【作者】 阮桢媛；

【导师】 陈晓鸣；

【作者基本信息】 中国林业科学研究院 ， 森林保护， 2013， 博士

【摘要】 角倍由于其中单宁含量丰富而被广泛应用于医药、食品、化工等各个行业。角倍是蚜虫与植物相互作用形成的虫瘿，了解蚜虫刺激植物后，植物对蚜虫的生理及分子应答具有重要的科学意义。本研究通过对保护酶及膜损伤相关化学成分的检测，判断角倍蚜对盐肤木造成氧化性损伤及细胞膜伤害的可能性，从侧面反映二者之间可能存在的生态关系。转化酶活性测定与转化酶基因的表达分析分别从蛋白与核酸水平讨论盐肤木叶片与倍子之间的营养分配情况以及转化酶在角倍形成中发挥的作用。通过cDNA-AFLP方法在转录组水平上筛选出参与应答反应的基因，用于对角倍形成相关基因的研究。以上三方面盐肤木生理及分子应答反应研究工作获得的主要结果如下：1.虫瘿形成过程中寄主植物保护酶等生理指标的反应：保护酶是植物遭受氧化性损伤时的防御性蛋白。对各时期虫瘿组与无虫瘿组之间超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）等保护酶活性的比较结果显示各指标在两组之间均无显著差别（P＜0.05）。可溶性总蛋白和脯氨酸在脂质膜遭到损坏时具有调节作用，而丙二醛反映出膜的氧化程度，三种活性物质比较分析结果显示两组叶片之间无显著差别（P＜0.05）。以上结果说明蚜虫取食以及数量的增加可能并为造成寄主植物严重的氧化性损伤或者对植物细胞膜的伤害，所以未能产生常见的防御性反应，角倍蚜与盐肤木之间的关系区别于其它寄主植物上的虫害关系，二者或许以“共生”方式存在。各生理指标在两组叶片中变化趋势的比较中发现，可溶性总蛋白含量在后期变化趋势上存在显著差异（P＜0.05），这说明角倍蚜取食造成了寄主植物生理代谢的改变，这一方面可能反映出叶片与角倍之间蛋白分配的改变，另一方面说明了它对叶片生长发育进程的影响。2.虫瘿形成过程中转化酶活性反应：转化酶是植物糖代谢过程中的关键酶，在植物生长发育调节中具有重要作用。对有倍叶、无倍叶以及倍子中液泡型酸性转化酶、细胞质型中性转化酶、细胞壁型酸性转化酶与细胞壁型中性转化酶四种类型转化酶活性进行比较，其中液泡型转化酶与细胞壁型转化酶活性在叶片整个生长发育过程中迅速降低（P＜0.05），说明叶片的生长发育与三种转化酶活性呈负相关；细胞壁型转化酶活性在角倍形成初期未能检测到，液泡型转化酶则随角倍生长显著下降，反映出角倍生长发育与液泡型转化酶之间的负相关关系。角倍细胞质型中性转化酶活性显著升高（P＜0.05），与其胞内酸性酶活性持续降低相对应，胞质中性转化酶或许作为维持酶存在。倍子中酸性转化酶活性始终高于叶片，且有倍叶中活性高于无倍叶，无倍叶可能以源叶形式存在，倍子从中获取营养增加库强。植物通过对四种转化酶活性的调节实现对角倍生长发育的调节，而酸性转化酶则在叶片与倍子的营养分配中发挥重要作用。3.盐肤木转化酶基因克隆及角倍形成过程中基因表达分析：转化酶基因的克隆与表达分析是实现角倍生长发育调控的基础。本研究克隆获得细胞质型转化酶基因INV1、细胞壁型转化酶基因INV2与液泡型转化酶基因INV3，三种转化酶基因在不同组织中的荧光定量结果显示INV2的表达具有组织与发育特异性，而且其表达可能对倍子形成以及快速生长发挥正调控作用。液泡型转化酶基因INV3的表达在有倍叶与倍子中呈现出相同的下降趋势，而无倍叶仅在倍子快速生长时显著下降，该基因可能对角倍形成的早期调控具有更为重要的意义。细胞质型转化酶基因INV1的表达在角倍形成晚期倍子中表达量才显著高于无倍叶，说明该基因可能也与角倍快速生长有关。由以上结果推测，INV1基因的表达可能促进角倍的快速生长，INV2基因的增量表达可能利于角倍的形成以及快速生长，而INV3基因可能参与角倍生长的早期调控。4.盐肤木分子应答相关基因筛选及功能分析：角倍蚜取食造成植物不同组织差异表达基因的筛选是对角倍形成相关基因的初步判断，对其功能的分析是虫瘿形成分子机制的基础。通过cDNA-AFLP方法在早期与角倍形成过程中分别筛选出64和71个参与应答的基因，涉及光合作用、氧化还原反应、基因表达调控、次生代谢、信号转导、跨膜转运、应激反应、细胞命运、细胞组分等多个方面，角倍蚜通过对植物代谢过程和细胞过程等的改变导致虫瘿组织的形成。各候选基因参与的生物调节、细胞组分生物形成、细胞组分的组织、细胞过程、发育过程、定位的建立、定位、代谢过程、多细胞有机体过程、色素沉着、复制和应激反应等生物过程反映出植物分子应答网络系统的框架。更多还原

【Abstract】 For the high content of tannin, horned gall is widely used in different fields such asmedicine, food and chemical industry. Horned gall is a special gall caused by the interactionbetween Rhus Chinensis and Schlechtendalia chinensis. Exploring physiologial and molecularresponses of the plant attacked by the aphids is significant for researches.With the detection of some protective enzymes and chemical components related tomembrane injury, we try to confirm the possibility of oxidative damage or membrane injury,and reflect the ecological relationship between them. Invertase activities and gene expressionwill show nutrient partitioning in the plant and their roles in gall formation at protein andnucletide level respectively. Responses of the plants are revealed by cDNA-AFLP attranscriptome level. Genes selected from this research will be useful for the detection of gallformation. The results of physiological and molecular responses mentioned above were asfollows:1. Protective enzyme and other physiological indicators in host plants during gallformation: Protective enzyme is a series of protevtive proteins when plants face with oxidativedamage. The comparision of SOD, POD and CAT between galled and ungalled leaves indifferent period showed no significant difference（P＜0.05）. Total soluable protein and prolineplays adjust role of membrane when it is damaged, while malonaldehyde (MDA) indicats theoxidative damage of lipid membrane. There was no significant difference found between galledand ungalled leaves for these three active substances. The results above showed us that afterfeeding of aphids and with the increase of aphid number, there seemed to be not any seriousoxidative damage or membrane injury. So the plants did not show usual protective activities. S.chinensis might be different frome other pests in plants, and the mutualistic relationship mightexist between this aphid and plant. In all of the results, there is only significant differercehappeded in the later stage for the change trend of soluable protein（P＜0.05）. This illustrated the changes of metabolism in plants induced by aphids, which might be caused by thesource-sink relationship or changes in the process of development.2. Activity of invertase during gall formation: Invertase is important in sucrosemetabolism, so it plays an impotant role in the adjustment of plant growth. In the detection offour kinds of invertase including vacuolar invertase, cytoplasmic invertase, cell wall acidinvertase and cell wall neutral enzyme, activities of vacuolar invertase and cell wall invertasein leaves decreased dramatically （P＜0.05）which proved that leaf growth negativelycorrelated with three kinds of enzymes. Cell wall invertase in galls was not detected whilevacuolar invertase decreased dramatically all the time. It indicated the negative correlationbetween gall development and vacuolar invertase. Different from the decrease of acid invertasein the cell, cytoplasmic invertase in galls increased with growth. Cytoplasmic invertase mightact as a sustain enzyme. Higher acid activies in galls and galled leaves might due to the sinkrole of galls. Ungalled leaves acted as souce tissue, and galls received nutrition frome them.Plant mediated the development by the control of different kinds of invertase, and acidinvertase should be quite important in partition.3. cDNA cloning and expression of invertase gene: cDNA cloning and analysis of geneexpression will be helpful for the control of gall growth and development. cDNA cloneencoding cytoplasmic invertase INV1, cell wall invertase INV2and vacuolar invertase INV3were isolated for the analysis of gene expression. Expression of INV2had its specificity intissue and development, and it played a positive role in gall formation. Expression of INV3ingalls and galled leaves decreased all the time, while it only decreased in ungalled leaves at thefast growth time of galls. This gene might be related to the fast growth and expansion of galls.The latter increase of INV1expression in galls might indicate its relationship with fast growthof galls. We can conclude that higher expression of INV2might be useful for the gall formation,while INV1and INV3should cause the fast growth of galls.4. Screening of genes in responses of the plant and analysis of gene function: Differentexpression of genes in different tissues can help us find the gall-formation-ralated genes, andanalysis of gene function is the base of the molecular mechanism of gall formation.64genes in early responses and71genes in responses during gall formation were selected by the methodof cDNA-AFLP respectively. These genes refered to photosynthesis, redox reaction, regulationof gene expression, secondary metabolism, signal transduction, transmembrane transport, stressreaction, cell fate, cell component and so on. Great changes in metabolic process and cellularprocess caused the formation of galls. Candidate genes taking part in biological regulation,cellular component biogenesis, cellular component organization, cellular process, developmentprocess, establishment of localization, localization, metabolic process, multicellular organismalprocess, pigmentation, reproduction and response to stimulus paved the way for researches onmolecular mechanism.更多还原