董桂書臺灣大學：分子與細胞生物學研究所呂冠箴Lu, Kuan-JenKuan-JenLu2007-11-252018-07-062007-11-252018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/49899摘要 植物暫存性澱粉在白天於葉片中合成，並在晚間進行降解。阿拉伯芥基因SEX4突變時，突變株會在夜晚累積過多的澱粉，已知SEX4蛋白質為一雙重特異性蛋白質磷解酵素 (dual-specificity protein phosphatase)，並參與在澱粉降解過程中，然而對於SEX4如何參與澱粉降解目前仍不清楚。近來研究指出，一群參與澱粉分解的基因包括SEX4，其基因的轉錄具有日夜週期的變化，白天這些基因的轉錄會增加，夜晚會減少。為進一步了解SEX4基因轉錄的調控機制，本論文利用SEX4基因啟動子片段做為探針進行電泳位移分析Electrophoretic mobility assay (EMSA)，鑑定出能與細胞核之蛋白萃取物專一結合的調節片段，並以所鑑定之調節片段進行調控因子的篩選。實驗結果顯示在SEX4基因轉譯起點上游具有四個調節片段，分別位於SEX4轉錄起點前-501~ -452bp (片段AA’), -456~ -407bp (片段BB’), -186~-137bp (片段HH’) 及 +37~ +57bp (片段LL’)。EMSA實驗中AA’和BB’片段能相互競爭葉片組織的某個核蛋白，推測可能與同一個未知的蛋白質結合。HH’調節片段能與葉片及根部的核蛋白結合，顯示出HH’片段上有組織特異性的調節序列。另外LL’片段能與大腸桿菌所表現之重組蛋白質CCA1和LHY結合，由微矩陣資料庫分析得知在cca1/lhy雙突變株中，SEX4 mRNA的轉錄會明顯的減弱，顯示CCA1和LHY可能參與調控SEX4的表現。調控因子篩選方面，則利用BB’片段進行yeast one-hybrid實驗，篩選出一個CCAAT序列結合因子B類似的蛋白質SCAT蛋白基因，並證實在酵母菌中SCAT能與SEX4基因轉錄起點前的AA’和BB’片段結合。Abstract Starch is synthesized in leaves during photosynthesis and is degraded during night. Arabidopsis sex4 mutants have a starch excess phenotype. SEX4 gene encodes a dual specificity protein phosphatase and is involved in starch degradation; however, its role is poorly understood. Recent evidence showed that circadian regulation plays a role in the oscillation of transcription level of starch degradation related genes, including SEX4 gene. The transcripts for these genes increased during the day and decreased at subsequently night. In this study, putative regulatory elements of SEX4 promoter were analyzed by electrophoretic mobility shift assay (EMSA). EMSA with nuclear extract prepared from Arabidopsis mature leaves and short DNA fragments of SEX4 promoter showed that there were four primary regulatory elements bound specifically with nuclear proteins. The positions at -501 to -452 (AA’ fragment) and -456 to -407 (BB’ fragment) upstream of SEX4 transcriptional start site showed a reciprocal competition in the in vitro binding assay, suggesting that AA’ and BB’ may share a specific unknown protein for interactions. The position at -186 to -137 (HH’ fragment) was identified as a tissue specific binding site. In addition, transcription factors CCA1 and LHY1 bound to AAAATATCT element at position +36 to +57bp 5’-UTR of SEX4 in vitro. mRNA oscillation levels of SEX4 were eliminated in cca1/lhy null mutants, suggesting that CCA1 and LHY may play roles to regulate SEX4 gene expression. Yeast one hybrid screening showed that a CCAAT box subunit protein, SCAT could interact with AA’ and BB’elements in SEX4 promoter. Possible mechanisms controlling SEX4 expression have been discussed in this study.TABLE OF CONTENTS 口試委員會審定書 Acknowledgements……………………………………………………………………...i中文摘要………………………………………………………………………………...ii Abstract……………………………………………………………………………..iii, iv Chapter 1 Introduction…………………………………….…………………………………….1 1-1 Transitory starch synthesis in Arabidopsis leaves………………..…….…1 1-2 The pathway of starch degradation in Arabidopsis…………..…………...1 1-3 SEX4 plays a role in starch degradation…………………...………...…….4 1-4 A circadian regulatory mechanism might be involved in regulating starch degradation related genes……………………………………………….…..6 1-5 A temporal and spatial coordinated regulation involved in expression of starch degradation related genes…………………………………….…..….8 2 Materials and Methods 2-1 Probes preparation………………………………………...……………...…...9 2-2 Double stranded short probes preparation……………………...………..…..10 2-3 Preparation of nuclear proteins prepared from leaves and roots….................10 2-4 Electrophoretic mobility shift assay……………………………………........11 2-5 Cloning of elements into the bait vector for Yeast One Hybrid system.........12 2-6 Transformation of baits into yeast AH109 strain……………………...….....13 2-7 Titration of 3-AT inhibitor required to reduce background growth of clones………………………………………………………………………...14 2-8 Preparations of cDNA libraries………...………………………………...….15 2-9 Identification of DNA-protein interaction using yeast one hybrid system……………………………………………………………………..…16 2-10 Lac Z test for eliminating false positive clones…………...………………..17 2-11 Southern blot analysis of scat-1 and wild type Col genomic DNA with TS probes…………………………………………...…………………...……..17 3 Results…………………………………………………………………………….….19 3-1 Expression of SEX4 and several starch degradation related genes has a similar diurnal oscillation pattern……………………………………………………19 3-2 DNA fragments were designed in this study to screen possible regulatory elements of SEX4………………………………………………………….....20 3-3 There are three regulatory elements in the upstream region of SEX4 transcriptional start site………………………………………………………21 3-4 HH’ element could be bound by root and leaf nuclear proteins respectively……………………………………………………………….….22 3-5 A point mutation at -175 of SEX4 affects the interaction between HF’ and root nuclear proteins……………………………………………...........…….23 3-6 A CCAAT binding transcription factor subunit B-like protein (SCAT) interacts with AA’ and BB’ elements in yeast…………………...………….25 3-7 scat-1 was a homozygote mutant as a member of a gene family in Arabidopsis determined by Southern blot analysis………………….………27 3-8 Possible interactions between SCAT-like proteins and elements of AA’ and BB’ may take place in Arabidopsis………………………………………….28 3-9 scat-1 mutant is late flowering……………………………………………....28 3-10 CCA1 and LHY proteins interact with LL’ elements in vitro…………...…29 3-11 CCA1/LHY might regulate expression of SEX4…………………….……..30 4 Discussion…………………………………………………………………………….31 4-1 AA’ and BB’ may interact with the same protein in the in vitro binding assay…………………………...……………………………………….……31 4-2 AA’ and BB’ fragments could interact with SCAT proteins in yeast……….32 4-3 scat-1 mutant is late flowering........................................................................33 4-4 HH’ fragment of -186 to -169 may be important for tissue specific proteins binding……………………………………………………………....…….....34 4-5 CCA1 and LHY might play roles in regulating SEX4 gene expression…......35 5 Summary and Conclusions……………………………………………………….......37 6 List of Reference……………………………………...………………………..…….59 LIST OF TABLES Table 1A Possible regulatory elements of SEX4 were divided into 12 DNA fragments, shown as AA’ through LL’.…..…………...………….………….…………..………….…38 1B Three shorter fragments were designed for the HH’ region , shown as HF through HL……………………………………………………….……….………….....…...39 2 Potential protein binding sites for AA’ and BB’ elements……...…..……….……..40 LIST OF FIGURES Figures Figure 1 Structural domains of SEX4…………………………………………………..41 Figure 2 Diurnal mRNA expression levels of genes participating in starch degradation………………………………………………………………......41 Figure 3 Genes involved in starch degradation were shown with a tissue specific property in transcription level…………………………………………….....42 Figure 4 Positions of putative regulatory elements of SEX4………………...……..…..43 Figure 5 DNA fragments at position -501 to -452 and -456 to -407 of SEX4 promoter might interact with the same protein…………………………..…..44 Figure 6 DNA fragment at position -186 to -137 of SEX4 promoter could be bound by leaves nuclear lysates specifically………...………………….……………...45 Figure 7 HH’ fragment could be bound by different factors in leaves and roots...…….46 Figure 8 HF’ fragment interacted with root and leaf nuclear proteins………...…….…47 Figure 9 The interaction of nuclear lysates and HF’ fragment…………...……...……..48 Figure 10A Reporter constructs………………………………………………………...50 Figure 10B Effector construct……………………………………………………...…..50 Figure 10C AH109 strain construct…………………………………………………….51 Figure 11A AH109 yeast cells containing reporter constructs or AH109 alone with GAD-SCAT effector constructs grew well on medium lacking Leu and Ura……………………………………………………………...…....……51 Figure 11B AH109 containing pHisi4-3BB’, pHisi4-7BB’ and pHisi4-3AA’ with GAD-SCAT grew well on medium lacking Leu, Ura, and His….....……52 Figure 11C GAD-SCAT proteins did not interact with GAL4-UAS and MEL1 promoter element………………………………………………...………..………...53 Figure 12A T-DNA was inserted at +477 of SCAT gene………………….…………...54 Figure 12B BamHI restriction enzyme map of Arabidopsis genomic DNA of wild type Col and scat-1 mutant…………………………………………...………...54 Figure 12C Hybridizations of Arabidopsis genomic DNA of wild type Col and scat-1 mutants with TS probes…………………………………………..……….55 Figure 13 T4 and T5 generation Arabidopsis plants of scat-1 mutants are late flowering……………………………………………………………….…...56 Figure 14 CCA1 and LHY proteins bind to LL’ elements in vitro………...……..……57 Figure 15 The mRNA oscillations of SEX4 were different in cca1/lhy and cca1-ox mutants from wild type under extended continuous light……………..…...581384145 bytesapplication/pdfen-USSEX4調節片段SCATCCA1LHYstarch excess 4 gene (SEX4)regulatory elementsCCAAT box binding factor subunit B-like protein SCATotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49899/1/ntu-96-R94b43024-1.pdf