Molecular Studies on Sweet Protein Mabinlin; Thermal Stability By LEUNG Chun-wah A Thesis Submitted in Partial fulfillment of the Requirement for the degree of Master ofPhilosophy in Biology ©The Chinese University ofHong Kong July 2000 The Chinese University ofHong Kong holds the copyright ofthis thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School. Thesis Committee Prof. Sun S. S. M. (Supervisor) Prof. Lam H. M. (hitemal examiner) Prof. Wong Y. S. (Internal examiner) Prof. Wayne M. Becker (External examiner) Statement All the experimental work reported in this thesis was performed by the author, unless specially stated otherwise in the text. LEUNG Chun-wah Acknowledgment First ofall, I would like to express my gratitude to my supervisor, Prof. Sun S.S.M. for getting me into the field of Biotechnology. I am indebted to his invaluable experience, guidance, understanding and tolerance all the way through. I am also grateful to Prof. Sun S.S.M. for his insightful comments and patient reviews (even during his vacation) to the drafts ofthis thesis. I would like to express my heartfelt appreciation for the valuable advice and encouragement to the members of my thesis committee, Prof. Wong Y.S. and Prof. Lam H.M. My gratitude should also go to my extemal examiner, Prof. Wayne M. Becker for his patience ofreviewing my thesis. I am also thankful for the generosity ofProf. Lam H.M. for translating the Chinese version of my abstract, providing the Agrobacterial strain and bringing about the vaccum infiltration technique in my research work. Particularly, I would like to acknowledge Dr. Linda Ooi for her teachings and technical suggestions and Prof. Ooi V.E.C. for his caring. Their research experience and attitude is valuable to me in the course of my research study. In addition, I am indebted to the continuous encouragement and technical assistance of my labmates (Marilyn Yu M.L., Simon Cheng M.K.， Donna Lee C.，Danny Ng W.K., Angela Yu W.S., Rosalyn Chen L., Paulus Fong M.K., Zhang Xiaodong, Stanley Cheung C.K., Jones Zhang S.Y., Gundam Wong H.W., Juon Juon Lee J.K., Yuan Dingyang, Smile Duan M.J., 111 Xiao Guoying). The quality of my thesis was greatly enhanced by the effort and cooperation from them. I would also like to express special thanks to Dr. Lucia Liu P.S. for her sharing of computer knowledge and constructive suggestions. My appreciation should also go to many parties in the department (Mr. Jack Lo C.S.，Miss May Yam K.M., Mr. Lung Chan Wl.，Mr. Chan Y.B.，Miss Grace Leung S.W.，Mr. Eric Suen P.K., Mr. Arthur Lee CL.，Miss Maggie Kwan P.C.，Miss Elizabeth Wilhnott, all technicians from team C and team A， members from Prof. Lam H.M.'s lab, Prof. Fung M.C.'s lab, Prof. Ge Wei's lab, Prof. Wong Y.S.'s lab, Prof. Kwan H.S.'s lab) for their lab-material support, technical and emotional sharing. Besides, the spiritual support and technical advice from my friends and previous colleagues (flatmates from HKU, a-dozen-people from HKU A&P Biotechnology, labmates from HKU Leung P.C.，s lab and HKIB, old friends from CACWGC, impossible to name all) is indispensible for the completion of my thesis. Last but not least, I would like to express my deepest gratitude to my dearest parents, sister (Jenny) and brother (Jimmy). Their unconditional love, infinite understanding, emotional support and caring have been the greatest help to finish my postgraduate study. IV Abstract Sweet plant proteins have the potential to substitute for sucrose as an alternative natural sweetener in low-calorie and diabetic products. However, most sweet plant proteins lose their sweetness upon high temperature treatment and cleavage of disulfide bridges is a main factor of their heat- instability. Mabinlin (MBL), a plant protein with four isoforms (MBL I，II，III & IV) and 400 times sweeter than sucrose, shows high heat-stability in three (MBL II，III & IV) of its four isoforms. Previous biochemical and molecular studies also revealed that each MBL isoform consists of two subunit polypeptides; the four MBLs possess highly similar amino acid sequences; and all four disulfide bridges are at the same positions in both the heat-stable (MBL II，III & IV) and unstable (MBL I) isoforms. The later structural feature suggests that the disulfide bridges may not be the sole determinant of heat stability in MBLs. A single amino acid difference, at position #47 in the large subunit, however, was found between the two groups, i.e. Arg in MBL II, III & IV while Gki in MBL I. hi view of these findings, we are attempting to explore the underlying determinant(s) ofheat stability in the MBLs. Site-directed mutagenesis was performed in the cDNAs encoding the heat-stable MBL III and unstable MBL I in an attempt to generate single amino acid mutations at position #47 of the large subunits, either to reverse (by Arg to Gln or vice versa) or to maintain (by similar amino acids, Gly & Asp) their heat-stability properties. Both the wild-type and mutant MBL III and MBL I cDNAs were used to construct chimeric genes under the control of the phaseolin regulatory regions of French bean. The chimeric genes were transferred into the Arabidopsis thaliana genome via Agrobacterium-rnQdmied transformation. Plants containing a single copy of the transgene were selected and propagated to obtain homozygous R3 generation for expression analysis, as a means of setting up a system to test the relationships between heat stability and MBL primary structure. For transgenic plants containing the chimeric genes MBL III Q)BI / phas / MBLIII-wt, -GLN, & -LYS)，the activity of the marker gene was detected by GUS assay. The integration of the MBL III transgenes was proved by polymerase chain reaction (PCR), and the MBL III RNA transcripts were detected by reverse transcritption-PCR (RT-PCR) and DNA sequencing. For plants transformed with the chimeric genes MBL I (pBl / phas / MBLI-wt, -ARG, -ASP), Ri seeds had been harvested for future analysis. To detect the MBL protein in transgenic seeds, antisera with very high titres against MBL were obtained. Tricine SDS-PAGE, protein sequencing, isoelectric precipitation, and westem blot and immunodetection had been attempted to demonstrate the stable accumulation of MBL III protein in transgenic seeds. However, despite the proven presence of their transcripts, detectable levels of MBL proteins have thus far not been confirmed in the transgenic seeds. The similar size and immuno-cross-reactivity of the 2S protein of the host plant Arabidopsis with the MBL protein compounds were the difficulties in detecting the MBL. Further experiments that could separate the two low molecular weight proteins and enhance the protein detection sensitivity would be desirable, in addition to experiments to determine if the MBLs are unstable in the transgenic Arabidopsis seeds. VI 摘要 「甜蛋白」有潛力替代鹿糖成爲一種低卡路里和可用於高血糖病 患者食品的天然甜味劑。然而，絕大多數植物甜蛋白在高溫處理下會喪 失甜味，二硫鍵被打斷是熱不穩定性的一個主要因素°馬檳榔甜蛋白 (MBL)比簾糖甜400倍，它是一群有四種同工型(MBL I，II，III及IV)的 植物甜蛋白，其中n，111及〜三種同工型表現出高熱穩定性。以往的生 化及分子生物硏究發現每種MBL同工型有兩個多駄亞基0所有馬檳榔 甜蛋白都擁有高度類近的氨基酸序列，而且在熱穩定及不穩定型MBL 內四個二硫鍵的位置都是相同的’這結構的特點顯示二硫鍵並不是決定 熱穩定性的唯一因素。然而’大亞基第47位氣基酸在這熱穩定及不穩 定型兩類MBL中卻有分別（Arg在MBL II，III及IV ； Gb在MBL I)。 基於此發現，我們試圖探索MBL熱穩定性背後的關鍵決定因素。 利用特點誘變技術，可以改變熱穩定型MBL III和熱不穩定型 MBL I的cDNAs。目的是通過在大亞基第47位的氨基酸產生突變，逆 轉（Arg改爲Ghi或倒轉）或維持（如Lys和Asp等同類氨基酸）其氣基酸 及特性，以測試其對熱穩定性的影響。MBL III及I原來的及誘變後的 cDNAs都會受菜豆蛋白基因調控區嵌合並受其調控。嵌合的基因以農杆 菌感染轉化至擬南芥菜基因組內。 歸選出被插入單拷貝基因的轉化植物後，經過增殖再獲得純合的 第三代轉基因植物，目標是建立一套測定熱穩定性與MBL —元結構關 係的糸統。 嵌合基因及其表達載體PBI / phas / MBLIII-wt, -GLN及-LYS，pBI / phas / MBLI-wt, -ARG及-ASP的構建已完成。這些嵌合基因亦已轉化 Vll 至擬南芥菜基因組。而被轉化MBL III (pBI / phas / MBLIII-wt, -GLN,- LYS)嵌合基因的轉基因純合植物，其標記基因的活性已用GUS測試來 確定；MBL III轉基因的整合通過聚合酶鏈式反應（PCR)來證實；RT- PCR及DNA序列測定證明了 MBL III RNA轉錄產物的存在。 此外’被轉化 MBL I Q)BI / phas / MBLI-wt, -ARG, -ASP)的尺！種 子已被收獲及貯藏以待進一步的分析° 高效價的抗血淸被用以檢測轉基因種子內的MBL 0 Tricine-SDS- PAGE，蛋白測序分析，等定點沉殿以及Westem蛋白印跡法則用來證明 MBL III蛋白的穩定表達。然而，到目前爲止，尙未能在轉基因植物種 子檢測到MBL蛋白。MBL檢測的另一困難是由於擬南芥菜內的2S蛋 白（AT2S3)與MBL的大小接近且能與其抗血淸產生交叉反應。因此’ 將來除了檢察MBL在轉基因種子內的穩定性外，還需要進一步實驗來 分離MBL及AT2S3兩種低分子量蛋白及提高檢測的敏感度° Vlll Table of Contents Thesis committee Statement Acknowledgment Abstract v Table of contents ix List of abbreviations xiv List offigures xvii List oftables xix T,ITF,RATIIRE REVIEW 1.1 INTRODUCTION 1 1.2 ARTIFICML SWEETENERS 3 1.2.1 SACCHARm 3 1.2.2 CYCLAMATE 4 1.2.3 ASPARTAME 4 1.2.4 ACESULFAME-K 5 1.2.5 SUCRALOSE 5 1.3 NATURAL SWEET PLANT PROTEINS 7 1.3.1 THAUMAim 7 IX 1.3.2 MONELLESf 10 1.3.3 CURCULES[ 11 1.3.4 PENTADD^AND BRAZZEDSf 11 1.3.5 MlRACUUN 12 1.3.6 MAmNUN 12 1.4 GENETIC ENGINEERING OF SWEET PLANT PROTEIN 19 1.4.1 BlOTECHNOLOGICAL STUDIES ON lHAUMATE^ 20 1.4.1.1 Protein modification and sweetness 20 1.4.11 Transgenic expression in microbes 21 1.4.1.3 Transgenic expression in higher plants 23 1.4.2 BlOTECHNOLOGICAL STUDIES ON MONEUJN 24 1.4.2.1 Gene modification and transgenic expression in microbes 24 1.4.2.2 Transgenic expression in plants 25 1.4.3 TRANSGENIC EXPRESSION OF MABD^LD^J ^ PLANTS 26 1.5 PHASEOLIN AND ITS REGULATORY SEQUENCES 27 1.6 ARABIDOPSIS 29 1.6.1 ARABIDOPSIS THAUANA AS A MODEL PLANT 29 1.6.2 TRANSFORMATION METHODS 29 1.6.2.1 Direct DNA uptake 30 1.6.2.2 Agrobacterium-mtdA2iiQ^ transformation 31 1.6.2.3 Inplanta transformation 31 2 CFNERAT TNTttOmirT!ON AND HYPOTHESIS 21 2.1 GENERAL INTRODUCTION 33 2.2 HYPOTHESIS 34 2 MOLECULAR STUDIES ON SWKET PROTEIN MAR!NT JN ； THERMAL STABILITY 28 3.1 INTRODUCTION 38 3.2 MATEWALS 40 3.2.1 LABORATORY WARES 40 3.2.2 EQUIPMENTS 40 3.2.3 CHEMICALS 40 3.2.4 COMMERICAL