【作者】 张建民；

【导师】 李学宝；

【作者基本信息】 华中师范大学 ， 农药学， 2014， 博士

【摘要】 棉蚜(Aphis gossypii Glover)是世界性经济作物棉花的重要害虫之一,刺吸式口器,以成虫、若虫群集于棉叶背面或嫩茎上刺吸汁液造成直接危害。同时,棉蚜还可通过传播植物病毒病和分泌蜜露引起煤污病造成间接危害。受到危害的棉花,不仅产量受到影响,而且棉纤维含糖量增加,严重影响棉纤维品质。棉蚜个体小,繁殖力强,生活周期短,世代重叠严重等特点,致使农药的使用十分频繁,再加上农药的不合理使用,导致棉蚜对多种农药产生较高水平的抗性,使棉蚜的防治更加困难。此外,伴随着Bt棉的推广使用,棉蚜的危害有进一步上升的趋势。而利用寄主植物对蚜虫的抗性控制蚜虫是一种有效而且环境友好型的控制蚜虫方式,它被认为是控制蚜虫危害的综合作物管理系统中的重要方法之一,受到植物保护研究人员的普遍重视。随着植物抗虫性研究的发展,许多抗蚜作物的培育已为农业生产带来了巨大生态和经济效益,许多研究结果还显示抗蚜品种可减少以蚜虫为媒介的植物病毒的传播。但是,新的问题亦开始出现,植物抗蚜基因的毒力在17种蚜虫中已经产生,其占所有展示毒力的节肢动物生物型的一半多。为了延迟和防止蚜虫生物型的出现,不断寻求新的抗蚜基因,揭示植物和蚜虫相互作用的分子机制,实现在生产系统中作物对蚜虫抗性的长期持久使用具有十分重要的意义。本项目从棉花对棉蚜的抗性入手,以抗蚜品种鸡脚红叶棉作为主要试验材料,采用抑制差减杂交法构建了棉蚜诱导的棉花叶片的抑制差减杂交cDNA文库。通过生物信息学分析,从文库中获得了253个与棉蚜诱导相关的表达序列标签(ESTs)。以其为信息来源,克隆了GhTCTP1和GhPSAK1两个与棉蚜诱导有关的基因,并对它们的抗蚜功能及其机制进行了研究。获得的主要研究结果如下：1.棉蚜诱导棉叶的抑制差减杂交cDNA文库构建与分析利用抑制差减杂交(Suppression subtractive hybridization, S SH)的方法构建了棉蚜诱导的棉花叶片的抑制差减杂交cDNA文库。从中随机选取253个克隆进行了分析,其片段大小约在250bp至2000bp之间。这些cDNA克隆测序后,对所获得的EST序列进行了生物信息学分析,从胁迫的角度归类,其中有38cDNA克隆只与生物胁迫有关的序列相匹配,95cDNA克隆只与非生物胁迫有关的序列匹配,93cDNA克隆同时和生物胁迫和非生物胁迫有关的序列匹配。以上结果暗示,棉蚜对棉花的诱导防御是相当复杂的,可能涉及多个信号诱导途径,并且各信号途径之间存在交叉对话。此外,在文库中还获得了一些有趣的基因信息,如糖原合成激酶(Glycogen synthase kinase3, GSK3),翻译控制肿瘤蛋白(‘Translationally controlled tumor protein, TCTP), MYB蛋白,植物细胞色素P450(Plant cytochromes450, P450), NAC蛋白,Rac蛋白和一些与光合作用有关的蛋白等。2. GhTCTP1序列和进化分析及在棉蚜诱导下或伤害诱导后的表达水平变化依据建立的鸡脚红叶棉叶片的抑制差减杂交cDNA文库所提供的信息,克隆了一个翻译控制肿瘤蛋白基因,将其命名为GhTCTP1(Accession number in GenBank:KC787689)。 GhTCTP1属于TCTP亚家族,编码一个168个氨基酸的蛋白质。该蛋白与水稻、小麦、拟南芥等植物的TCTP蛋白具有高度的相似性,具有相同的氨基酸95个,在进化上GhTCTP1与油桐TCTP亲缘关系较近。此外,该基因的表达水平在棉蚜攻击的棉花根、下胚轴、子叶和真叶中被抑制,而在受到伤害诱导后上调,暗示着GhTCTP1的表达水平与棉蚜的诱导或伤害诱导有关。3.在拟南芥中过量表达GhTCTP1可提高转基因植株对桃蚜的抗性GhTCTP1过量表达转基因拟南芥植株通过农杆菌介导法获得。选取其中三个株系Line1, Line2和Line10进行抗蚜性试验。选择性试验证明桃蚜对转基因拟南芥选择性较低,而对野生型拟南芥的选择性较高。非选择性试验表明转基因拟南芥叶片上桃蚜的数量低于野生型拟南芥叶片。这些结果表明,与野生型拟南芥相比,转基因拟南芥对桃蚜具有更高的抗性,暗示着过量表达GhTCTP1可以提高转基因拟南芥对桃蚜的抗性。4. GhTCTP1过量表达转基因拟南芥对桃蚜的抗性机制研究在拟南芥中过量表达GhTCTP1可以提高拟南芥对桃蚜的抗性,故对其抗蚜机制进行研究是很有必要的。为此,我们测定了可溶性糖和总游离氨基酸两种营养物质的含量,检测了苯丙氨酸解氨酶、过氧化物酶和超氧化物歧化酶三种酶的活性,用苯胺蓝染色法观察了叶片胼胝质的沉积情况。结果表明,野生型拟南芥和转基因拟南芥之间可溶性糖含量和总游离氨基酸含量均发生了变化；与野生型拟南芥相比,转基因拟南芥中三种酶的活性均有所提高；而且,转基因拟南芥中有更多的胼胝质沉积。这些结果暗示在拟南芥中过量表达GhTCTP1提高了部分拟南芥对桃蚜的抗性指标,进而提高了拟南芥对桃蚜的抗性。此外,在转基因拟南芥中,水杨酸防御响应途径相关基因转录水平上调,显示GhTCTP1可能通过水杨酸途径调节转基因拟南芥对蚜虫的抗性。5. GhTCTP1转基因棉花对棉蚜的抗性研究选择性试验在过量表达GhTCTP1转基因棉花(Line2, Line4, Line5和Line6)和野生型棉花(对照)之间进行。试验结果表明,在棉蚜危害6h、12h和24h后,转基因棉花Line4和Line6叶片上棉蚜的数量均低于野生型棉花,二者之间存在显著或极显著差异,而转基因植株Line2, Line5叶片上棉蚜的数量均略低于野生型棉花。以上结果暗示转基因棉花比野生型棉花更不适宜于棉蚜的生存。非选择性试验在棉花品种Coker312和鸡脚红叶棉及四个转基因棉花株系(Line2, Line4, Line5和Line6)上进行。在棉蚜危害24h和48h后,抗蚜品种鸡脚红叶棉和四个转基因株系(Line2, Line4, Line5和Line6)叶片上棉蚜的数量均不同程度的低于Coker312。以上结果表明,过量表达GhTCTP1转基因棉花T1代植株比野生型棉花Coker312植株对棉蚜具有更高的抗性。6. GhPSAK1序列和进化分析及在棉蚜诱导下或伤害诱导后的表达水平变化序列分析表明,GhPSAK1属于PSI_PSAK超基因家族,编码一个162个氨基酸的与光合作用相关的蛋白。GhPSAK1与苜蓿、大豆、小麦和拟南芥的GhPSAK蛋白具有高度的同源性。系统发育分析显示,GhPSAK1在进化关系上与大豆和苜蓿亲缘关系较近。棉花受到棉蚜危害时或受到轻度伤害12h和24h后,GhPSAK1在叶片中的转录水上升,在24h达到最高水平,其后逐步下降。在此过程中,受到棉蚜诱导时GhPSAK1的上升水平明显快于伤害,但其表达水平在降低时却明显慢于伤害。7.过量表达GhPSAK1转基因拟南芥对桃蚜的抗性研究对桃蚜的抗性分析在过量表达GhPSAK1转基因拟南芥株系(Line1, Line2和Line6)和野生型拟南芥(对照)之间进行。选择性实验结果表明,转基因拟南芥叶片上桃蚜种群在6h、12h和24h后均小于野生型拟南芥,表明桃蚜对转基因拟南芥具有较低的选择性。非选择性实验显示,在转基因拟南芥上桃蚜的数量均少于野生型拟南芥。以上实验结果暗示过量表达GhPSAK1可以改善拟南芥对桃蚜的抗性。8.过量表达GhPSAK1转基因拟南芥的抗蚜机制过量表达GhPSAK1转基因拟南芥胼胝质沉积多于野生型拟南芥。此外,其可溶性糖含量和游离氨基酸含量也发生了一定变化。而转基因拟南芥中SOD和POD的活性均高于野生型。这些结果暗示,过量表达GhPSAK1改善了拟南芥的抗蚜条件,从而使转基因拟南芥比野生型对桃蚜表现出更高的抗性。此外,水杨酸防御响应途径相关基因表达分析显示GhPSAK1可通过水杨酸途径调节转基因拟南芥对蚜虫的抗性。更多还原

【Abstract】 Cotton aphid (Aphis gossypii Glover) is one of the most important pests of the commercial crop cotton in the world. The adults and nymphs of cotton aphids collectively cause direct damage to leaves or young stems of cotton with their piercing-sucking mouths to acquire plant phloem liquids. At the same time, they also lead to indirect damage to cotton by transmitting plant viruses and triggering soot disease from honeydew secreted by aphids. The damage from cotton aphids has effects on cotton output and cotton fiber quality as result of the increased sugar content. The insecticides are used frequently because of cotton aphids characterized by small size, strong fertility, short lifespan and serious overlapping generation and so on. On account of the unreasonable use of insecticides, cotton aphids have produced resistance to many insecticides, which further make the control of cotton aphids more difficult. In addition, cotton aphid damage has a tendency to rise further with the extension and application of Bt cotton. It is regarded as an effective and environmentally friendly strategy to control aphids using plant resistance to them, and one of the most important methods of integrated management system of aphid damage, so it is focused on by worker on plant protection. With the development of research on plant resistance to insects, many of the aphid-resistant crop cultivation have brought huge ecological and economic benefits to agricultural production. Many research results also showed that aphids-resistant crops can reduce to the transmission of plant aphids-mediated virus. However, new problems also began to appear that the virulence of plant resistant genes to aphid has been produced in17kinds of aphids, which accounts for more than half of all arthropod biological type with virulence. To delay and prevent the occurrence of aphid biological type, it is of great significance to actively seek new aphid-resistant genes, reveal the molecular mechanism of interaction between plants and aphids, and realize long-lasting use of aphids-resistant gene in a production system of crop. From the perspective of cotton resistance to cotton aphids, as the main test materials with red-chicken-feet-leaf cotton (aphid-resistant varieties), we constructed the subtracted cDNA library of cotton leaves induced by cotton aphids with SSH (Suppression subtractive hybridization) method. The bioinformatics analysis showed that a total of253ESTs (Expression Sequence Tag) on cotton aphid induction were acquired from the cDNA subtracted library. As the source of their information, GhTCTP1and GhPSAK1, which were related to cotton aphid attacks, were cloned, and the functions and mechanisms of their resistance to aphids were studied. The main results were as follows:1. Construction and analysis on the subtracted cDNA library of cotton leaves induced by cotton aphidsA subtracted cDNA library of cotton leaves induced by cotton aphids was constructed with SSH method.253independent clones from the subtracted cDNA library were randomly chosen and analyzed. The size of the inserts varied from250to2000bp. The clones were sequenced and the expressed sequence tags (ESTs) were analyzed through homology search. From opinion of stress, there were38cDNA clones on biotic stress,95cDNA clones on abiotic stress,93cDNA clones on biotic and abiotic stress, and27cDNA clones on others. This suggested that plant induced defense to cotton aphids was relatively complex and involved several signal transduction pathway, and there were cross-talk among different signal pathways. In addition, we identified some interesting genes, Such as Glycogen Synthase Kinase3(GSK3), Translationally Controlled Tumor Protein (TCTP), MYB proteins, Plant cytochrome P450(P450s), NAC, Rac protein, and some proteins on photosynthesis.2. GhTCTP1sequence, phylogenetic analysis and its expression level under the cotton aphid attack or after wounding inductionA translationally controlled tumor protein gene, GhTCTP1(Accession number in GenBank: KC787689), was identified according to information from the subtracted cDNA library. GhTCTP1belonged to TCTP subfamily and edcoded a putative protein of168amino acids. GhTCTPl shared high similarity to plant TCTPs from rice, wheat and Arabidopsis and so on, with95same amino acids, and GhTCTPlwas close relative to TCTP of Vernicia fordii during their evolution. GhTCTP1expression level was suppressed in cotton roots, hypocotyls, cotyledons and leaves under cotton aphid attack, but its expression level was up-regulated in the wounded cotton leaves, which suggested that GhTCTP1expression level was relative to the induction from cotton aphids or wounding.3. GhTCTP1overexpressing in Arabidopsis thaliana can improve the resistance of transgenic plants to green peach aphidsGhTCTP1overexpression transgenic Arabidopsis plants were acquired by Agrobacterium-mediated transformation. We chose three Lines (Line1, Line2and Line10) from them to study their resistance to green peach aphids (Myzus persicae). The choice test demonstrated that the green peach aphids had lower choice for transgenic Arabidopsis, but higher for wild type Arabidopsis. No-choice test showed that the aphid number from transgenic Arabidopsis was more than that from wild type. These results suggested that transgenic Arabidopsis was higher resistant to green peach aphids than wild type, and GhTCTP1overexpression may improve the Arabidopsis resistance to green peach aphids.4. Overexpressing GhTCTP1transgenic Arabidopsis resistance mechanisms research on green peach aphidsSince GhTCTP1overexpression may improve Arabidopsis resistance to green peach aphids, the study of its mechanisms of aphid-resistance was necessary. To attain this aim, we measured of two kinds of important nutrient content (Soluble sugar and total free amino acid), activities of three enzymes (Phenylalanine ammonia enzyme, PAL; peroxidase, POD; superoxide dismutase, SOD), and observed the leaf callus deposition with aniline blue staining. The content of two nutrient substance was different in leaves between transgenic Arabidopsis and wild type Arabidopsis. The activities of PAL, SOD and POD in leaves from transgenic Arabidopsis were higher than wild type Arabidopsis. The callose amount in transgenic Arabidopsis leaf was more than wild type Arabidopsis leaf. These results suggested that GhTCTPl overexpression in Arabidopsis improved some aphid-resistant indexes and increased Arabidopsis resistance to green peach aphids. In addition, the expression levels of key genes in SA-related pathway were upregulated in transgenic Arabidopsis under aphid attack in comparison with wild type, suggesting GhTCTPl might regulated plant resistance to aphid by SA-related pathway.5. Analysis on GhTCTPl overexpressing transgenic cotton resistance to cotton aphidsChoice tests were carried out between GhTCTPl overexpression transgenic cotton Line2, Line4, Line5, Line6and wild type cotton Coker312. Choice tests showed that the number of cotton aphids from leaves of Line4or Line6was lower than that from leaves Coker312at6h,12h,24h under cotton aphid attack, and there was significant or very significant difference between them. The number of cotton aphids from leaves of Line2or Line5was no significant difference than from leaves of Coker312at6h,12h,24h under cotton aphid attack. However, the number of the former were little lower than the latter. No-choice tests were performed on leaves from Coker312, red-chick-feet-leaf cotton (JJ, aphid-resistant cultiyar), and four transgenic lines (Line2, Line4, Line5, Line6). The results confirmed that the cotton aphid number on leaves from red-chick-feet-leaf cotton, Line2, Line4, Line5, Line6was lower than that from Coker312at24h,48h under cotton aphid infestation. All these results suggested that T1generation of GhTCTP1overexpression transgenic cotton were higher resistant to cotton aphids than Coker312.6. GhPSAK1sequence, phylogenetic analysis and its transcripts under the cotton aphid infestation or after wounding inductionA photosynthesis-related cotton gene, GhPSAK1(Accession number in GenBank:KF246683), was identified. It belonged to PSI-PsaK family and encoded a putative protein of162amino acids. GhPSAKl was highly homologous with GhPSAKl of alfalfa, soybeans, wheat and Arabidopsis thaliana. Phylogenetic tree showed that GhPSAK1was closely relative to soybean and alfalfa during their evolutions. Cotton GhPSAKl expression levels rised at0h,12h,24h under cotton aphid attack or after wounding induction and reached highest level at24h. However, GhPSAKl transcripts declined gradually at48h and72h. In this process, GhPSAK1expression levels of cotton infested by cotton aphids raised more quickly than that induced by wounding, but the former decreased more slowly than the latter.7. Effects on resistance to green peach aphids in overexpressing GhPSAK1Arabidopsis.The analysis on resistance to green peach aphids (Myzus persicae) was performed between GhPSAK1overexpression Arabidopsis thaliana lines (Line1, Line2and Line10) and wild type (WT). Choice assays showed that aphid population on transgenic Arabidopsis leaf was smaller than that on WT at6h,12h,24h under attack from green peach aphids, which confirmed that green peach aphids had lower choice for transgenic Arabidopsis than WT. No-choice assay demonstrated that the number of green peach aphids on transgenic Arabidopsis was less than that on WT at24h,48h under attack from green peach aphids. All these results suggested that overexpression of GhPSAK1in Arabidopsis improved plant resistance to green peach aphids.8. Aphid-resistance mechanism of GhPSAK1overexpressing transgenic ArabidopsisThe callose amount in transgenic Arabidopsis leaves was more than that of wild type. In addition, The contents of the soluble sugars and total amino acids were also altered in leaves of transgenic Arabidopsis plants in comparison with wild type. Activities of SOD (superoxide dismutase) and POD (peroxidase) in transgenic leaves were higher than those of wild type. These results suggested that GhPSAK1overexpression could improve aphid-resistant condition of Arabidopsis, and the transgenic Arabidopsis showed higher resistance to green peach aphids than wild type. In addition, the transcriptions of key genes related on SA pathway were upregulated in transgenic Arabidopsis under aphid infection, suggesting GhPSAK1might regulate plant resistance to aphid by SA-related pathway.更多还原