本文選題：饅頭柳 + 鎘　； 參考：《中國林業(yè)科學研究院》2017年博士論文

【摘要】：人類活動和工業(yè)的發(fā)展導致土壤Cd污染日益嚴重。Cd是植物生長非必需元素,其化合物溶解度高,在土壤——植物系統(tǒng)中具有很強的遷移能力。植物修復是利用植物將重金屬污染物從土壤中清除,或使之無害,具有費用低廉、安全有效、原位修復、可持續(xù)發(fā)展等特征。木本植物具有生物量大、生長迅速、根系拓展的深度和范圍廣、能有效地將重金屬轉運至地上部等優(yōu)勢,是重金屬污染土壤修復的優(yōu)良候選材料。本研究以旱柳的栽培變型饅頭柳為研究對象,旱柳為對照,探索二者在Cd脅迫后的形態(tài)、生理生化和分子方面的差異,隨后通過轉錄組測序技術從分子層面挖掘Cd吸收、轉運、解毒等過程相關的基因,進而解析分子調控機制。主要研究結果如下:1.從分子系統(tǒng)進化方面證實了饅頭柳和旱柳分類學上的親緣關系,二者葉綠體基因matK和核糖體基因ITS同源性高達99%以上。100μmol L-1 CdCl2脅迫后,饅頭柳和旱柳在植物生長、根系形態(tài)、葉綠素含量、光合作用等方面都表現(xiàn)出一定的差異。2.饅頭柳和旱柳在地上部Cd積累方面存在顯著差異,同一脅迫時間點饅頭柳相同組織中的Cd含量高于旱柳,且隨脅迫時間的延長差異越顯著。脅迫1天后,在饅頭柳根尖有較強的Cd2+流入,并且其空間動態(tài)變化趨勢同旱柳基本一致,但饅頭柳同一根尖位置吸收量高于旱柳,并隨脅迫時間的延長而遞增。雙硫腙組化染色定位發(fā)現(xiàn)饅頭柳在根、莖、葉及主脈中積累了大量的Cd-雙硫腙復合物,同位置積累復合物明顯多于旱柳。透射電鏡和EDX能譜觀測證實Cd主要積累在液泡內、細胞壁和細胞間隙。Cd脅迫下,饅頭柳、旱柳的根系、樹皮、木質部和葉片F(xiàn)TIR特征圖譜存在不同程度的差異,是由于與Cd發(fā)生離子交換、絡合、轉運的基團、載體或大分子物質存在不同,而饅頭柳的根、皮、木質部和葉片能通過羥基、羧基等官能團的結合來緩解Cd積累所造成的毒害。以上結果表明,饅頭柳具有相對較強的Cd吸收、轉運和積累能力,是優(yōu)良的植物修復候選材料。3.Cd脅迫后,饅頭柳和旱柳體內膜質過氧化水平和活性氧自由基(ROS)含量隨脅迫時間的延長而增加。根系活力、抗氧化酶、非酶類抗氧化物等表現(xiàn)出了時間、組織的特異性。綜合對比各項生理指標,饅頭柳具備更強的適應能力和抗氧化脅迫能力。植物內源激素參與了重金屬脅迫響應等許多生理過程的調節(jié),本研究中,生長素、脫落酸、茉莉酸和水楊酸在響應Cd脅迫后都發(fā)生了變化,其中茉莉酸顯著升高且趨勢明顯,可能在響應Cd脅迫方面起到重要的作用。4.利用Illumina HiSeqTM2500測序平臺對Cd脅迫后不同時間的兩種柳樹根系進行轉錄組測序。經過測序質控共得到高質量的152.46 Gb Clean Data,隨后組裝、拼接共得到76 194條Unigenes,N50為1 427 bp。通過對比不同脅迫時間饅頭柳和旱柳根系內差異基因數(shù)量變化,發(fā)現(xiàn)Cd脅迫初期,饅頭柳中響應的差異基因數(shù)量雖少于旱柳,但后期參與的基因數(shù)量顯著高于旱柳。差異基因在饅頭柳、旱柳中的瞬時表達模式及其GO注釋分類存在明顯差異,饅頭柳根系中有更多的基因參與了Cd的轉運、解毒等生物學過程。通過對Cd轉運、解毒、信號傳導等相關的12個基因q-PCR分析,結果表明這些基因在饅頭柳根中的表達量顯著高于旱柳,初步證實ABC轉運因子(ATP-binding cassette transponer,ABC)、bZIP轉運因子(bZIP transcription factor family,bZIP)、金屬硫蛋白(Metallothionein,MT)、植物螯合肽合成酶(Phytochelatin syntheses,PCS)、金屬轉運因子(Natural resistance-associated macrophage protein,Nramp)等基因在饅頭柳根系中起到了重要的作用,也為進一步探索其功能提供了依據(jù)。5.基于轉錄組測序數(shù)據(jù),通過5種運算統(tǒng)計方法對饅頭柳11個候選內參基因在干旱、鹽、重金屬3種非生物脅迫及6個不同組織器官中的表達穩(wěn)定性分析,篩選出2個表達最穩(wěn)定的內參基因α-TUB2和DnaJ。利用功能基因SmCAT檢測了這兩個內參基因的穩(wěn)定性,證明α-TUB2和DnaJ在饅頭柳11個候選內參基因中是最穩(wěn)定、可靠的內參基因。本研究中內參基因的篩選將促進柳屬植物在各類非生物脅迫下相關基因的表達分析和比較。
[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.
【學位授予單位】：中國林業(yè)科學研究院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S792.12

【參考文獻】

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1 胡博華;徐R，

本文編號：2099774