【作者】 周贝贝；

【导师】 裴东；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2014， 博士

【摘要】 嫁接一方面保持了品种性状相对一致性，另一方面不同砧木也会明显改变树体的表型。砧木如何影响接穗表型性状，其分子机制有待深入研究。本研究将‘上宋-14’核桃品种分别嫁接到‘中宁奇’和‘宁优’砧木上，发现核桃嫁接苗树体在表型性状和生理特性（如生长量、光合特性等）发生明显改变，进而对基因组DNA甲基化研究发现，同为‘上宋-14’，嫁接到‘中宁奇’和‘宁优’砧木上，其基因组DNA甲基化水平和模式发生了变化，对甲基化模式差异位点进行测序发现，有些位点序列与光合作用和代谢密切相关。该研究证实了核桃砧木通过影响基因组DNA甲基化水平和模式，进而对树体生长发育产生影响，结果丰富了人们对砧木影响树体生长势的认识。本研究所取得的主要结果如下：（1）建立了优化的核桃基因组DNA甲基化研究实验体系。以核桃子叶为试材，比较了3种DNA提取方法，发现改良高盐CTAB法获得的基因组DNA质量最好，从90对引物组合中筛选出了43对多态性高、电泳谱带清晰、谱带分离状态好的MSAP引物组合，建立优化了核桃基因组DNA甲基化研究的MSAP实验体系。应用建立的优化MSAP实验体系对核桃叶片基因组DNA的甲基化修饰水平进行了分析，共扩增出1060条清晰可辨且可重复条带，对14个核桃基因组DNA甲基化修饰位点进行回收、克隆与测序，BLASTn分析表明核桃叶片包括转录因子在内的多种类型的DNA序列中均存在甲基化修饰现象；（2）利用MSAP技术对核桃同树龄同品种不同组织（成熟叶片组织、当年生嫩茎韧皮部组织、当年生根韧皮部组织、花粉组织、子叶组织和青皮组织）全基因组DNA甲基化进行分析，共扩增出972条清晰可辨的谱带，核桃同一品种不同组织之间基因组DNA甲基化水平差异不显著，其甲基化模式变异频率在0.199~2.493%之间（表3-5），其中，子叶组织相对于花粉组织的CG超甲基化（CG-hyper）变异频率最高（2.493%，表3-5），叶片组织相对于茎韧皮部组织的CHG超甲基化（CG-hyper）变异频率最低（0.199%，表3-5），表明相同树龄的同一核桃品种不同组织间基因组DNA甲基化水平相对稳定；（3）将‘上宋-14’核桃接穗分别嫁接到‘中宁奇’（‘上宋-14/中宁奇’）和‘宁优’（‘上宋-14/宁优’）砧木上，测定了3年生树体的树高、树径、冠幅、生长枝长、生长枝粗和发育枝长，不同砧木对树体生长具有显著影响。‘上宋-14/中宁奇’和‘上宋-14/宁优’树高平均值分别为5.33m和4.18m，‘上宋-14/中宁奇’明显优于‘上宋-14/宁优’，其它反应生长势的表型性状指标如树径、冠幅、生长枝长、生长枝粗和发育枝长，‘上宋-14/中宁奇’也明显高于‘上宋-14/宁优’（P<0.01，表4-2），表明‘中宁奇’和‘宁优’两种砧木对‘上宋-14’接穗的生长和发育具有显著影响；（4）采用Li-6400光合仪测量了分别嫁接在‘中宁奇’和‘宁优’砧木上的‘上宋-14’的光合特性。在相同立地条件和环境下，光合特性各项指标在两组砧穗组合上存在显著差异。‘上宋-14/中宁奇’最大净光合速率（21.52μmol m-2s-1）显著高于‘上宋-14/宁优’（19.96μmol m-2s-1），‘上宋-14/中宁奇’的光饱和点（LSP (B)）、光补偿点（LCP (C)）和暗呼吸速率（Rd(D)）指标（平均1191.6μmol m-2s-1、8.03μmol m-2s-1和0.52μmol m-2s-1）显著低于‘上宋-14/宁优’核桃树数值（平均1257.87μmol m-2s-1、9.8μmol m-2s-1和0.64μmol m-2s-1，P<0.05，图4-2）；（5）采用MSAP技术分析了嫁接到‘中宁奇’和‘宁优’砧木上的‘上宋-14’品种的新梢韧皮部组织全基因组DNA甲基化状态，共扩增出1607条清晰可辨的电泳谱带。‘上宋-14/中宁奇’组合的整体甲基化水平（25.39％）、CG全甲基化位点甲基化水平（17.67％）和CHG半甲基化位点甲基化水平（7.72％）虽然均略低于‘上宋-14/宁优’组合（分别为27.57％、18.73%和8.84%，表4-3），但差异不显著（Utotal=0.72，<1.96），甲基化模式变异频率在0.85-1.95%之间。因此，鉴定甲基化改变的特异位点，了解其生物学功能，成为更重要和实质性的研究。（6）对存在于两组砧穗组合的甲基化变异条带，进行克隆和测序，得到73条反应甲基化模式变异的变异条带序列， Blast分析表明，49.32%的条带序列与已知功能的蛋白编码基因具有同源性，12.33%的基因片段与光合作用相关基因同源，6.85%的片段序列与质体基因组序列同源，其余为假定蛋白编码基因（16.44%）和转座子/逆转座子（6.85%）同源序列，与NCBI数据库序列无明显相似性序列占20.55%。（7）本研究揭示出嫁接可以改变树体生理和代谢、影响树体生长，嫁接也会引起基因组DNA特异位点甲基化改变，而这些位点的基因序列又与树体生长和生理代谢密切相关。因此，砧木改变树体基因组甲基化模式可能是改变其生理和代谢进而影响生长发育的重要原因之一。更多还原

【Abstract】 Graftingmaintains the relative consistency of characters in varieties, while the phenotypesof the tree are significantly changed through different rootstocks. However, how the rootstockaffects phenotypic traits of scions, and its molecular mechanism remains unclear. In thisresearch, the walnut variety ‘SHS’ were respectively grafted on the rootstocks ‘ZNQ’ and ‘NY’,and the results showed that the phenotypic traits and physiological characteristics of walnutgrafts (such as growth, photosynthetic characteristics, etc.) changed obviously. Also, thegenomic DNA methylation level and patterns of walnut grafts (SHS/ZNQ and SHS/NY) werechanged, and some sequences in variated sites showing methylation pattern were closelyrelated to photosynthesis and metabolism. The research confirmed that walnut scion growthwas affected by the rootstocks influencing the genomic DNA methylation levels and patterns,which would enrich the understanding of tree growth potential affected by rootstocks.The main results are as follows:1. The optimized experiment system of walnut genomic DNA methylation was established.The walnut cotyledons were adopted as materials to compare three methods for extraction ofgonomic DNA, and the DNA isolated by modified high-salt CTAB protocol showed bestquality. After that, forty-three pairs of primer combinations, with clear and reproducible bands,were screened out in ninty pairs of primer combinations.The modifed MSAP experimentalsystem was established for walnut genomic DNA methylation analyses. Using modified MSAPexperimental system, the methylation level of genomic DNA in walnut leaves were analyzedand1060clear and reproducible bands were amplified. What is more, fourteen methylated siteswere recycled, cloned and sequenced, and BLASTn analysis showed that many types of DNAsequences, including transcription factors, presented methylation phenomenon in walnutleaves. 2. Genomic DNA methylation was analyzed using MSAP technology in various tissues ofthe same walnut variety (mature leaf, stem, root, pollen, cotyledon and husk), and972clearand reproducible bands were amplified. The methyltion levels of genomic DNA variedbetween different tissues of the same variety of walnut with nonsignificance, and themethylation patterns of variation ranged from0.199%~2.493%, with the highest CG-hyperrate (2.493%) in cotyledon tissue compaired with pollen tissue, and the lowest CG-hyper rate(0.199%) in leaf tissue compaired with steem tissue. Which demonstrated that the genomicDNA methylation levels kept relatively stable between different tissues in the same walnutvariety.3. Variances of growth vigour in walnut were assessed by measuring tree height, diameterat breast height, crown diameter, shoot length, shoot diameter and developing branch length of3-year-old SHS scions grafted on ZNQ and NY. The average height of SHS/ZNQ was5.33m,which was significantly higher than SHS/NY (4.18m). Other phenotypic traits of growthpotential (diameter at breast height, crown diameter, shoot length, shoot diameter anddeveloping branch length) of SHS/ZNQ were as well significantly higher than that of SHS/NY(P<0.01). The result showed that rootstocks had a significant impact on growth anddevelopment of the scions grafted on them.4. The photosynthetic characteristics were measured by Li-6400portable gas exchangesystem. At the same site conditions and the environment, the variations of photosyntheticcharacteristics between two scion combinations were different significantly. The light-saturatedrate of photosynthesis of SHS/ZNQ (21.52μmol m-2s-1) was significantly higher than that ofSHS/NY(19.96μmol m-2s-1). However, the photosynthetic parameters of LSP (B), LCP (C),and Rd(D) of SHS/ZNQ were significantly lower than that of SHS/NY(P<0.05).5. Genomic DNA methylation was analyzed using MSAP technology in phloem tissues ofSHS/ZNQ and SHS/NY, and1607clear and reproducible bands were amplified. The relativetotal methylation levels, CG and CHG on SHS/ZNQ (25.39%,17.67%, and7.72%respectively) were slightly lower than that on SHS/NY (27.57%,18.73%, and8.84% respectively. Nevertheless, the difference detected between SHS/ZNQ and SHS/NY was notsignificant (Utotal=0.72,<1.96), and the variation of methylation patterns ranged from0.85%to1.95%. Hence, it would be an important and substantive research for identifing specific sites ofmethylation changes and understanding its biological functions.6. In order to explore possible functional relevance of the loci with DNA methylationalterations, we isolated and sequenced a subset of variant MSAP bands from SHS walnutscions grafted on ZNQ and NY rootstocks. Based on BlastX analysis, we found that of the73variant MSAP bands that gave quality sequencing,49.32%showed significant homology toprotein-coding genes with known functions,12.33%fragments were homologous tophotosynthesis related genes and6.85%fragments were homologous to plastid sequences.7. This research demonstrated that the grafting can change the physiology and metabolismof the tree and affect tree growth. Also, grafting can cause genomic DNA methylation changesin specific sites, which were closely related to the growth and physiological metabolism.Therefore, the change of genomic DNA methylation patterns induced by rootstocks may be animportant reason to change its physiological and metabolic characteristics, which affected thegrowth and development of the grafts.更多还原