馒头柳镉吸收转运的分子生理学基础研究

发布时间：2018-07-05 09:37

本文选题：馒头柳 + 镉　；参考：《中国林业科学研究院》2017年博士论文

【摘要】：人类活动和工业的发展导致土壤Cd污染日益严重。Cd是植物生长非必需元素,其化合物溶解度高,在土壤——植物系统中具有很强的迁移能力。植物修复是利用植物将重金属污染物从土壤中清除,或使之无害,具有费用低廉、安全有效、原位修复、可持续发展等特征。木本植物具有生物量大、生长迅速、根系拓展的深度和范围广、能有效地将重金属转运至地上部等优势,是重金属污染土壤修复的优良候选材料。本研究以旱柳的栽培变型馒头柳为研究对象,旱柳为对照,探索二者在Cd胁迫后的形态、生理生化和分子方面的差异,随后通过转录组测序技术从分子层面挖掘Cd吸收、转运、解毒等过程相关的基因,进而解析分子调控机制。主要研究结果如下:1.从分子系统进化方面证实了馒头柳和旱柳分类学上的亲缘关系,二者叶绿体基因matK和核糖体基因ITS同源性高达99%以上。100μmol L-1 CdCl2胁迫后,馒头柳和旱柳在植物生长、根系形态、叶绿素含量、光合作用等方面都表现出一定的差异。2.馒头柳和旱柳在地上部Cd积累方面存在显著差异,同一胁迫时间点馒头柳相同组织中的Cd含量高于旱柳,且随胁迫时间的延长差异越显著。胁迫1天后,在馒头柳根尖有较强的Cd2+流入,并且其空间动态变化趋势同旱柳基本一致,但馒头柳同一根尖位置吸收量高于旱柳,并随胁迫时间的延长而递增。双硫腙组化染色定位发现馒头柳在根、茎、叶及主脉中积累了大量的Cd-双硫腙复合物,同位置积累复合物明显多于旱柳。透射电镜和EDX能谱观测证实Cd主要积累在液泡内、细胞壁和细胞间隙。Cd胁迫下,馒头柳、旱柳的根系、树皮、木质部和叶片FTIR特征图谱存在不同程度的差异,是由于与Cd发生离子交换、络合、转运的基团、载体或大分子物质存在不同,而馒头柳的根、皮、木质部和叶片能通过羟基、羧基等官能团的结合来缓解Cd积累所造成的毒害。以上结果表明,馒头柳具有相对较强的Cd吸收、转运和积累能力,是优良的植物修复候选材料。3.Cd胁迫后,馒头柳和旱柳体内膜质过氧化水平和活性氧自由基(ROS)含量随胁迫时间的延长而增加。根系活力、抗氧化酶、非酶类抗氧化物等表现出了时间、组织的特异性。综合对比各项生理指标,馒头柳具备更强的适应能力和抗氧化胁迫能力。植物内源激素参与了重金属胁迫响应等许多生理过程的调节,本研究中,生长素、脱落酸、茉莉酸和水杨酸在响应Cd胁迫后都发生了变化,其中茉莉酸显著升高且趋势明显,可能在响应Cd胁迫方面起到重要的作用。4.利用Illumina HiSeqTM2500测序平台对Cd胁迫后不同时间的两种柳树根系进行转录组测序。经过测序质控共得到高质量的152.46 Gb Clean Data,随后组装、拼接共得到76 194条Unigenes,N50为1 427 bp。通过对比不同胁迫时间馒头柳和旱柳根系内差异基因数量变化,发现Cd胁迫初期,馒头柳中响应的差异基因数量虽少于旱柳,但后期参与的基因数量显著高于旱柳。差异基因在馒头柳、旱柳中的瞬时表达模式及其GO注释分类存在明显差异,馒头柳根系中有更多的基因参与了Cd的转运、解毒等生物学过程。通过对Cd转运、解毒、信号传导等相关的12个基因q-PCR分析,结果表明这些基因在馒头柳根中的表达量显著高于旱柳,初步证实ABC转运因子(ATP-binding cassette transponer,ABC)、bZIP转运因子(bZIP transcription factor family,bZIP)、金属硫蛋白(Metallothionein,MT)、植物螯合肽合成酶(Phytochelatin syntheses,PCS)、金属转运因子(Natural resistance-associated macrophage protein,Nramp)等基因在馒头柳根系中起到了重要的作用,也为进一步探索其功能提供了依据。5.基于转录组测序数据,通过5种运算统计方法对馒头柳11个候选内参基因在干旱、盐、重金属3种非生物胁迫及6个不同组织器官中的表达稳定性分析,筛选出2个表达最稳定的内参基因α-TUB2和DnaJ。利用功能基因SmCAT检测了这两个内参基因的稳定性,证明α-TUB2和DnaJ在馒头柳11个候选内参基因中是最稳定、可靠的内参基因。本研究中内参基因的筛选将促进柳属植物在各类非生物胁迫下相关基因的表达分析和比较。
[Abstract]:The development of human activities and industry leads to the increasing pollution of soil Cd, which is a non essential element for plant growth. The solubility of the compound is high, and its compound has high mobility in the soil plant system. Phytoremediation is the use of plants to remove heavy metal pollutants from the soil, or make it harmless, with low cost, safe and effective. It is a good candidate material for the remediation of heavy metal contaminated soil. This study takes the cultivated and transformed steamed willow of willow as the research object. The study of dryland willow is the control and exploration. The two changes in morphology, physiology, biochemistry and molecular aspects after Cd stress, and then using the transcription group sequencing technology to excavate the related genes of Cd absorption, transport and detoxification from the molecular level, and then analyze the molecular regulation mechanism. The main results are as follows: 1. from the molecular phylogenetic evolution, the taxonomy of steamed willow and dryland willow was confirmed. The homology of the two chloroplast gene matK and ribosome gene ITS was more than 99%.100 Mu mol L-1 CdCl2 stress, the steamed willow and the dry willow in the plant growth, the root morphology, the chlorophyll content, the photosynthesis and so on, there were some differences between the.2. steamed willow and the dry willow, and the same stress was found on the accumulation of Cd in the upper part of the ground, and the same stress. The Cd content in the same tissue of steamed steamed willow was higher than that in the dry willow, and the difference was more significant with the prolongation of the stress time. After 1 days of stress, there was a strong Cd2+ inflow in the root tip of the steamed bread, and the dynamic trend of the steamed willow was basically the same as that of the dry willow, but the absorption amount of the same root tip in the steamed bread was higher than that of the dry willow, and increased with the prolonged stress time. A large amount of Cd- dithizone complex was accumulated in the root, stem, leaf and main vein of steamed buns, and the accumulation complex in the same position was more than that of the dry willow. The transmission electron microscope and EDX spectrum observation confirmed that Cd was mainly accumulated in the vacuole, under the.Cd stress of the cell wall and cell gap, the root of steamed bread, the bark, xylem and leaf of the willow. There are different degrees of difference in the feature map of FTIR. It is due to the difference of ion exchange with Cd, complex, transport group, carrier or macromolecular substance, while the root, skin, xylem and leaf of steamed bread can be combined with the hydroxyl and carboxyl groups to alleviate the toxicity caused by the accumulation of Cd. The above results show that the steamed bread has a phase. For the strong Cd absorption, transport and accumulation ability, it is a good candidate for phytoremediation of.3.Cd. The content of endocardial peroxidation and reactive oxygen free radicals (ROS) in steamed steamed and drya body increases with the prolongation of stress time. Root activity, antioxidant enzymes, non enzyme antioxidants, etc., show time and tissue specificity. In this study, auxin, abscisic acid, jasmonic acid and salicylic acid have been changed in response to Cd stress, and jasmonic acid is significantly increased and trend is obvious in this study. Obviously, it may play an important role in response to Cd stress..4. uses Illumina HiSeqTM2500 sequencing platform to sequence the two species of willow roots at different time after Cd stress. After sequencing quality control, a high quality 152.46 Gb Clean Data is obtained. Then, the assembly is assembled to 76194 Unigenes, and N50 is 1427 bp. through comparison. The number of different genes in steamed steamed willow and dry willow root system was changed at different stress times. It was found that the number of differentially expressed genes in the steamed bread willow was less than that of the dry willow in the early stage of Cd stress, but the number of genes involved in the later period was significantly higher than that of the dryland willow. The difference gene was in the steamed willow, and the instantaneous expression pattern and the GO annotation in the willow were distinct, steamed willow More genes were involved in the transport of Cd, detoxification and other biological processes. Through the analysis of 12 genes related to Cd transport, detoxification and signal transduction, the results showed that the expression of these genes in the steamed bread was significantly higher than that of the dry willow, and the ABC transport factor (ATP-binding cassette transponer, ABC) and bZIP transport factor were preliminarily confirmed. (bZIP transcription factor family, bZIP), metallothionein (Metallothionein, MT), plant chelating peptide synthetase (Phytochelatin syntheses, PCS), metal transport factor (Natural resistance-associated) and other genes play an important role in the root of steamed bread, and also provide a further exploration of its function. Based on.5. based on the sequence of transcriptional sequence data, the expression stability of 11 candidate internal reference genes in steamed bread was analyzed by 5 kinds of operational statistics in drought, salt, heavy metal 3 abiotic stresses and 6 different tissues and organs, and 2 most stable internal reference genes, alpha -TUB2 and DnaJ. using functional gene SmCAT, were screened for the detection of these two. The stability of the ginseng gene has proved that alpha -TUB2 and DnaJ are the most stable and reliable internal reference genes in 11 candidate internal reference genes of steamed willow. The screening of the internal reference genes in this study will promote the analysis and comparison of the related genes in the genus willow under various abiotic stresses.
【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】：S792.12

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