侧柏古树端粒相关基因克隆及其抗逆功能研究

发布时间：2018-03-20 13:51

本文选题：侧柏　切入点：古树　出处：《中国林业科学研究院》2017年博士论文　论文类型：学位论文

【摘要】：端粒与寿命、衰老关系密切;而端粒相关蛋白调控端粒酶活性,从而影响端粒长度。然而,端粒或其相关蛋白在植物生长发育以及胁迫响应中的作用机制尚不清楚。因此,本研究以长寿、抗逆的侧柏(Platycladus orientalis)为研究材料,通过对20、500、1 000和1 700年树龄叶片进行RNA-seq分析,发掘PoWHY1、PoKub3和PoTRF1等端粒相关基因;采用实时定量qRT-PCR技术,研究侧柏幼苗在NaCl、H2O2、ABA胁迫处理下,这些端粒相关基因的响应模式变化;并在侧柏古树(500年)中克隆获得PoWHY1、PoKub3和PoTRF1基因全长cDNA序列的基础上,分析它们在不同胁迫条件下的表达模式;最后,利用转基因技术在拟南芥(Arabidopsis thaliana)中验证上述三个基因对高盐、干旱等胁迫的抵御作用。主要结论如下:(1)采用RNA-seq(Illumina HiSeqTM 2000)对不同年龄侧柏叶片转录组测序,获得48 044个Unigene,平均长度为896 bp,N50为1 714 bp。与公共数据库进行比对,注释到NR、NT、SwissProt、PFAM、GO、COG和KEGG数据库的Unigene分别为24080、8 380、18 483、18 546、20 029、9 877和7 783个。此外,运用MISA软件发现侧柏转录组中具有2~6个核苷酸重复类型的SSR位点1 256个。从转录组中筛选出4个端粒相关基因PoWHY1、PoKub3、PoTRF1和PoPOT1,且qRT-PCR结果显示,随着侧柏年龄的增加,PoWHY1和PoTRF1表达水平呈先升后降的趋势,而PoPOT1则呈相反的趋势,但PoKub3是逐渐升高的。这表明4个端粒相关基因表达水平变化与年龄存在密切关系,为侧柏衰老相关基因功能的研究提供了参考。(2)结合活性氧代谢相关生理指标,通过对活性氧代谢相关基因(Cu/Zn-SOD、CAT、GR、APX、MDAR和GST)和端粒相关基因(WHY1、Kub3、TRF1和POT1)在NaCl和H2O2胁迫和外源ABA处理下的基因表达分析表明,NaCl胁迫能诱导叶片活性氧代谢相关基因和端粒相关基因(除POT1外)表达水平增加,但外施ABA可改变NaCl胁迫对这些基因表达水平的作用效果;H2O2胁迫引起叶片活性氧代谢和端粒相关基因表达变化,但外施低浓度0.5μmol/L ABA 48 h能进一步提高活性氧代谢和端粒相关基因的表达水平。结果初步表明,端粒相关基因可能与侧柏的胁迫响应密切相关。(3)采用RACE技术克隆PoWHY1基因全长cDNA序列,并利用生物信息学方法对PoWHY1、PoKub3和PoTRF1基因进行分析。结果显示,PoWHY1基因全长cDNA序列为1 295 bp,开放阅读框ORF为723 bp,编码240个氨基酸;蛋白质分子量为26.47 kDa,等电位点为9.19;其二级结构以无规则卷曲为主,并含有一个Whirly结构域;与芝麻(Sesamum indicum)有较高的同源性。PoKub3蛋白质分子量为20.92 kDa,等电位点为6.05;二级结构以无规则卷曲和α-螺旋为主,并含有Peptidase_M76结构域;与拟南芥、琴叶拟南芥(Arabidopsis lyrata)、毛果杨(Populus trichocarpa)和可可(Theobroma cacao)有较高的同源性。PoTRF1蛋白质分子量为34.06 kDa,等电位点为9.07;二级结构以α-螺旋和无规则卷曲为主,并含有SANT_TRF和H15结构域;与无油樟(Amborella trichopoda)有较高的同源性。定量分析表明,NaCl、高温、PEG6000、UV-C和ABA处理诱导PoWHY1、PoKub3和PoTRF1基因表达水平变化。(4)构建PoWHY1、PoKub3和PoTRF1过表达载体,采用农杆菌介导转化拟南芥,分别获得了各基因的过表达植株;结合生理和分子指标,研究它们在非生物胁迫中的作用机理。结果显示,与野生型和空载体植株相比,PoWHY1过表达提高了盐胁迫下种子发芽率、降低了丙二醛积累、提高脯氨酸含量,同时JAZ1、LOX1、ABI1和ABI2表达水平降低,而RAB18、APX1、GSTF6和DREB2A表达水平增加,表明PoWHY1过表达增强了拟南芥对盐胁迫的耐受性;此外,PoWHY1过表达也增强了拟南芥的抗旱性。同样,PoKub3过表达提高了拟南芥对盐和干旱胁迫的抗性,以及种子对ABA的敏感性。通过以上结果可以推断,高龄侧柏古树叶片中维持PoWHY1和PoKub3较高表达水平可能有助于增强它们对环境的适应能力。然而PoTRF1过表达降低了盐胁迫下拟南芥种子发芽率,但目前尚未能证实其对盐胁迫的抵御作用,表明植物体内PoTRF1在不同时期所起的作用可能有所不同。综上所述,本文围绕着“端粒相关蛋白是如何调节古树端粒动态平衡从而影响其长寿机制”这一主题,研究了侧柏古树三个端粒相关基因在抵御逆境胁迫中的作用机理,为端粒相关基因在植物抗性方面的功能研究奠定一定的基础,并为端粒在古树上的研究起到积极的推动作用。
[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.

【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2;S791.38

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