本文選題：TOR + 突變體��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：TOR(雷帕霉素的靶標(biāo))激酶在酵母、植物、動(dòng)物和人類進(jìn)化中是一個(gè)保守的主調(diào)節(jié)因子,它整合營(yíng)養(yǎng)和能量信號(hào)來(lái)促進(jìn)細(xì)胞增殖和生長(zhǎng),TOR信號(hào)通路與生物的很多生理機(jī)能密切相關(guān),如細(xì)胞生長(zhǎng)、細(xì)胞分裂、細(xì)胞的新陳代謝等進(jìn)程都有著密不可分的關(guān)系。通過(guò)對(duì)出芽酵母抗雷帕霉素的遺傳突變篩選,TOR首先被鑒定出來(lái)。雷帕霉素是一種能夠阻斷人類T細(xì)胞激活和增殖的免疫抑制劑,雷帕霉素可以與FKBP12結(jié)合,形成RAPA-FKBP12二元復(fù)合物,然后RAPA-FKBP12復(fù)合物作用于TOR的FRB結(jié)構(gòu)域以此抑制TOR激酶活性。雖然在酵母中鑒定出了兩個(gè)TOR基因,但隨后在擬南芥、綠藻、大多數(shù)動(dòng)物和人類中只鑒定出了一個(gè)TOR基因。擬南芥和人類的TOR基因具有高度相似的氨基酸序列,特別是在蛋白激酶區(qū)(75%的相似性),表明這些蛋白激酶具有相似的功能和蛋白基底。在動(dòng)物和酵母中關(guān)于TOR信號(hào)通路已經(jīng)進(jìn)行了深入的研究,但由于擬南芥FKBP12無(wú)法與雷帕霉素形成二元聚合物,導(dǎo)致雷帕霉素不能與擬南芥TOR的FRB結(jié)構(gòu)域結(jié)合,從而不能抑制TOR蛋白的活性,所以關(guān)于擬南芥的TOR通路研究較少,本文通過(guò)甲基磺酸乙酯(EMS)誘導(dǎo)Columbia-0(Col-0、Col,WT)型擬南芥篩選出了具有TOR抑制劑抗性的突變體,對(duì)這些突變體進(jìn)行表型觀察分析和基因定位等相關(guān)研究。主要結(jié)論如下:1.突變體篩選結(jié)果實(shí)驗(yàn)中所用Columbia(Col)擬南芥都已轉(zhuǎn)入酵母中的FKBP12蛋白,轉(zhuǎn)入植株被命名為BP12-2。TOR的一代抑制劑為雷帕霉素(RAP),二代抑制劑為KU、AZD、Torin1等,分別用RAP和AZD將TOR的FRB結(jié)構(gòu)域和Kinase結(jié)構(gòu)域抑制,用EMS誘變Col型擬南芥,利用RAP+AZD雙重抑制TOR活性,篩選出9個(gè)對(duì)RAP+AZD抑制劑不敏感的突變體,命名為TOR抑制劑抗性突變體(tor-inhibitor-insensitive,trin),分別為trin-1、trin-2、trin-6、trin-8、trin-10、trin-12、trin-16、trin-17、trin-19。2.表型觀察結(jié)果9個(gè)突變體生理成熟后,所表現(xiàn)出的外觀性狀有所不同,且大致分為三類:(1)野生表型單株:trin-1、trin-6、trin-8、trin-10、trin-12;(2)雙果莢、生長(zhǎng)緩慢單株:trin-2;(3)雙果莢、不育單株:trin-16、trin-17、trin-19。3.鉀敏感度實(shí)驗(yàn)結(jié)果用不同濃度鉀培養(yǎng)基進(jìn)行鑒定,將鉀元素濃度按照MS培養(yǎng)基的配方從低到高設(shè)置了7個(gè)等級(jí),分別是0%、10%、20%、30%、40%、50%和100%,每個(gè)突變體在不同鉀濃度下又設(shè)置了兩次重復(fù),對(duì)9個(gè)突變體做不同濃度鉀培養(yǎng)基的直接培養(yǎng)和先在正常1/2MS培養(yǎng)基上鋪種,長(zhǎng)10天左右轉(zhuǎn)苗到不同濃度梯度鉀培養(yǎng)基的間接培養(yǎng)實(shí)驗(yàn),9個(gè)突變體中發(fā)現(xiàn)了有對(duì)低鉀或無(wú)鉀敏感的突變體trin-2突變體。4.遺傳分析統(tǒng)計(jì)和觀察突變體和野生型Lansberg(Ler)雜交組合中的F1代和F2的植株中在培養(yǎng)基上的表型,發(fā)現(xiàn)所有的F1表現(xiàn)正常,都表現(xiàn)出萎蔫枯黃,表明9個(gè)突變體均為隱性突變;而F2代中的苗子在篩選培養(yǎng)基上出現(xiàn)正常生長(zhǎng)和萎蔫枯黃不正常生長(zhǎng)兩種表型,統(tǒng)計(jì)不同雜交組合的表型,?2測(cè)驗(yàn)顯示正常植株與突變植株的分離比均符合3:1,表明每個(gè)突變體的突變性狀均受1對(duì)隱性核基因控制。5.初步定位結(jié)果利用突變體和野生型Lansberg(Ler)雜交組合中F2群體對(duì)TOR抑制劑抗性系列突變體進(jìn)行定位。結(jié)果表明,trin-1定位在第3條染色體上9.3M(1M=1024kb,1kb=1024b)處的ciw11分子標(biāo)記附近;trin-2定位在第5條染色體上13.4M處的PHYC和16.3M處的ciw9分子標(biāo)記之間;trin-6定位在第1條染色體上20.0M處的nga280和26.1M處的nga111分子標(biāo)記之間;trin-8定位在第3條染色體上18.0M處的ciw4和22.0M處的nga6分子標(biāo)記之間;trin-10定位在第4條染色體0.7M處的ciw5和7.5M處的ciw6分子標(biāo)記之間;trin-12定位在第4條染色體0.7M處的ciw5和7.5M處的ciw6分子標(biāo)記之間;trin-16定位在第5條染色體上13.4M處的PHYC和16.3M處的ciw9分子標(biāo)記之間;trin-17定位在第5條染色體上13.4M處的PHYC和16.3M處的ciw9分子標(biāo)記之間;trin-19定位在第5條染色體上13.4M處的PHYC和16.3M處的ciw9分子標(biāo)記之間。6.候選基因分析將初步定位后確定的分子標(biāo)記之間的序列進(jìn)行了測(cè)序,確定了各突變體的候選基因范圍。其中trin-1的候選基因?yàn)?個(gè);trin-2的候選基因?yàn)?個(gè);trin-6的候選基因?yàn)?個(gè);trin-8的候選基因?yàn)?個(gè);trin-10的候選基因?yàn)?個(gè);trin-12的候選基因?yàn)?0個(gè);trin-16的候選基因?yàn)?0個(gè);trin-17的候選基因?yàn)?個(gè);trin-19的候選基因?yàn)?個(gè)。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 杜亞琳;;擬南芥缺銅敏感突變體的鑒定分析[J];貴州農(nóng)業(yè)科學(xué);2016年08期

2 張愛(ài)利;;釀酒酵母TOR信號(hào)通路研究進(jìn)展[J];化學(xué)工業(yè)與工程;2011年05期

3 何俊平;阮松林;祝水金;馬華升;;圖位克隆技術(shù)在農(nóng)作物基因分離中的應(yīng)用與評(píng)價(jià)[J];遺傳;2010年09期

4 侯仙慧;丁茂予;劉賽男;李林川;瞿禮嘉;;擬南芥MeIAA抗性突變體的篩選和初步圖位克隆分析[J];植物學(xué)報(bào);2009年01期

5 王澤立,戴景瑞,王斌;植物基因的圖位克隆[J];生物技術(shù)通報(bào);2000年04期

，

本文編號(hào)：2026772