FT基因是高等植物开花的信号调控中一个重要的整合因子，它通过调控花原基特异性基因的表达来启动花原基的分化。本研究通过遗传学和分子生物学的技术研究了MADS-box基因CAL、FUL对开花时间的影响以及FT对CAL、FUL的调控，另外也深入探讨了FT蛋白在内质网的定位情况以及影响其定位的因素。实验主要得到了以下结果： 1、发现了超表达MADS-box基因CAL、FUL能诱导拟南芥植株早花，而CAL与FUL诱导的早花依赖于FT基因。 2、证明了FT能够抑制CAL、FUL蛋白通过泛素蛋白酶体途径的降解。 3、证实了FT能在内质网形成复合物。 4、发现了FT的C-末端融合HA标签会影响复合物的...FT gene is an critical integrator in the flowering signal regulation of higher plant, FT protein initiates the development of floral meristem by activating the expression of floral meristem identity genes. We use genetics and molecular biotechnologic methods to investigate how FT protein regulate MADS-box genes CAL、FUL function the flowering time control. We also study the location of FT on ER and...学位：理学硕士院系专业：生命科学学院生物学系_细胞生物学学号：2172009115215

冯玲玲

Xiamen University Institutional Repository

  学校编码：10384                                 分类号      密级        学号：21720091152157                                        UDC         硕  士  学  位  论  文  FT 对 CAL 与 FUL 基因表达调控的研究 The regulation of CAL and FUL expression by FT  冯玲玲   指导教师姓名：黄 涛 教授 专  业 名 称： 细胞生物学 论文提交日期： 2012 年  月 论文答辩时间： 2012 年  月 学位授予日期： 2012 年  月  答辩委员会主席：陶懿 教授 评    阅    人：_           2012 年   月   厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库  厦门大学学位论文原创性声明  本人呈交的学位论文是本人在导师指导下,独立完成的研究成果。本人在论文写作中参考其他个人或集体已经发表的研究成果，均在文中以适当方式明确标明，并符合法律规范和《厦门大学研究生学术活动规范（试行）》。 另外，该学位论文为（                        ）课题（组）的研究成果，获得（               ）课题（组）经费或实验室的资助，在（               ）实验室完成。（请在以上括号内填写课题或课题组负责人或实验室名称，未有此项声明内容的，可以不作特别声明。）  声明人（签名）：           年   月   日                厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库  厦门大学学位论文著作权使用声明  本人同意厦门大学根据《中华人民共和国学位条例暂行实施办法》等规定保留和使用此学位论文，并向主管部门或其指定机构送交学位论文（包括纸质版和电子版），允许学位论文进入厦门大学图书馆及其数据库被查阅、借阅。本人同意厦门大学将学位论文加入全国博士、硕士学位论文共建单位数据库进行检索，将学位论文的标题和摘要汇编出版，采用影印、缩印或者其它方式合理复制学位论文。 本学位论文属于： （  √ ）1.经厦门大学保密委员会审查核定的保密学位论文，于 2013 年 12 月 1 日解密，解密后适用上述授权。 （     ）2.不保密，适用上述授权。 （请在以上相应括号内打“√”或填上相应内容。保密学位论文应是已经厦门大学保密委员会审定过的学位论文，未经厦门大学保密委员会审定的学位论文均为公开学位论文。此声明栏不填写的，默认为公开学位论文，均适用上述授权。）                                声明人（签名）：  年  月   日   厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库目录  I  目录 摘  要 ........................................................................................................ 1 ABSTRACT ............................................................................................... 2 第一章 前 言 ............................................................................................ 3 1  开花素 FT 与高等植物开花的信号调控 ....................................................... 3 1.1  高等植物开花的信号调控 ..................................................................... 3 1.2  FT 在拟南芥开花中的调控作用 ........................................................... 5 1.3  FT 的定位及长距离运输 ....................................................................... 6 2  MADS-box 基因 CAL、FUL 及其泛素化降解 ............................................ 8 2.1  ABCDE 模型 .......................................................................................... 9 2.2  CAL、FUL 对植物开花的调控 ........................................................... 10 2.3  泛素蛋白酶体途径调节的蛋白质降解 ............................................... 12 3  研究目的和意义 ............................................................................................ 15 第二章    材料与方法 .......................................................................... 16 1  实验材料 ........................................................................................................ 16 1.1  植物材料及菌株 ................................................................................... 16 1.2  培养基及抗生素 ................................................................................... 17 1.3  常用溶液 ............................................................................................... 18 1.4  主要试剂 ............................................................................................... 20 1.5  引物序列 ............................................................................................... 21 1.6  实验仪器 ............................................................................................... 21 2  实验方法 ........................................................................................................ 22 2.1 生理实验操作 ......................................................................................... 22 2.2 分子克隆实验操作 ................................................................................. 24 2.3 蛋白实验操作 ......................................................................................... 29 第三章   实验结果与分析 .................................................................... 36 1  拟南芥植株中 FT 基因对 CAL、FUL 基因表达的调控 ........................... 36 厦门大学博硕士论文摘要库目录  II  1.1  在 WT 植株体内超表达 CAL、FUL 能诱导植物早花………………36 1.2  在 ft-10 植株体内超表达 CAL、FUL 不能恢复其晚花表型……… 38 1.3  在 WT 与 ft-10 植株背景中都能检测到 CAL-HA、FUL-HA 蛋白 ……………………………………………………………… ………… 40 1.4  植株中 FT 对 CAL、FUL 表达的影响………………………………41 2 离体实验中 FT 对 CAL、FUL 的调控……………………………………42 2.1  pMBP-C-CAL与 pMBP-C-FUL的测序结果…………………………43 2.2  MBP-CAL、MBP-FUL 蛋白的体外诱导表达………………………44 2.3  MBP-CAL、MBP-FUL 蛋白的纯化………………………………………45 2.4  CAL、FUL 蛋白在 ft-10 植株的无细胞提取液中会发生降解 ……46 2.5  FT 能够抑制 CAL、FUL 蛋白的降解 ………………………………47 3  C-末端融合 HA 标签对 FT 蛋白在内质网定位的影响 ............................. 48 3.1  C-末端融合 HA 标签不影响 FT 蛋白进入内质网腔 ......................... 49 3.2  C-末端融合 HA 标签阻碍 FT 蛋白在内质网形成转运复合物 ......... 49 4  FD、SOC1 基因对 FT 蛋白在内质网定位的影响 ..................................... 50 4.1  FD、SOC1 的缺失不影响 FT 蛋白进入内质网腔 ............................ 51 4.2  FD、SOC1 的缺失不影响 FT 蛋白在内质网形成转运复合物 ........ 51 第四章 讨论 ............................................................................................ 53 1  MADS-box 基因在控制开花时间早晚中的作用 ........................................ 53 2  FT 对 AP1、CAL 和 FUL 表达的调控 ....................................................... 53 3  FT 通过抑制泛素蛋白酶体途径抑制蛋白的降解 ...................................... 54 4  FT 蛋白在内质网定位与形成复合物的检测 .............................................. 54 第五章 总结与展望 ................................................................................ 56 参考文献 .................................................................................................. 57 致谢 .......................................................................................................... 67   厦门大学博硕士论文摘要库CONTENTS III   CONTENTS Abstract (in Chinese) ········································· 1 Abstract (in English) ········································· 2 Chapter 1 Introduction ······································· 3 1  FT gene and Flowering regulation of higher plants ···················· 3 1.1 Flowering regulation of higher plants ······························· 3 1.2 The role of FT in Arabidopsis thaliana flowering regulation ············· 5 1.3 Location and long-distance signaling of FT gene ······················ 6 2  MADS-box genes CAL、FUL and their degradation via ubiquitin – proteasome pathway ················································· 8 2.1 The ABCDE model ············································· 9 2.2 The role of CAL and FUL in Arabidopsis thaliana flowering regulation ····10 2.3 Protein degradation via the ubiquitin - proteasome pathway ·············12 3  Purpose and significance of the research ···························· 15 Chaper 2 Materials and methods ······························ 16 1  Materials ······················································ 16 1.1 Plants materials and plasmids ····································16 1.2 Medium and antibiotics ·········································17 1.3 Common solutions ·············································18 1.4 Main reagents ················································20 1.5 Primer sequences ··············································21 1.6 Main instruments ··············································21 2  Methods ······················································· 22 2.1 Physiological experiment ·······································22 2.2 Molecular cloning ·············································24 2.3 Protein experiments ············································29 厦门大学博硕士论文摘要库CONTENTS IV  Chaper 3 Results and analysis ································ 36 1  The regulation of CAL and FUL expression by FT in vivo ·············· 36 1.1 Overexpression of CAL and FUL causes early flowering in WT plant ······36 1.2 Overexpression of CAL and FUL fails to cause early flowering in ft-10 plant ······························································38 1.3 The CAL and FUL protein can be detected in the WT and ft - 10 plants ····40 1.4 The regulation of CAL and FUL protein by FT in vivo ·················41 2  The regulation of CAL and FUL protein by FT in vitro ················ 42 2.1 The sequencing results of pMBP-C-CAL and pMBP-C-FUL ·············43 2.2 The expression of CAL and FUL in vitro ····························44 2.3 The purification of MBP-CAL and MBP-FUL protein ·················45 2.4 CAL and FUL protein are degraded in cell-free extraction prepared from    ft -10 ··························································46 2.5 FT inhibits the degradation of CAL and FUL in cell-free extraction ·······47 3  The fusion HA tags in C-terminous of FT affects its location in the ER  ·· 48 3.1 FT:HA fusion protein can enter the ER ·····························49 3.2 FT:HA fusion protein fails to bind to the ER membrane ················49 4  The effect of FD and SOC1 on FT’s location of ER ··················· 50 4.1 Loss function of FD and SOC1 dose not affect the entry of FT in the ER lumen ·························································51 4.2 Loss function of FD and SOC1 does not affect the binding of FT protein to the ER membrane ················································51 Chaper 4 Discussion ········································· 53 1  The formation of FT protein complex in the ER ······················ 53 2  The role of MADS-box genes in the initiatim of floral merestem ········ 53 3  The regulation of AP1、CAL and FUL expression by FT ··············· 54 4 FT inhibit CAL and FUL protein degradation via the ubiquitination-proteasome pathway ··································· 54 Chaper 5 Summary and Outlook ······························ 56 厦门大学博硕士论文摘要库CONTENTS V  References ················································· 57 Acknowledgement ·········································· 67   厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库ABSTRACT 1  摘  要 FT 基因是高等植物开花的信号调控中一个重要的整合因子，它通过调控花原基特异性基因的表达来启动花原基的分化。本研究通过遗传学和分子生物学的技术研究了 MADS-box 基因 CAL、FUL 对开花时间的影响以及 FT 对 CAL、FUL的调控，另外也深入探讨了 FT 蛋白在内质网的定位情况以及影响其定位的因素。实验主要得到了以下结果： 1、发现了超表达 MADS-box 基因 CAL、FUL 能诱导拟南芥植株早花，而CAL 与 FUL 诱导的早花依赖于 FT 基因。 2、证明了 FT 能够抑制 CAL、FUL 蛋白通过泛素蛋白酶体途径的降解。 3、证实了 FT 能在内质网形成复合物。 4、发现了 FT 的 C-末端融合 HA 标签会影响复合物的形成但是不影响 FT 蛋白进入内质网； FD、SOC1 的缺失既不影响 FT 进入内质网也不影响其形成复合物。     本实验证实了 FT 对 MADS-box 基因稳定性的调控，这对植物的遗传操作有着重要的意义，另外也为寻找参与 FT 复合物形成的其他因子进一步提供了依据。 关键词：FT；CAL；FUL；花原基分化          厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库ABSTRACT 2  ABSTRACT FT gene is an critical integrator in the flowering signal regulation of higher plant, FT protein initiates the development of floral meristem by activating the expression of floral meristem identity genes. We use genetics and molecular biotechnologic methods to investigate how FT protein regulate MADS-box genes CAL、FUL function the flowering time control. We also study the location of FT on ER and the affectors that regulate FT localization. The main results are following: 1、Overexpression of MADS-box gene CAL and FUL causes early flowering, which is dependent on FT gene. 2、  FT is able to inhibit the degradation of CAL、FUL protein via the ubiquitination -proteasome pathway. 3、FT protein is recruited into a complex by bind to ER membrane. 4、Fusion HA tags in C-terminus of FT protein will affect the complex formation, but does not affect the entry of FT into the ER. Loss of FD and SOC1 function affect neither the location of FT protein in the ER nor the formation of complex. Our results suggest that FT regulate the stability of MADS-box protein, which has an important significance for genetic manipulation of plants. It also provides the basis for seeking the other components in FT complex.  Key words: FT; CAL; FUL；Floral meristem     厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库Degree papers are in the “Xiamen University Electronic Theses and Dissertations Database”. Fulltexts are available in the following ways: 1. If your library is a CALIS member libraries, please log on http://etd.calis.edu.cn/ and submitrequests online, or consult the interlibrary loan department in your library. 2. For users of non-CALIS member libraries, please mail to etd@xmu.edu.cn for delivery details.厦门大学博硕士论文摘要库

The regulation of CAL and FUL expression by FT

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The regulation of CAL and FUL expression by FT

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