuonidase (GUS) gene driven by different regions of Mh-ABC1 promoter were transformed into tobacco and the transgenic plants were used to study the promoter activity of Mh-ABC1. Only pBI121Bid, containing 5’-flanking sequence without putative intron 1 and 5’-UTR, expressed GUS activity in seedling stage. The position of its GUS activity lies in the hyposotyl and the apical meristem. In flower bud, pBI121B, pB2, pBid2
transgenic plant only express GUS activity in anther. Complete 4.5 Kb 5’-flanking sequence of Mh-ABC1 express of GUS gene in bract, stigma, style,ovary and anther of transgenic tobacco. pBI121Bid expressed GUS activity in
anther and ovary. Furthermore, analysis of promoter region from Mh-ABC1 revealed that several conserved cis-acting elements responsive to abscisic acid, gibberellic acid, jasmonic acid, salicylic acid (SA), light, pathogen, salt,
drought/dehydration, cold and elicitor, are present. SA treatment enhanced the promoter activity in pBI121Bid transgenic seedlings. Promoter activity of transgenic plants was induced by 50 mM CuSO4 and expression of GUS
activity was found at the border of hypocotyl and root in all of the five constructs used for this study. Moreover, promoter activity in pBI121Bid transgenic plant was elevated by Ca2+ treatment. Upon treatment with Al2(SO4)3, the level of GUS expression was increased in entire of pB2
transgenic plants and in apical meristem of pBI121Bid transgenic plants.

並列關鍵字

ABC (ATP-binding cassette) transporter

參考文獻

Biemans-ldehinkel, E., M. K. Doeven, and B. Poolman. 2006. ABC transporter architecture and regulatory roles of accessory domains. FEBS

Bovet, L., U. Feller, and E. Martinoia. 2005. Possible involvement of plant ABC transporters in cadmium detoxification: a cDNA sub-microarray approach. Environ Int. 31: 263-267.

Ducos, E., S. Fraysse, and M. Boutry. 2005. NtPDR3, an iron-deficiency inducible ABC transporter in Nicotiana tabacum. FEBS Lett. 579: 6791-6795.

Eichhorn, H., M. Klinghammer, P. Becht, and R. Tenhaken. 2006. Isolation of a novel ABC-transporter gene from soybean induced by salicylic acid. J Exp. Bot. 57: 2193-2201.

Gaedeke, N., M. Klein, U. Kolukisaoglu, C. Forestier, A. Muller, M. Ansorge, D. Becker, Y. Mamnun, K. Kuchler, B. Schulz, B. Mueller-Roeber, and E. Martinoia. 2001. The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement. EMBO J. 20: 1875-1887.