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  本文選題：側(cè)柏　切入點：古樹　出處：《中國林業(yè)科學(xué)研究院》2017年博士論文　論文類型：學(xué)位論文

【摘要】：端粒與壽命、衰老關(guān)系密切;而端粒相關(guān)蛋白調(diào)控端粒酶活性,從而影響端粒長度。然而,端粒或其相關(guān)蛋白在植物生長發(fā)育以及脅迫響應(yīng)中的作用機制尚不清楚。因此,本研究以長壽、抗逆的側(cè)柏(Platycladus orientalis)為研究材料,通過對20、500、1 000和1 700年樹齡葉片進行RNA-seq分析,發(fā)掘PoWHY1、PoKub3和PoTRF1等端粒相關(guān)基因;采用實時定量qRT-PCR技術(shù),研究側(cè)柏幼苗在NaCl、H2O2、ABA脅迫處理下,這些端粒相關(guān)基因的響應(yīng)模式變化;并在側(cè)柏古樹(500年)中克隆獲得PoWHY1、PoKub3和PoTRF1基因全長cDNA序列的基礎(chǔ)上,分析它們在不同脅迫條件下的表達模式;最后,利用轉(zhuǎn)基因技術(shù)在擬南芥(Arabidopsis thaliana)中驗證上述三個基因?qū)Ω啕}、干旱等脅迫的抵御作用。主要結(jié)論如下:(1)采用RNA-seq(Illumina HiSeqTM 2000)對不同年齡側(cè)柏葉片轉(zhuǎn)錄組測序,獲得48 044個Unigene,平均長度為896 bp,N50為1 714 bp。與公共數(shù)據(jù)庫進行比對,注釋到NR、NT、SwissProt、PFAM、GO、COG和KEGG數(shù)據(jù)庫的Unigene分別為24080、8 380、18 483、18 546、20 029、9 877和7 783個。此外,運用MISA軟件發(fā)現(xiàn)側(cè)柏轉(zhuǎn)錄組中具有2~6個核苷酸重復(fù)類型的SSR位點1 256個。從轉(zhuǎn)錄組中篩選出4個端粒相關(guān)基因PoWHY1、PoKub3、PoTRF1和PoPOT1,且qRT-PCR結(jié)果顯示,隨著側(cè)柏年齡的增加,PoWHY1和PoTRF1表達水平呈先升后降的趨勢,而PoPOT1則呈相反的趨勢,但PoKub3是逐漸升高的。這表明4個端粒相關(guān)基因表達水平變化與年齡存在密切關(guān)系,為側(cè)柏衰老相關(guān)基因功能的研究提供了參考。(2)結(jié)合活性氧代謝相關(guān)生理指標,通過對活性氧代謝相關(guān)基因(Cu/Zn-SOD、CAT、GR、APX、MDAR和GST)和端粒相關(guān)基因(WHY1、Kub3、TRF1和POT1)在NaCl和H2O2脅迫和外源ABA處理下的基因表達分析表明,NaCl脅迫能誘導(dǎo)葉片活性氧代謝相關(guān)基因和端粒相關(guān)基因(除POT1外)表達水平增加,但外施ABA可改變NaCl脅迫對這些基因表達水平的作用效果;H2O2脅迫引起葉片活性氧代謝和端粒相關(guān)基因表達變化,但外施低濃度0.5μmol/L ABA 48 h能進一步提高活性氧代謝和端粒相關(guān)基因的表達水平。結(jié)果初步表明,端粒相關(guān)基因可能與側(cè)柏的脅迫響應(yīng)密切相關(guān)。(3)采用RACE技術(shù)克隆PoWHY1基因全長cDNA序列,并利用生物信息學(xué)方法對PoWHY1、PoKub3和PoTRF1基因進行分析。結(jié)果顯示,PoWHY1基因全長cDNA序列為1 295 bp,開放閱讀框ORF為723 bp,編碼240個氨基酸;蛋白質(zhì)分子量為26.47 kDa,等電位點為9.19;其二級結(jié)構(gòu)以無規(guī)則卷曲為主,并含有一個Whirly結(jié)構(gòu)域;與芝麻(Sesamum indicum)有較高的同源性。PoKub3蛋白質(zhì)分子量為20.92 kDa,等電位點為6.05;二級結(jié)構(gòu)以無規(guī)則卷曲和α-螺旋為主,并含有Peptidase_M76結(jié)構(gòu)域;與擬南芥、琴葉擬南芥(Arabidopsis lyrata)、毛果楊(Populus trichocarpa)和可可(Theobroma cacao)有較高的同源性。PoTRF1蛋白質(zhì)分子量為34.06 kDa,等電位點為9.07;二級結(jié)構(gòu)以α-螺旋和無規(guī)則卷曲為主,并含有SANT_TRF和H15結(jié)構(gòu)域;與無油樟(Amborella trichopoda)有較高的同源性。定量分析表明,NaCl、高溫、PEG6000、UV-C和ABA處理誘導(dǎo)PoWHY1、PoKub3和PoTRF1基因表達水平變化。(4)構(gòu)建PoWHY1、PoKub3和PoTRF1過表達載體,采用農(nóng)桿菌介導(dǎo)轉(zhuǎn)化擬南芥,分別獲得了各基因的過表達植株;結(jié)合生理和分子指標,研究它們在非生物脅迫中的作用機理。結(jié)果顯示,與野生型和空載體植株相比,PoWHY1過表達提高了鹽脅迫下種子發(fā)芽率、降低了丙二醛積累、提高脯氨酸含量,同時JAZ1、LOX1、ABI1和ABI2表達水平降低,而RAB18、APX1、GSTF6和DREB2A表達水平增加,表明PoWHY1過表達增強了擬南芥對鹽脅迫的耐受性;此外,PoWHY1過表達也增強了擬南芥的抗旱性。同樣,PoKub3過表達提高了擬南芥對鹽和干旱脅迫的抗性,以及種子對ABA的敏感性。通過以上結(jié)果可以推斷,高齡側(cè)柏古樹葉片中維持PoWHY1和PoKub3較高表達水平可能有助于增強它們對環(huán)境的適應(yīng)能力。然而PoTRF1過表達降低了鹽脅迫下擬南芥種子發(fā)芽率,但目前尚未能證實其對鹽脅迫的抵御作用,表明植物體內(nèi)PoTRF1在不同時期所起的作用可能有所不同。綜上所述,本文圍繞著“端粒相關(guān)蛋白是如何調(diào)節(jié)古樹端粒動態(tài)平衡從而影響其長壽機制”這一主題,研究了側(cè)柏古樹三個端粒相關(guān)基因在抵御逆境脅迫中的作用機理,為端粒相關(guān)基因在植物抗性方面的功能研究奠定一定的基礎(chǔ),并為端粒在古樹上的研究起到積極的推動作用。
[Abstract]:Telomere and life, senescence; and telomere associated proteins regulate telomerase activity, thus affecting telomere length. However, telomere associated protein or its growth and stress response mechanism is still not clear in plants. Therefore, the study on longevity, arborvitae resistance (Platycladus orientalis) was studied by RNA-seq analysis of 20500,1 000 and PoWHY1 PoKub3 to explore 1700 years old leaves, PoTRF1 and telomere related genes; using quantitative real-time qRT-PCR technology, the research of Platycladus orientalis Seedlings in NaCl, H2O2, ABA stress, in response to changes in patterns of these genes and telomere; in Platycladus orientalis trees (500 years) was cloned based PoWHY1. The full-length cDNA of PoKub3 and PoTRF1 gene sequences, and analyze them in different stress conditions expression patterns; finally, the use of transgenic technology in Arabidopsis (Arabidopsis thaliana) verification These three genes of high salt, drought stress resistance. The main conclusions are as follows: (1) using RNA-seq (Illumina HiSeqTM 2000) of different age leaves of Platycladus orientalis transcriptome sequencing, 48044 Unigene, the average length of 896 BP, N50 1714 bp. and the public data base for comparison, notes to NR NT, SwissProt, PFAM, GO, COG and KEGG respectively. The Unigene 24080,8 380,18 database 483,18 546,20 029,9 877 and 7783. In addition, using the MISA software with 2~6 nucleotide repeat type transcription in the SSR group of Platycladus orientalis. 1256 loci screened 4 telomere related gene PoWHY1, transcribed from group PoKub3, PoTRF1 qRT-PCR and PoPOT1, and the results show that with the increase of the age of Platycladus orientalis and the expression of PoTRF1, PoWHY1 decreased, while PoPOT1 showed the opposite trend, but PoKub3 is gradually increased. This shows that 4 telomere related gene expression There is a close relationship between the level of change with age, provide a reference for the study of Platycladus orientalis senescence related gene function. (2) combined with the related physiological indexes of active oxygen metabolism, based on active oxygen metabolism related genes (Cu/Zn-SOD, CAT, GR, APX, MDAR and GST) and telomere related genes (WHY1, Kub3, TRF1 and POT1) analysis showed that expression in NaCl and H2O2 stress and exogenous ABA treatment gene, NaCl stress can induce active oxygen metabolism related genes and telomere related genes in leaves (except POT1) expression level increased, but the addition of ABA can change the expression level of NaCl stress effect on these genes; H2O2 stress expression changes of leaf active oxygen telomere metabolism and related genes, but the application of low concentration of 0.5 mol/L ABA 48 h can further improve the expression level of telomerase activity and oxygen metabolism related genes. The results show that telomere associated genes may be associated with the stress response density of Platycladus orientalis Cut. (3) the full-length cDNA RACE cloning PoWHY1 gene sequence and Bioinformatics Method to analyze PoWHY1, PoKub3 and PoTRF1 genes. The results showed that the full-length cDNA sequence of PoWHY1 gene was 1295 BP, the open reading frame of ORF was 723 BP, encoding 240 amino acids; protein molecular weight of 26.47 kDa. At the isoelectric point was 9.19; the secondary structure with random coil, and contains a Whirly domain (Sesamum indicum); and the sesame seeds have high homology of.PoKub3 protein with molecular weight of 20.92 kDa, isoelectric point was 6.05; the two level structure with random coil and alpha helix, and contains Peptidase_M76 domain; Arabidopsis thaliana, Arabidopsis lyrata (Arabidopsis lyrata), poplar (Populus trichocarpa) and cocoa (Theobroma cacao) have high homology of.PoTRF1 protein with molecular weight of 34.06 kDa, isoelectric point was 9.07; two level structure The alpha helix and random coil, and contains SANT_TRF and H15 domain; and the (Amborella trichopoda) have high homology. The quantitative analysis showed that NaCl, PEG6000, PoWHY1 induced by high temperature, and ABA UV-C, the expression of PoKub3 and PoTRF1 gene. (4) construction of PoWHY1, PoKub3 and PoTRF1 over expression vector by Agrobacterium mediated transformation of Arabidopsis overexpressing plants were obtained by the combination of molecular marker and gene; physiological study on their abiotic stress mechanism. The results showed that compared with wild type plants and empty vector, overexpression of PoWHY1 increased the germination rate of seeds under salt stress, reduce the accumulation of MDA, proline content was increased, while JAZ1, LOX1, the expression level of ABI1 and ABI2 decreased, while RAB18, APX1, the expression level of GSTF6 and DREB2A increased, showed that over expression of PoWHY1 conferred enhanced tolerance to salt stress; in addition, P Overexpression of oWHY1 also enhanced the drought resistance of Arabidopsis thaliana. Similarly, overexpression of PoKub3 in Arabidopsis increased resistance to salt and drought stress, and the sensitivity of seeds to ABA. The above results can be inferred that PoWHY1 and PoKub3 maintain higher expression level in leaves of Platycladus orientalis aged trees may help to enhance their ability to adapt to the environment. However, overexpression of PoTRF1 reduces the rate of seed germination of Arabidopsis thaliana under salt stress, but has yet to confirm its role against the salt stress, plants showed that PoTRF1 played a role at different times may be different. To sum up, this paper focuses on the "telomere associated protein was the theme of how to adjust the dynamic balance of telomere trees the mechanism of longevity", three telomere related genes of Platycladus orientalis trees in resisting the mechanism of stress and telomere related genes in plant resistance. It lays a foundation for the functional study of surface and plays a positive role in the research of telomere in ancient trees.

【學(xué)位授予單位】：中國林業(yè)科學(xué)研究院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S791.38

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