【作者】 董杰；

【导师】 詹亚光；

【作者基本信息】 东北林业大学 ， 森林生物工程， 2012， 博士

【摘要】 白蜡属(Fraxinus)植物全世界约有70余种,广泛分布于北半球。我国白蜡属植物约20余种,其中有水曲柳(F. mandshurica)、绒毛白蜡(F. velutina)、小叶白蜡(F.sogdiana)及大叶白蜡(即美国白蜡,F. americana)等多种重要的珍贵用材树种,具有很高的经济价值。随着林业的发展和社会需求的提高,选育生长性能好,适应性强的优质树种尤为重要。在生物界杂种优势是存在的一个普遍的重要遗传现象。种间杂交能将优良性状集中在一起,显著改良品质、提高抗逆性,因而能创造出显著的经济效益和社会效益。然而,由于树木生长慢,生长周期长,树木的种间杂种优势利用不如玉米和水稻那样广泛,白蜡属种间杂种抗旱性优势的研究机理目前研究还很少,国内外对白蜡属种间杂种抗旱性优势在DNA甲基化水平和基因表达谱的研究尚未见报道。因此,迫切需要研究白蜡属种间杂种抗旱性优势的遗传机理以促进优良杂交树种的选育。本研究以水曲柳作为母本分别与大、小叶白蜡进行种间杂交得到杂种F1,通过干旱胁迫处理,对种间杂种F1抗旱性优势进行了研究,采用MASP方法,探索干旱胁迫对种间杂种及其亲本材料所引起的DNA甲基化的变化规律,发生DNA甲基化变化的基因,并对这些基因的抗旱功能进行进一步研究。利用数字基因表达谱深度测序,了解干旱胁迫下,种间杂种F1和亲本材料的基因表达的情况；通过基因表达差异分析,发掘影响植物抵抗干旱胁迫过程中的基因。揭示种间杂种抗旱优势产生的分子机理,探索种间杂种F1代抗旱优势形成的分子基础。主要研究结论如下：1.水曲柳×大、小叶白蜡种间杂种F1抗旱性生理研究(1)胁迫处理抑制了植株的生长量、光合作用、及光合色素的合成。同时,在干旱胁迫处理下各植株的SOD活性、POD活性均有不同程度的提高。复水处理3天后,大多数生理指标能得到恢复,但仍都大于处理前水平。(2)种间杂种F1对干旱胁迫的响应：以各干旱胁迫下生理指标为主,研究了水曲柳×大叶白蜡、小叶白蜡的杂种抗旱优势。干旱胁迫处理下,对于亲本材料来说,种间杂种F1的生长量、光合作用、相对电导率、光合色素含量及SOD、POD活性等生理指标表现出不同程度的抗旱性优势,但因母本家系的不同而有所差异。(3)对杂种F1在耐受力抗旱性方面进行了综合评价与排序,并最终得到综合抗旱能力排序为：110>135>137>36>149>195>14>76>62>142>171>65>136>16>169>72>32>58>131>24>114>29>156>47>15>115>87,其中种间杂种F1110、36、149、195、14排在前五位,其抗旱性综合评价指标超过其母本材料20%-35.48%,说明种间杂种Fl在抗旱性方面显示出明显的优势。其中初步选出优良种间杂种F1为：水曲柳与北京大叶白蜡种间杂种F1110,其抗旱性综合评价指标超过其母本材料达31.57%。2.水曲柳×大叶白蜡种间杂种F1干旱胁迫中DNA甲基化的变异机制(1)未经干旱处理的对照组：母本材料(母本自由授粉子代)113、父本材料(父本自由授粉子代)4-3甲基化程度分别为20.12%、24.61%；种间杂种F1110、115甲基化程度为15.48%、16.81%。种间杂种F1甲基化水平低于亲本材料,仅为亲本材料的72%。表明种间杂种F1比相应的亲本在形成杂合体时某些位点发生了去甲基化作用。(2)干旱胁迫处理：导致DNA甲基化平均水平明显增加,其中种间杂种F1115、母本材料113和父本材料4-3增加幅度尤为明显(分别由16.81%增至29.10%,20.12%至31.91%,24.61%至32.70%)；而优良种间杂种F1110DNA甲基化水平胁迫前后无明显增幅,表明DNA甲基化水平变化具有一定的种属特异性。种间杂种F1115、母本材料113和父本材料4-3幼苗基因组的DNA甲基化模式主要以内侧胞嘧啶的甲基化为主,种间杂种F1110幼苗基因组的DNA甲基化模式内、外侧胞嘧啶的甲基化相差不多。种间杂种F1DNA甲基化模式经历了一定的改变与调整,推测种间杂种抗旱优势的产生与种间杂种F1基因组DNA甲基化模式的改变和重新调整有关。与对照相比,干旱处理3d都是以甲基化出现为主,且母本材料113的变化较大。干旱9d处理以去甲基化为主,且种间杂种Fl115和父本材料4-3的变化较大,这都与干旱处理的甲基化水平的变化的结果相似。(3)对干旱胁迫特异诱导DNA甲基化片段序列分析结果表明：其中一些条带与功能基因具有一定的同源性,包括氨基酸代谢相关蛋白、跨膜运输载体、细胞色素氧化酶、蛋白激酶、DNA结合蛋白、ATP结合蛋白、叶绿体PetD、多聚嘧啶结合蛋白、果胶酶的前体、解旋酶、葡萄糖-6-磷酸脱氢酶、热休克蛋白-70、脱氧核糖核酸外切酶、线粒体ort214、氧化还原酶、整合酶和三磷酸腺苷酶等。可以推知其中一些基因表达可能与干旱胁迫相关。3.水曲柳×大叶白蜡干旱胁迫中数字基因表达谱分析(1)通过对3个样品CleanTag(测序标签)拷贝数分布统计分析,大约占基因种类4%的基因,表达量占总表达量的65%,而占Tag种类60-70%的Tag表达量仅仅占总表达量的5-9%。这意味着在杂种F1和其亲本材料中,某些基因的高表达量,意味着其在抵抗干旱胁迫中可能发挥着重要的作用。(2)对差异表达基因进行pathway显著富集分析发现：种间杂种F1110与母本材料113、父本材料4-3差异均集中在对刺激的响应(包括外界刺激响应、化学刺激响应、水应答、压力响应等),代谢和能量前体合成,光合作用,物质运输系统。差异代谢过程,主要包括淀粉与蔗糖代谢、植物生理节律、光合作用、植物激素信号转导、苯基丙氨酸代谢等,暗示着这些过程可能在植物生长发育及抵抗干旱胁迫过程中起着重要作用。在这些过程中发现了一些关键的上调、下调表达的基因。综上所述,种间杂种F1在抗旱性能方面,从生长量、光合作用、相对电导率、光合色素含量及SOD、POD(?)舌性等方面都体现出了优于亲本的特点,从DNA甲基化水平分析,在干旱胁迫时种间杂种F1较其亲本材料DNA甲基化的程度低,这可能使与抗旱相关的基因表达量更高,进一步从转录水平分析时,得到了相似的结论,种间杂种F1与亲本材料对干旱胁迫表现出不同代谢途径差异,并且推测这些途径可能与抗旱性能有关。更多还原

【Abstract】 The genus Fraxinus contains about70species throughout the world They are widely distributed in the Northern Hemisphere, among which20species are distributed in China, including F.mandshurica, F.velutina, F.sogdiana and F.americana etc.Hybrid plant could greatly inerease quality, modestly improve and raise tolerance to stresses. Accordingly, hybrid forestry trees could create remarkable economic and social benefits. However, heterosis of trees has not been used as widely as that of rice and corn. Deficiency of slowly growing of trees and the expense of producing F1seeds are two main reasons leading to this present situation. Therefore, it is urgent to study the genetic mechanism of trees heterosis to promote selection of elite hybrideotton.Based on the hybridization between F.mandshurica and F.sogdiana/F.americana, this paper studied the heterosis of F.mandshurica×F.sogdiana and F.mandshurica×F.americana under drought stress. The main conclusions are as follows:1. F. mandshurica X F. americana, F. sogdiana interspecific hybrid F1drought resistance physiological research(1)Research on drought resistance indicators of F.mandshurica×F.sogdiana and F.mandshurica×F.americana:the hybrid combination were gradual wilting under drought stress and some were withered like burnt. All of them can resume normal after rehabilitation treatment. Drought stress inhibited plant growth, photosynthesis and the synthesis of pigment for photosynthesis. Furthermore, the SOD activity, POD activities increased in different degree under drought stress.3days after rewater, most of the physiological characters can be restored, but still greater than the level before treatment.(2)Research on heterosis of F.mandshurica×F.sogdiana and F.mandshurica×F.americana: The physiological character under drought stress were studied and used to analysis the heterosis of hybrids. For F.mandshurica×F.americana and Fmandshurica×Fsogdiana, growth, photosynthesis, POD activity, SOD activity, the relative conductivity, and photosynthetic pigment content showed heterosis in different degree and varied with their female parents.(3)Comprehensive evaluation of drought resistance on hybrids:39hybrids were evaluated on drought resistance. The order of integrated drought resistance was:110>135>137>36>149>195>14>76>62>142>171>65>136>16>169>72>32>58>131>24>114>29>156>47>15>115>87, of which the best drought resistance heterosis is F.mandshurica×F.americana F1110.2. DNA methylation variation mechanism of F. mandshurica X F. americana interspecific hybrid F1under drought stress (1) CK team:The MSAP ratio, which was the ratio of MSAP sites to the totally amplified sites, in two hybrids (110,115) were15.48%,16.81%, respectively, all of which were lower than those of either their parents(20.12%,24.61%). The results suggested that demethylation was occurred in some amplified5’CCGG sites.(2)Results showed that the average level of methylation was increased under drought stress treatment, especially in115,113,4-3. But the alterations of the DNA methylation state and level induced by drought stress were not increased obviously, and were species specificity.Genomic DNA methylation patterns in parental (113and4-3) and hybrids (115) seedlings were mainly inside cytosine methylation. Genomic DNA methylation patterns in hybrid (110) seedlings were mainly outside of cytosine methylation. The DNA methylation patterns of hybrid F1have been changed and adjusted, suggesting that heterosis of hybrids F1appeared because of the genomic methylation patterns’ change and re-adjust.Results showed that the average of DNA methylation in seedlings were increased under drought stress treatment after three days, while the main MSAP pattern was decreasing bands, and female parent(113) changed obviously. The average of DNA methylation in seedlings were increased under drought stress treatment after nine days, while the main MSAP pattern was also decreasing bands, and male parent(4-3) and hybrids (115)changed obviously. The result was similar with the genomic methylation level change.(3)Some variable MSAP profiles were isolated and sequenced, which were analyzed with Blast. Blast analysis results showed that sequences have homology with some functional genes.3. F. mandshurica×F. americana in drought stress digital gene expression profiling analysis(1)About4%of Tag types accounted for nearly65%of the total Tags, but60-70%of Tags type was less than5-9%of the total Tag number. This meant some specific genes play a significant role in the process of the resistance drought stress.(2)Through analysising the differential expressed genes in hybrids110and parent were significant differences in Photosynthesis、Photosynthesis-antenna proteins、Metabolic pathways、Biosynthesis of secondary metabolites、Biosynthesis of unsaturated fatty acids、 Carbon fixation in photosynthetic organisms、Pyruvate metabolism. Nitrogen metabolism、 Starch and sucrose metabolism、Glycolysis/Gluconeogenesis、Circadian rhythm-plant、 Phenylpropanoid biosynthesis、Plant hormone signal transduction etc.更多还原