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杨树无性系交互嫁接对干旱胁迫的生理响应及分子机制
Alternative TitlePhysiological responses and molecular mechanism of reciprocal grafting of Populus clones to drought stress
韩清泉
Subtype博士
Thesis Advisor李春阳 ; 张胜
2018
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline生态学
Keyword杨树种间嫁接 水分利用效率 抗旱性 转录组学 蛋白质组学
Abstract杨树(Populus)是广泛分布在北半球温带和寒温带的森林树种,因具有生长迅速、适应性强、容易繁殖以及遗传多样性等优点,成为研究的主要树种。随着全球环境的变化干旱的发生也越来越频繁,嫁接被证明是一种提高植物生产力和抗逆性的有效方法,但关于抗旱性不同的杨树进行种间嫁接后是否能产生最优嫁接组合及由嫁接引起的生理响应差异的可能分子机制的研究还未见报道。本研究以青杨(Populus cathayana)和美洲黑杨(Populus deltoides)无性系为试验材料,通过自身嫁接和种间交互嫁接处理共得到四种不同嫁接组合:C/C(青杨自身嫁接)、C/D(青杨做接穗美洲黑杨做砧木)、D/D(美洲黑杨自身嫁接)、D/C(美洲黑杨做接穗青杨做砧木),四种嫁接组合的成活率均达到95%以上并生长良好。经过两种水分梯度(100%和30%田间持水量)处理,研究不同嫁接组合对干旱胁迫的生理响应差异,从而得到最优嫁接组合;同时借助Illumina转录组学测序技术和iTRAQ定量蛋白质组学技术对正常生长条件下的四种不同嫁接组合分别进行叶片转录组学和叶绿体蛋白质组学研究及两组学的关联分析,探究嫁接影响植物生长的分子机制。本研究获得的主要结果如下:(1) 青杨无性系和美洲黑杨无性系交互嫁接对干旱胁迫的生理响应不同嫁接组合对干旱胁迫的生理响应差异较大。试验结果表明:干旱胁迫显著降低了所有嫁接组合的生长速率、生物量积累、净光合作用速率(Pn)、黎明前叶片水势(Ψwp)、叶片的相对含水量(RWC)和总可溶性糖(TSS),显著增加了内在水分利用效率(WUEi)、叶片和根的脯氨酸含量。美洲黑杨做砧木的嫁接组合(C/D和D/D),其幼苗的生长、光合速率和细胞超微结构在干旱胁迫下受到的影响较小;相比之下,青杨做砧木的嫁接组合(C/C和D/C)对干旱胁迫更敏感,其水分利用效率和渗透调节能力较低,说明嫁接植物的抗干旱胁迫能力主要取决于砧木而不是接穗。此外,与其他嫁接组合相比,把青杨接穗嫁接到美洲黑杨的砧木(C/D)上被证明是在两种水分条件下最好的:100%田间持水量条件下,C/D嫁接幼苗具有较快的生长速率和较高的地上部分生物量积累;30%田间持水量条件下,C/D的光合速率和水分利用效率较高、非结构性碳水化合物含量较高、渗透性调节能力更强,生长较好。由上可知,种间嫁接能提高杨树的抗旱性,我们的试验结果为杨树的栽培提供了新方法和理论基础。 (2) 青杨无性系和美洲黑杨无性系交互嫁接引起的差异表达基因研究 转录组学结果显示与青杨自身嫁接相比,把青杨接穗嫁接到美洲黑杨砧木上,检测到277个差异表达基因(239个表达上调,38个表达下调),差异表达基因的功能与细胞壁、激素合成与信号转导、次级代谢产物的生物合成、转录调控、脂代谢、氨基酸代谢等相关;与美洲黑杨自身嫁接相比,把美洲黑杨接穗嫁接到青杨砧木上,检测到287个差异表达基因(181个表达上调,106个表达下调);差异表达基因的功能与光合作用、激素合成与信号转导、转录调控、次级代谢产物的生物合成、脂代谢、蛋白质的合成与修饰等相关。总之,青杨和美洲黑杨的种间嫁接主要通过调控与植物激素合成与信号转导、细胞壁的修饰与合成、次级代谢产物的生物合成、转录因子的调控、脂代谢和蛋白质的合成与修饰等方面的基因表达来促进杨树幼苗的生长发育,并且各个过程之间相互协调和促进,这为种间嫁接促进植物生长发育提供了分子基础。(3) 青杨无性系和美洲黑杨无性系交互嫁接引起的差异表达蛋白质研究青杨和美洲黑杨的种间嫁接幼苗的叶绿体蛋白质组学结合其叶绿体的超微结构、叶绿素浓度及光合生长等方面分析发现:种间嫁接幼苗叶绿体中的基粒类囊体数目和叶绿素浓度增加,为植物较高的光合生长提供生理基础。叶绿体蛋白质组学分析表明,与自身嫁接相比,种间嫁接能引起叶绿体蛋白质的差异表达。其中与青杨自身嫁接相比,把青杨嫁接到美洲黑杨砧木上,共有13个蛋白质出现差异表达(11个表达上调,2个表达下调);与美洲黑杨自身嫁接相比,把美洲黑杨嫁接到青杨砧木上,共检测到23个差异表达蛋白质(13个表达上调,10个表达下调)。两者检测到的共同差异表达蛋白质,尤其是与光合作用相关的如ATP合酶蛋白质(ATPA、ATPC)、光系统II反应中心蛋白质(PSBB)、放氧复合体蛋白质(PSBP、PSBO)和捕光叶绿素a-b结合蛋白质(LHCO)等的共同上调表达,可能是种间嫁接提高植物的光合速率、促进嫁接幼苗生长的分子基础。另外,转录组学和蛋白质组学的关联分析显示,两者共关联到49个基因/蛋白质,说明两者的关联性较低,这可能与细胞器层面的蛋白质较少及存在丰富的转录后调控有关。通过GO注释分析表明关联到的基因/蛋白质主要集中在细胞过程、代谢过程、细胞组分、细胞器、结合和催化活性等条目上。其中,与叶绿体和光合作用相关的如光系统II CP47蛋白、叶绿素a-b结合家族蛋白和叶绿体内膜导入蛋白Tic22等在种间嫁接幼苗的转录和蛋白质水平的上调表达,也为嫁接幼苗较高的光合能力提供了理论依据。 
Other AbstractPopulus are mainly distributed in the temperate and cool temperate zones of the northern hemisphere, and it has great practical and research value because of its fast growing, strong adaptability, easy breeding and genetic diversity. Drought is becoming more and more frequent due to global climate change, grafting has been proved to be an effective mean to modulate stress resistance of economically important plants, but there is limited information of optimum grafting combinations of different poplar species with different drought tolerance. In this study, reciprocal grafts between Populus cathayana (less drought resistant) and P. deltoides (more resistant) were used to investigate whether interspecific grafting results in improved resistance to drought when compared to intraspecific grafting. Altogether, four grafting combinations were created, including two intraspecific combinations, i.e., P. cathayana scion with P. cathayana rootstock (C/C) and P. deltoides scion with P. deltoides rootstock (D/D), and two interspecific combinations, i.e., P. cathayana scion with P. deltoides rootstock (C/D) and P. deltoides scion with P. cathayana rootstock (D/C). The grafting compatibility between P. cathayana and P. deltoides was very high with the survival rate of all grafted seedlings exceeding 95%. The growth rate, gas exchange capacity, water use efficiency, osmotic adjustment and cell ultrastructure were studied to evaluate differences among different grafting combinations in response to two watering regimes (100% and 30% of field capacity). At the same time, leaf transcriptome and chloroplast proteomics research were carried out for the four different grafting combinations which under control growth condition, to explore the molecular mechanism of grafting on plant growth. The main results and conclutions obtained in this study are as follows:(1) Physiological responses of interspecific grafted seedings of different poplar clones to drought stressThe physiological responses of different grafting combinations to drought stress were different. Drought significantly decreased growth rate, biomass accumulation, net photosynthesis rate (Pn), predawn leaf water potential (Ψwp), relative water content (RWC) and total soluble sugars (TSS), and increased the intrinsic water use efficiency (WUEi) and leaf and root proline contents of all grafting combinations. Drought reduced growth and photosynthesis and cellular ultrastructure less in the grafting combinations (C/D and D/D) with P. deltoides rootstock. In contrast, grafting combinations with a P. cathayana rootstock (C/C and D/C) appeared more sensitive to drought stress and had a lower water use efficiency and osmotic adjustment ability. These results indicated that the resistance of grafting plants to drought stress mainly depends on the rootstock rather than the scion. Moreover, grafting a P. cathayana scion onto a P. deltoides rootstock (C/D) was the best grafting combination under both watering regimes: under 100% field capacity conditions, C/D had a higher growth rate and aboveground biomass accumulation, and under 30% field capacity conditions, the growth, photosynthetic rate, water use efficiency, non structural carbohydrate content and osmotic adjustment ability of C/D were better than the others. To sum up, our results collectively indicate that grafting is a promising strategy to enhance drought resistance and achieve high yield for key forest plantation trees.(2) Transcriptional profiling analysis in different grafting combinations reveals the molecular mechansim of interspecific grafting to promote growthIllumina RNA-Seq of different grafting combination leaves was performed to determine the molecular mechanism of interspecific grafting to promote growth of seedlings. The results showed that compared with the self-grafting of Populus cathayana, 277 different expressed genes (239 up-regulated and 38 down-regulated) was detected in grafting Populus cathayana scion onto Populus deltoides rootstock. The function of different expressed genes is mainly related to cell wall, hormone synthesis and signal transduction, biosynthesis of secondary metabolites, transcriptional regulation, lipid metabolism and amino acid metabolism. On the other hand, compared with the self-grafting of Populus deltoides, 287 different expressed genes (181 were up-regulated and 106 were down-regulated) was detected in grafting Populus deltoides scion onto the Populus cathayana rootstock. These different expressed genes were involved in photosynthesis, hormone synthesis and signal transduction, transcriptional regulation, biosynthesis of secondary metabolites, lipid metabolism and protein synthesis and modification. All in all, interspecific grafting was mainly through regulating gene expression that related to regulation of plant hormone synthesis and signal transduction, cell wall modification and synthesis, secondary metabolite biosynthesis, transcription factor regulation, lipid metabolism and protein synthesis and modification to promote the growth and development of grafted poplar seedlings. And all processes are coordinated and promoted, which provides a molecular basis for interspecific grafting to promote plant growth and development. (3) Chloroplast proteomic analysis reveals the molecular mechansim of interspecific grafting to promote growthITRAQ-based quantification proteomics was used to determine differential expression of chloroplast proteins caused by interspecific grafting. Combined with the chloroplast ultrastructure, chlorophyll concentration and photosynthetic growth, we found that the number of thylakoids and chlorophyll concentration were higher in interspecific grafted seedlings than self-grafted ones, which provided physiological basis for higher photosynthetic growth of interspecific grafted seedlings. Chloroplast proteome analysis results showed that interspecific grafting can lead to the difference expression of chloroplast protein. Compared with the self-grafting of Populus cathayana, 13 proteins were detected to be differentially expressed (11 up-regulated and 2 down-regulated) in grafting Populus cathayana scion onto Populus deltoides rootstock; compared with the self-grafting of Populus deltoides, a total of 23 differentially expressed proteins (13 up-regulated and 10 down-regulated) were found in grafting Populus deltoides scion onto the Populus cathayana rootstock. The common up-regulated protein in both interspecific grafted seedlings, such as ATP synthase protein (ATPA, ATPC), photosystem II reaction center protein (PSBB), oxygen-evolving complex protein (PSBP, PSBO), chlorophyll a-b binding protein (LHCO), may be the molecular basis for interspecific grafting to increase the photosynthetic rate, thereby promoting the growth of grafted seedlings.In addition, the correlation analysis of transcriptomics and proteomics showed that 49 genes / proteins were associated, indicating that the association was low, which may be related to less protein at the organelle level and a rich post transcriptional regulation. The GO annotation analysis showed that the associated genes / proteins were mainly concentrated on the cellular process, metabolic process, cell part, organelle, binding and catalytic activity. Among them, proteins related to chloroplast and photosynthesis, such as photosystem II CP47 protein, chlorophyll a-b binding family protein and chloroplast inner membrane import protein Tic22 were up-regulated and expressed in the transcriptional and protein levels of interspecific grafted seedlings, and these also provide a theoretical basis for higher photosynthetic capacity of grafted seedlings. 
Pages166
Language中文
Document Type学位论文
Identifierhttp://ir.imde.ac.cn/handle/131551/24801
Collection山地表生过程与生态调控重点实验室
Affiliation中国科学院成都山地灾害与环境研究所
First Author Affilication中国科学院水利部成都山地灾害与环境研究所
Recommended Citation
GB/T 7714
韩清泉. 杨树无性系交互嫁接对干旱胁迫的生理响应及分子机制[D]. 北京. 中国科学院大学,2018.
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