發(fā)布時(shí)間：2018-05-21 10:52

  本文選題：側(cè)柏(Platycladus + orientalis(L.))��； 參考：《西北農(nóng)林科技大學(xué)》2017年博士論文

【摘要】：側(cè)柏(Platycladus orientalis(L.))耐干旱瘠薄、壽命長(zhǎng)、抗病蟲、具有較強(qiáng)的適應(yīng)性,并且耗水量低,是干旱半干旱地區(qū)的造林先鋒樹種。側(cè)柏在中國(guó)分布較廣,歷來作為名地古剎的重要栽培樹種;在中國(guó)許多地區(qū)均有樹齡超過千年的側(cè)柏,目前對(duì)古側(cè)柏的研究還較為滯后,許多科學(xué)問題亟待解決,如側(cè)柏生長(zhǎng)千年而不衰的分子機(jī)制和近年來一些千年古側(cè)柏出現(xiàn)衰退跡象的影響機(jī)制問題。作為一個(gè)長(zhǎng)壽樹種,側(cè)柏對(duì)環(huán)境脅迫的抗性基礎(chǔ)可能是其生長(zhǎng)千年而不衰的重要因素之一,同時(shí)近年來極端不良的環(huán)境因素也可能是使古樹出現(xiàn)衰退的主要原因。本論文深入研究了水分脅迫條件下側(cè)柏的抗逆分子機(jī)制,同時(shí)開展了古側(cè)柏低溫環(huán)境響應(yīng)的分子機(jī)理研究,為揭示側(cè)柏的抗衰老機(jī)制、衰退機(jī)理奠定基礎(chǔ)。主要的研究結(jié)果如下:1.側(cè)柏幼苗干旱脅迫根系轉(zhuǎn)錄組測(cè)序研究與差異基因篩選對(duì)側(cè)柏幼苗開展溫室模擬干旱實(shí)驗(yàn),經(jīng)過兩個(gè)月的干旱脅迫處理,采集側(cè)柏根系建立c DNA文庫(kù),得到了53.1-63.4百萬clean reads。經(jīng)過對(duì)高質(zhì)量clean reads進(jìn)行de novo組裝和去除冗余最終得到148,392條unigene。對(duì)最終保留的116,716條unigene序列做后續(xù)分析,注釋結(jié)果表明29.9%(34,845/116,716)的基因注釋到NCBI nr數(shù)據(jù)庫(kù),20.2%(23,573/116,716)注釋到Swiss-Prot蛋白數(shù)據(jù)庫(kù)。綜合分析,一共有36,900 unigenes(31.62%)至少成功注釋到一個(gè)數(shù)據(jù)庫(kù)中(NCBI nr,Swiss-Prot,KEGG,GO和Pfam),有7998 unigenes(6.85%)同時(shí)注釋到上述5個(gè)數(shù)據(jù)庫(kù)。通過GO數(shù)據(jù)庫(kù)比對(duì),116716條unigene中有17012條基因被賦予4441個(gè)GO條目,劃分為60個(gè)功能組。對(duì)116716條unigene做進(jìn)一步的KEGG通路分析表明一共有13312(11.41%)條unigene被分配到5個(gè)功能通路大類和98個(gè)KEGG通路中。通過基因差異分析,得到3930個(gè)差異表達(dá)基因,其中881個(gè)基因發(fā)生上調(diào)表達(dá),3049個(gè)基因發(fā)生下調(diào)表達(dá)。這其中檢測(cè)到194個(gè)轉(zhuǎn)錄因子在干旱脅迫下表現(xiàn)出顯著表達(dá)變化。通過差異基因分析以及基因功能富集分析得到了和側(cè)柏干旱脅迫防御/響應(yīng)相關(guān)的候選基因,這些差異基因中具有信號(hào)轉(zhuǎn)導(dǎo)、生物合成、ROS清除、離子/能量轉(zhuǎn)運(yùn)、蛋白酶、大分子保護(hù)等功能。本研究在轉(zhuǎn)錄水平得出側(cè)柏干旱分子防御/響應(yīng)機(jī)制是基于眾多的基因直接參與側(cè)柏干旱脅迫的保護(hù)過程,這包括大量信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)基因、調(diào)控基因和功能基因的參與,基于此得到了側(cè)柏干旱脅迫分子響應(yīng)復(fù)雜調(diào)控機(jī)制:包括,干旱脅迫第一階段,根系細(xì)胞通過膜和/或細(xì)胞的變化感受水分脅迫,接著細(xì)胞激活Ca2+信號(hào)、ROS信號(hào)和激素信號(hào)(信號(hào)感知階段)引起下游脅迫響應(yīng)基因的表達(dá),包括轉(zhuǎn)錄因子、離子/能量轉(zhuǎn)運(yùn)蛋白、大分子的防護(hù)因子、解毒作用因子、滲透物質(zhì)合成關(guān)鍵酶和蛋白酶類�？傮w來說,這些基因可以分成兩組:第一組包含的基因(蛋白質(zhì))主要參與基因表達(dá)的調(diào)控(調(diào)控蛋白/轉(zhuǎn)錄因子),第二組包含基因(蛋白質(zhì))主要在脅迫適應(yīng)性過程起直接防御/修復(fù)作用(功能蛋白),通過這些被激活的脅迫響應(yīng)基因表達(dá)最終使細(xì)胞重新恢復(fù)穩(wěn)定和平衡,修復(fù)和保護(hù)被干旱脅迫破壞的細(xì)胞膜和蛋白質(zhì)。2.側(cè)柏根系、葉組織干旱脅迫的生理與蛋白質(zhì)組學(xué)分析基于側(cè)柏轉(zhuǎn)錄組測(cè)序的完成,為進(jìn)一步理解側(cè)柏干旱脅迫響應(yīng)/防御機(jī)制,采集四個(gè)水分控制條件下(正常水分供應(yīng):80±5%的田間持水量;輕度的水分脅迫:70±5%的田間持水量;中等水分脅迫:50±5%的田間持水量;嚴(yán)重干旱脅迫:35±5%的田間持水量)側(cè)柏幼苗根系和葉組織樣本開展生理學(xué)和蛋白質(zhì)組學(xué)分析。生理研究表明:輕度的水分脅迫不會(huì)對(duì)側(cè)柏產(chǎn)生顯著影響,側(cè)柏根系和葉組織可溶性蛋白、葉片葉綠素含量未見顯著變化。中等和嚴(yán)重水分脅迫導(dǎo)致了幼苗在形態(tài)上和生理上均有較顯著的變化,包括顯著減少葉RWC、根系WC和凈生長(zhǎng)。從凈生長(zhǎng)來看,20天的嚴(yán)重干旱脅迫和25天的中等干旱脅迫已經(jīng)對(duì)側(cè)柏幼苗生長(zhǎng)產(chǎn)生顯著的影響。蛋白質(zhì)組學(xué)研究揭示了水分梯度脅迫下根系和葉組織蛋白質(zhì)表達(dá)差異特征。研究分別對(duì)根系和葉組織97個(gè)和65個(gè)具有顯著差異表達(dá)的蛋白質(zhì)進(jìn)行質(zhì)譜鑒定。從這些蛋白質(zhì)的功能分類來看這些蛋白質(zhì)主要參與脅迫響應(yīng)/防御、碳水化合物代謝過程和氮代謝。從整體的蛋白質(zhì)變化特征可以得出,側(cè)柏在干旱環(huán)境下生存需要許多的策略;如,保持細(xì)胞內(nèi)蛋白質(zhì)穩(wěn)定、活性和折疊;ROS解毒過程、調(diào)節(jié)細(xì)胞的滲透勢(shì)、維持細(xì)胞壁完整性、能量代謝和細(xì)胞骨架穩(wěn)定等。綜合根系轉(zhuǎn)錄組和蛋白質(zhì)組學(xué)結(jié)果總體來看,當(dāng)脅迫發(fā)生轉(zhuǎn)錄和蛋白質(zhì)水平調(diào)控具有較大的一致性。3.側(cè)柏干旱脅迫響應(yīng)基因在干旱后復(fù)水表達(dá)分析驗(yàn)證對(duì)得到的干旱脅迫防御/響應(yīng)相關(guān)基因進(jìn)一步的表達(dá)驗(yàn)證,篩選了轉(zhuǎn)錄因子WRKY53、NAC72和AP2/ERF;蛋白激酶CDPK34、CDPK16和CDPK1;活性氧清除相關(guān)基因Fe-SOD、CAT3、POD3、APX3、APX6;滲透調(diào)節(jié)相關(guān)基因SUS1、SUS3、SUS6、TRE6PS、P5CS2和P5CR;保護(hù)因子蛋白s HSP(HSP18.1、HSP23.5和HSP25.3)、HSP70和LEA;14-3-3like蛋白、皮部?jī)?chǔ)藏蛋白(BSP)基因進(jìn)行了干旱和復(fù)水后基因的表達(dá)量分析。基因表達(dá)分析得出:1.轉(zhuǎn)錄因子NAC72參與側(cè)柏干旱脅迫調(diào)控,NAC72基因的高表達(dá)可能對(duì)下游基因的激活有重要調(diào)控作用;轉(zhuǎn)錄因子AP2/ERF表現(xiàn)出明顯的復(fù)水補(bǔ)償特征,可能和側(cè)柏復(fù)水后生長(zhǎng)、組織損傷修復(fù)有關(guān)。2.APX3、Tre6PS、LEA、14-3-3蛋白在干旱下顯著上調(diào)表達(dá)可能參與了側(cè)柏干旱響應(yīng)的相關(guān)調(diào)控。3.脯氨酸合成關(guān)鍵基因(P5CS2)表達(dá)水平分析得出,P5CS2在側(cè)柏干旱脅迫防御/響應(yīng)過程中可能起到促進(jìn)滲透調(diào)節(jié)物質(zhì)合成的作用。4.s HSP和HSP70在側(cè)柏干旱脅迫不同時(shí)期高豐度上調(diào)表達(dá),可能參與了干旱不同程度的脅迫修復(fù)或響應(yīng);研究還發(fā)現(xiàn)部分基因在根系和葉組織響應(yīng)干旱脅迫中具有協(xié)同性和不同步性。4.古側(cè)柏葉冬季自然低溫響應(yīng)的生理與差異蛋白質(zhì)組學(xué)研究對(duì)樹齡20±5,500±100和1200±200年側(cè)柏的葉組織開展自然低溫脅迫響應(yīng)的生理學(xué)與蛋白質(zhì)組學(xué)研究,結(jié)果證實(shí)古樹尤其以千年古樹受低溫的影響更大,葉組織葉綠素、可溶性蛋白質(zhì)含量的顯著下降,MDA含量的升高,SOD、POD活性的下降都成為古樹在低溫下的重要標(biāo)志,也預(yù)示著古樹葉的早衰可能和環(huán)境的影響有重要關(guān)系。經(jīng)過對(duì)三個(gè)樹齡葉組織蛋白質(zhì)電泳分析和差異蛋白質(zhì)MALDI TOF/TOF MS/MS鑒定,77個(gè)差異蛋白質(zhì)凝膠點(diǎn)得到成功鑒定和注釋。這些成功鑒定的蛋白質(zhì)可以劃分為多個(gè)功能組,包括防御/脅迫相關(guān)、能量和碳水化合物代謝相關(guān)、光合作用和激素相關(guān)等。從蛋白質(zhì)組學(xué)的對(duì)比分析得出成年樹和古樹間存在較大的蛋白質(zhì)響應(yīng)差異,如在低溫下參與光合作用、防御/脅迫響應(yīng)蛋白豐度在古樹中(尤其是千年古樹中)顯著下降。然而,功能分類為能量和碳水化合物代謝相關(guān)蛋白在古樹中多為高豐度表達(dá)。從蛋白質(zhì)組學(xué)分析總體來看,千年古樹表現(xiàn)出較為明顯的低溫下葉衰退跡象;但在古樹的葉組織中也篩選到一些高豐度脅迫防御、大分子保護(hù)物質(zhì)、激素合成等相關(guān)蛋白,這些蛋白質(zhì)可能是古側(cè)柏抗衰老的重要因子。生理和蛋白組學(xué)分析展示了古樹低溫響應(yīng)的一個(gè)復(fù)雜的生理和分子差異變化特征框架。
[Abstract]:Platycladus orientalis (L.) is a forestation pioneer tree in arid and semi-arid areas with drought and barren resistance, long life, strong adaptability and low water consumption. It is widely distributed in China and has always been used as an important planting tree in the famous ancient temples; in many areas there are more than 1000 year old Platycladus orientalis in China. The study of the ancient Platycladus orientalis is still lagging behind, and many scientific problems need to be solved, such as the molecular mechanism of the unfailing growth of the Platycladus orientalis and the mechanism of the decline of some millennial Platycladus orientalis in recent years. As a long-lived tree, the resistance basis of the Platycladus orientalis to environmental stress can be one of the important factors for its growth for a thousand years. At the same time, extreme adverse environmental factors may also be the main reasons for the decline of ancient trees. This paper studies the anti inverse molecular mechanism of Platycladus orientalis under water stress, and studies the molecular mechanism of the environmental response of Platycladus orientalis at low temperature, which lays the foundation for revealing the mechanism of anti aging and the mechanism of decline of the Platycladus orientalis. The results are as follows: 1. the root transcriptional group of Platycladus orientalis seedling drought stress was sequenced and the differential gene screening was used to carry out simulated drought experiment in the greenhouse. After two months of drought stress treatment, the C DNA library was set up by the root system of Platycladus orientalis, and 53.1-63.4 million clean reads. was assembled and gone through the high quality clean reads. In addition to redundancy, 148392 unigene. sequences were eventually obtained for the final 116716 UniGene sequences that were retained. The annotation results showed that 29.9% (34845/116716) gene was annotated to the NCBI NR database, 20.2% (23573/116716) annotated to the Swiss-Prot protein database. A total of 36900 unigenes (31.62%) had been annotated to at least one In the database (NCBI NR, Swiss-Prot, KEGG, GO and Pfam) with 7998 unigenes (6.85%) simultaneously annotated to the above 5 databases. 17012 genes in 116716 UniGene are assigned to 4441 GO entries and 60 functional groups by the GO database alignment. Further KEGG path analysis for 116716 UniGene shows that there are 13312 (11.41%) entries in all. Gene was assigned to 5 functional pathways and 98 KEGG pathways. Through genetic diversity analysis, 3930 differentially expressed genes were obtained, of which 881 genes were up-regulated and 3049 genes were down regulated. Among them, 194 transcriptional factors showed significant changes under drought stress. Gene function enrichment analysis has obtained candidate genes related to the defense / response of Platycladus orientalis drought stress. These genes have signal transduction, biosynthesis, ROS scavenging, ion / energy transport, protease, macromolecule protection and other functions. This study shows that the mechanism of defense / response of Platycladus orientalis drought molecules is based on numerous genes Direct participation in the protection process of Platycladus orientalis drought stress, which includes a large number of signal transduction system genes, regulatory genes and functional genes involved. Based on this, the complex regulation mechanism of the response of droughts stress molecules in the Platycladus orientalis is obtained: the first stage of drought stress, the root cells feel water stress through the changes of membrane and / or cells, and then cell excitation Live Ca2+ signal, ROS signal and hormone signal (signal perception stage) cause the expression of downstream stress response gene, including transcription factor, ion / energy transporter, macromolecule protective factor, detoxification factor, osmotic substance synthesis key enzyme and protease. Generally speaking, these genes can be divided into two groups: the first group of genes ( Protein) is mainly involved in the regulation of gene expression (regulatory protein / transcription factor), and the second groups of genes (proteins) mainly play a direct defense / repair function (functional protein) in the process of stress adaptation, which eventually restores and balances the cells through these activated stress response genes, and repairs and protects drought stress. The damaged cell membrane and the protein.2. of Platycladus orientalis root, the physiological and proteomic analysis of leaf tissue drought stress were based on the complete sequence of the Platycladus orientalis transcriptional group. In order to further understand the response / defense mechanism of Platycladus orientalis drought stress, under four water control conditions (normal water supply: 80 + 5% field water holding capacity; mild water stress: 70 + 5%. Field water holding capacity, medium water stress: 50 + 5% field water capacity, severe drought stress: 35 + 5% field water capacity), physiological and proteomics analysis of root and leaf tissue samples of Platycladus orientalis Seedlings. Physiological studies show that mild water stress does not significantly affect the yield of Platycladus orientalis, root and leaf tissue soluble protein of Platycladus orientalis, There was no significant change in leaf chlorophyll content. Medium and severe water stress resulted in significant changes in the morphology and physiology of seedlings, including significantly reducing leaf RWC, root WC and net growth. From the net growth, 20 days of severe drought stress and 25 days of medium drought had a significant effect on the growth of the seedlings of the Platycladus orientalis. Proteomics studies revealed differences in protein expression in root and leaf tissues under water gradient stress. 97 and 65 proteins with significant differences in root and leaf tissues were identified by mass spectrometry. These proteins were mainly involved in stress response / defense and carbon hydration from the functional classification of these proteins. Metabolic processes and nitrogen metabolism. From the overall characteristics of protein change, it can be concluded that the Platycladus orientalis needs a lot of strategies in the drought environment, for example, maintaining protein stability, activity and folding in the cell; ROS detoxification process, regulating cell osmotic potential, maintaining cell wall integrity, energy metabolism and cytoskeleton stability. Integrated root transcription. The results of the group and proteomic analysis showed that when the transcription and protein level of stress were regulated, the response genes for drought stress response genes of.3. in the drought stress were verified to verify the further expression of defense / response related genes of drought stress, and the transcription factor WRKY53, NAC72 and AP2/ERF were screened and the eggs were screened. CDPK34, CDPK16 and CDPK1; active oxygen scavenging related genes Fe-SOD, CAT3, POD3, APX3, APX6; osmotic regulation related genes SUS1, SUS3, SUS6, TRE6PS, etc. Analysis. Gene expression analysis showed that the 1. transcription factor NAC72 participates in the regulation of drought stress of Platycladus orientalis, the high expression of NAC72 gene may play an important role in regulating the activation of the downstream genes; the transcription factor AP2/ERF shows obvious rehydration characteristics and may grow after the rehydration of the Platycladus orientalis, and the tissue damage repair is related to the.2.APX3, Tre6PS, LEA, 14-3-3 protein. Significant up-regulated expression in drought stress may participate in the related regulation of the response of Platycladus orientalis drought response.3. proline synthesis key gene (P5CS2) expression level analysis, P5CS2 may play the role of promoting the synthesis of osmotic regulators in the process of drought stress and response of Platycladus orientalis,.4.s HSP and HSP70 are high abundance at different periods of drought stress in the Platycladus orientalis. Up-regulated expression may be involved in the drought stress repair or response to different degrees of drought. The study also found that some genes have synergism and asynchrony in response to drought stress in root and leaf tissues. The physiological and differential proteomics of the natural low temperature response of.4. palaeo cypress leaves in winter is 20 + 5500 + 100 and 1200 + 200 years old. The physiological and proteomics studies of the response to natural low temperature stress showed that the ancient trees were more affected by the low temperature, the chlorophyll, the content of soluble protein, the content of MDA and the decrease of the activity of SOD and POD were all the important signs of the ancient trees at low temperature and the ancient trees. The premature senility of leaves may have an important relationship with the environmental impact. After the protein electrophoresis analysis of three tree age leaves and identification of the differential protein MALDI TOF/TOF MS/MS, the 77 differentially identified protein gel points are successfully identified and annotated. The proteins identified successfully can be divided into several functional groups, including defense / stress correlation, and energy. Correlation of carbohydrate metabolism, photosynthesis, and hormone related. Comparative analysis of proteomics from proteomics shows that there are significant differences in protein responses between adult and ancient trees, such as photosynthesis at low temperatures, and the abundances of defense / stress response proteins are significantly decreased in ancient trees (especially in Millennium ancient trees). The proteins associated with energy and carbohydrate metabolism are highly expressed in ancient trees. From the analysis of proteomic analysis, the millennial ancient trees showed obvious signs of lower hypothermia, but in the leaf tissues of the ancient trees, some high abundance stress defenses, large sub protections, hormone synthesis and other related proteins were also screened in the leaf tissues of the ancient trees. Protein may be an important factor in antiaging of Platycladus orientalis. Physiological and proteomics analysis shows a complex framework of physiological and molecular variation in the low temperature response of ancient trees.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S791.38
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本文編號(hào)：1918899