拟南芥TOR抑制剂抗性突变体的筛选及初步定位

发布时间：2018-06-16 12:55

本文选题：TOR + 突变体　；参考：《西南大学》2017年硕士论文

【摘要】：TOR(雷帕霉素的靶标)激酶在酵母、植物、动物和人类进化中是一个保守的主调节因子,它整合营养和能量信号来促进细胞增殖和生长,TOR信号通路与生物的很多生理机能密切相关,如细胞生长、细胞分裂、细胞的新陈代谢等进程都有着密不可分的关系。通过对出芽酵母抗雷帕霉素的遗传突变筛选,TOR首先被鉴定出来。雷帕霉素是一种能够阻断人类T细胞激活和增殖的免疫抑制剂,雷帕霉素可以与FKBP12结合,形成RAPA-FKBP12二元复合物,然后RAPA-FKBP12复合物作用于TOR的FRB结构域以此抑制TOR激酶活性。虽然在酵母中鉴定出了两个TOR基因,但随后在拟南芥、绿藻、大多数动物和人类中只鉴定出了一个TOR基因。拟南芥和人类的TOR基因具有高度相似的氨基酸序列,特别是在蛋白激酶区(75%的相似性),表明这些蛋白激酶具有相似的功能和蛋白基底。在动物和酵母中关于TOR信号通路已经进行了深入的研究,但由于拟南芥FKBP12无法与雷帕霉素形成二元聚合物,导致雷帕霉素不能与拟南芥TOR的FRB结构域结合,从而不能抑制TOR蛋白的活性,所以关于拟南芥的TOR通路研究较少,本文通过甲基磺酸乙酯(EMS)诱导Columbia-0(Col-0、Col,WT)型拟南芥筛选出了具有TOR抑制剂抗性的突变体,对这些突变体进行表型观察分析和基因定位等相关研究。主要结论如下:1.突变体筛选结果实验中所用Columbia(Col)拟南芥都已转入酵母中的FKBP12蛋白,转入植株被命名为BP12-2。TOR的一代抑制剂为雷帕霉素(RAP),二代抑制剂为KU、AZD、Torin1等,分别用RAP和AZD将TOR的FRB结构域和Kinase结构域抑制,用EMS诱变Col型拟南芥,利用RAP+AZD双重抑制TOR活性,筛选出9个对RAP+AZD抑制剂不敏感的突变体,命名为TOR抑制剂抗性突变体(tor-inhibitor-insensitive,trin),分别为trin-1、trin-2、trin-6、trin-8、trin-10、trin-12、trin-16、trin-17、trin-19。2.表型观察结果9个突变体生理成熟后,所表现出的外观性状有所不同,且大致分为三类:(1)野生表型单株:trin-1、trin-6、trin-8、trin-10、trin-12;(2)双果荚、生长缓慢单株:trin-2;(3)双果荚、不育单株:trin-16、trin-17、trin-19。3.钾敏感度实验结果用不同浓度钾培养基进行鉴定,将钾元素浓度按照MS培养基的配方从低到高设置了7个等级,分别是0%、10%、20%、30%、40%、50%和100%,每个突变体在不同钾浓度下又设置了两次重复,对9个突变体做不同浓度钾培养基的直接培养和先在正常1/2MS培养基上铺种,长10天左右转苗到不同浓度梯度钾培养基的间接培养实验,9个突变体中发现了有对低钾或无钾敏感的突变体trin-2突变体。4.遗传分析统计和观察突变体和野生型Lansberg(Ler)杂交组合中的F1代和F2的植株中在培养基上的表型,发现所有的F1表现正常,都表现出萎蔫枯黄,表明9个突变体均为隐性突变;而F2代中的苗子在筛选培养基上出现正常生长和萎蔫枯黄不正常生长两种表型,统计不同杂交组合的表型,?2测验显示正常植株与突变植株的分离比均符合3:1,表明每个突变体的突变性状均受1对隐性核基因控制。5.初步定位结果利用突变体和野生型Lansberg(Ler)杂交组合中F2群体对TOR抑制剂抗性系列突变体进行定位。结果表明,trin-1定位在第3条染色体上9.3M(1M=1024kb,1kb=1024b)处的ciw11分子标记附近;trin-2定位在第5条染色体上13.4M处的PHYC和16.3M处的ciw9分子标记之间;trin-6定位在第1条染色体上20.0M处的nga280和26.1M处的nga111分子标记之间;trin-8定位在第3条染色体上18.0M处的ciw4和22.0M处的nga6分子标记之间;trin-10定位在第4条染色体0.7M处的ciw5和7.5M处的ciw6分子标记之间;trin-12定位在第4条染色体0.7M处的ciw5和7.5M处的ciw6分子标记之间;trin-16定位在第5条染色体上13.4M处的PHYC和16.3M处的ciw9分子标记之间;trin-17定位在第5条染色体上13.4M处的PHYC和16.3M处的ciw9分子标记之间;trin-19定位在第5条染色体上13.4M处的PHYC和16.3M处的ciw9分子标记之间。6.候选基因分析将初步定位后确定的分子标记之间的序列进行了测序,确定了各突变体的候选基因范围。其中trin-1的候选基因为8个;trin-2的候选基因为5个;trin-6的候选基因为6个;trin-8的候选基因为6个;trin-10的候选基因为2个;trin-12的候选基因为10个;trin-16的候选基因为10个;trin-17的候选基因为5个;trin-19的候选基因为9个。
[Abstract]:TOR (rapamycin target) kinase is a conservative principal regulator in the evolution of yeast, plants, animals and human beings. It integrates nutritional and energy signals to promote cell proliferation and growth. The TOR signaling pathway is closely related to many biological functions, such as cell growth, cell division, and cell metabolism. TOR is first identified by screening of the genetic mutation of the buds yeast against rapamycin. Rapamycin is an immunosuppressant that blocks the activation and proliferation of human T cells. Rapamycin can combine with FKBP12 to form a RAPA-FKBP12 two element complex, and the RAPA-FKBP12 complex acts on the FRB structure of TOR. This domain inhibits TOR kinase activity. Although two TOR genes are identified in yeast, only one TOR gene is identified in Arabidopsis, green algae, most animals and humans. The Arabidopsis and human TOR genes have a highly similar sequence of amino acids, especially in the egg white kinase region (75% similarity), indicating that these proteins are stimulated. The enzyme has similar functions and protein substrates. The TOR signaling pathway in animals and yeast has been studied in depth. But because Arabidopsis FKBP12 is unable to form two yuan polymer with rapamycin, rapamycin can not bind to the FRB domain of Arabidopsis TOR, and thus can not inhibit the activity of TOR protein, so about Arabidopsis thaliana. There are few studies in the TOR pathway. In this paper, EMS (Col-0, Col, WT) induced Columbia-0 (Col-0, Col, WT) type Arabidopsis thaliana mutants with TOR inhibitor resistance were screened in this paper. The phenotypic observation analysis and gene localization of these mutants were studied. The main conclusions are as follows: the Columbia (Col) of the 1. mutant screening results is proposed. The southern mustard has been transferred to the FKBP12 protein in yeast and transferred into the plant named BP12-2.TOR as a generation inhibitor of rapamycin (RAP), the two generation inhibitor is KU, AZD, Torin1 and so on. The FRB domain and Kinase domain of TOR are suppressed by RAP and AZD. ZD inhibitors are insensitive mutants named TOR inhibitor resistant mutants (tor-inhibitor-insensitive, Trin), which are trin-1, trin-2, trin-6, trin-8, trin-10, trin-12, trin-16, and trin-17. After physiological maturation, the appearance characteristics of the 9 mutants are different and are roughly divided into three categories: (1) wild. Phenotypic strains: trin-1, trin-6, trin-8, trin-10, trin-12; (2) double fruit pods, slow growth single plant: trin-2; (3) double fruit pods, sterile single plant: trin-16, trin-17, trin-19.3. potassium sensitivity test results were identified with different concentration potassium medium, and the potassium concentration was set from low to high according to the MS culture medium from low to high, which were 0%, 10%, 20, respectively. %, 30%, 40%, 50% and 100%, each mutant was set up two times at different potassium concentrations, and the 9 mutants were cultured directly with different concentrations of potassium medium and first planted on the normal 1/2MS medium. The seedlings were transferred to different concentration gradient potassium medium for 10 days for 10 days, and 9 mutants found a low potassium or no one. Genetic analysis of the potassium sensitive mutant trin-2 mutant.4. and observation of the phenotype on the medium of F1 and F2 in the hybrid combination of the mutant and the wild type Lansberg (Ler) hybrid combination, found that all the F1 manifestations were normal, all showed wilting and yellow, indicating that 9 mutants were all recessive, and the seedlings in F2 generation were screened on the medium. Two phenotypes of normal growth and wilting and yellowing abnormal growth were found, and the phenotypes of different hybrid combinations were counted, and the 2 tests showed that the separation ratio of normal plants and mutant plants were all conformed to 3:1, indicating that the mutant traits of each mutant were all controlled by 1 pairs of recessive genes for.5. preliminary localization and wild type Lansberg (Ler) hybridization. The central F2 population localize the TOR inhibitor resistance series. The results show that trin-1 is located near the ciw11 molecular markers at 9.3M (1M=1024kb, 1kb=1024b) on the third chromosomes, and trin-2 localize between PHYC and 16.3M ciw9 molecular markers at 13.4M at the fifth chromosomes, and located on the first chromosomes. Between 280 and nga111 markers at 26.1M; trin-8 located between the nga6 molecular markers at ciw4 and 22.0M at 18.0M on the third chromosomes; trin-10 located between ciw5 and 7.5M ciw6 molecular markers at 0.7M at fourth chromosomes; Ciw9 molecular markers located at 13.4M and 16.3M on Fifth chromosomes; trin-17 is located between the ciw9 molecular markers at PHYC and 16.3M at 13.4M on the fifth chromosomes; trin-19 is located on the PHYC of 13.4M on the fifth chromosomes and the markers of the molecular markers in the region. The sequence between the molecular markers was sequenced to determine the candidate gene range of the mutants, including 8 candidate genes for trin-1, 5 candidate genes for trin-2, 6 candidate genes for trin-6, 6 candidate genes for trin-8, 2 candidate genes for trin-10, 10 candidate genes for trin-12, and 10 candidate genes for trin-16. The candidate gene for trin-16 was 10. There are 5 candidate genes for trin-17 and 9 candidate genes for trin-19.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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