【作者】 何俊平；

【导师】 陈素梅；

【作者基本信息】 南京农业大学 ， 园林植物与观赏园艺， 2010， 硕士

【摘要】 菊花[Chrysanthemum grandiflorum (Ramat.) Kitamura]是我国十大传统名花、世界四大切花之一,在园林、家庭绿化美化中占有十分重要的地位。然而,菊花病虫害危害严重,蚜虫、蓟马、黑斑病、褐斑病等是其常见病虫害,在幼苗至成苗期时有发生,多危害芽心嫩尖,抑制植株的生长发育,严重影响观赏品质和应用,蚜虫不仅直接为害菊花,而且是TAV、PVP、CMV病毒的媒介者。目前,蚜虫的防治多采用化学方法,不仅消耗人力物力、造成环境污染,而且导致农药残留,使得蚜虫群体产生抗药性。因此,蚜虫的防治已成为菊花产业化生产中的一大难题,创造抗蚜虫菊花新种质已成为菊花抗性育种亟待解决的问题。目前关于切花菊抗蚜虫的研究较少,从切花菊中鉴定抗蚜虫性资源和利用转基因技术提高切花菊蚜虫抗性的研究尚未见报道。本研究旨在探讨切花菊抗蚜虫性鉴定方法,阐述蚜虫抗性机理及通过基因工程创造抗蚜虫切花菊新种质,进行了室内人工蚜虫接种的抗性鉴定,比较分析了不同蚜虫抗性品种间相关叶片结构特征、蚜虫接种后抗氧化酶与防御酶活性变化,并尝试了石蒜凝集素基因LLA转化切花菊的研究,主要研究结果如下：1.切花菊抗蚜虫性的抗性等级及不同品种的抗蚜虫性对35个切花菊品种进行了室内人工接种蚜虫的抗性鉴定研究,结果显示不同的菊花品种对蚜虫的抗性表现出明显差异,蚜虫接种21 d时各品种上虫口数量差异甚大,为0～267头不等；根据蚜量比值可将切花菊抗蚜虫性分为5级,分别为：高抗,0～0.25；中抗,0.26～0.50；抗,0.51～0.75；低抗,0.76～1.25；不抗：>1.25。其中,‘神马’、‘风车菊1’、‘南农红枫’等15个品种蚜量比值在1.25以上,为不抗品种；‘小丽’、‘韩1’、‘南农茶香’等4个品种蚜量比值为0.76～1.25,为低抗品种；‘阳光’、‘馒头菊’、‘优香’(3个品种)蚜量比值为0.51～0.75,为抗虫品种；‘韩5’、‘韩6’(2个品种)蚜量比值为0.26-0.50,为中抗品种；‘韩4’、‘南农紫勋章’、‘精云’等11个品种蚜量比值为0～0.25,为高抗品种。2.抗感切花菊品种的叶片结构特征、抗氧化酶及防御酶活性以高抗品种‘精云’、中抗品种‘韩6’和不抗品种‘神马’为材料,比较分析了三者相关叶片表面结构特征(表皮毛、腺体、蜡质等)及蚜虫接种对抗氧化酶、防御酶活性变化的影响,结果发现,‘精云’的表皮毛致密,单个表皮毛长度和高度、腺体大小及蜡质含量均高于‘韩6’和‘神马’,初步说明叶片表皮毛、腺体、蜡质含量等与切花菊抗蚜虫性具有一定的相关性；蚜虫接种后,抗氧化酶和防御酶活性水平变化均存在显著差异。在抗性品种中,超氧化物岐化酶(SOD)、过氧化物酶(POD)、抗坏血酸酶(APX)活性水平上升速度快,出现峰值较不抗品种早。APX活性变化幅度亦明显大于不抗品种。POD、POD活性在蚜虫接种后期(3d-7d)保持上升趋势。多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)活性均有所提高,抗性品种响应快,上升幅度大,且接种后期保持较高的活性水平。说明蚜虫接种前后切花菊抗感材料叶片的活性氧系统和防御酶系统与抗虫性密切相关,可作为其苗期蚜虫抗性评价的生理指标之一。3.农杆菌介导的LLA基因转化切花菊的研究以目前生产应用比较广泛的切花菊‘神马’为材料,对其遗传转化中合适的抗生素筛选浓度进行了研究,结果表明叶片对潮霉素、羧苄青霉素十分敏感,10 mg·L-1的潮霉素能够完全抑制叶片分化,20 mg·L-1的潮霉素完全抑制了不定芽生根；确定了‘神马’叶盘再生筛选以8～10 mg·L-1的潮霉素为宜,生根筛选以18 mg·L-1的潮霉素为宜。羧苄青霉素对菊花叶片的再生有一定的抑制作用,随着浓度的升高,抑菌效果增加,但芽的分化率逐渐下降,500 mg·L-1的羧苄青霉素能够完全抑制农杆菌的滋生,但严重抑制了叶片的分化,浓度降至300 mg·L-1时,能够完全抑制农杆菌的滋生并保证50%的再生率,故确定羧苄青霉素抑菌浓度早期以500 mg·L-1为宜,后期以250～300 mg-L-1为宜。通过农杆菌介导的叶盘法将长筒石蒜凝集素LLA基因转入‘神马’,获得了105个抗性芽,生根筛选后获得54个潮霉素抗性植株；PCR及RT-PCR分子检测显示有7个株系检测到目的条带的整合与表达；对该7个转基因株系采用室内人工蚜虫接种试验进行鉴定,结果表明转基因切花菊幼苗对蚜虫密度有明显的抑制作用,且不同转化株系的抗蚜性差异较大,蚜口密度抑制率为12.2%～76.8%不等,平均蚜口密度抑制率为41.8%。更多还原

【Abstract】 Chrysanthemum is one of ten traditional famous flowers in China and four cut flowers in the world, it play an important position on gardens and greening. However, chrysanthemum is seriously infested by pests from the time of its seedling establishment to its flowering stage. Aphid infestation usually hurt tender bud tip, inhibiting the plant growth and development, results in dwarf plant, yellowing leaf and yield loss, effectting the quality and ornamental value seriously, it as a result of their direct jeoprridize chrysanthemum itself, and their indirect action as vectors of TAV, PVP, CMV viruses. Currently, chemical pesticide was the major method to control aphid during horticultural practices, which not only consumed higher agrochemical costs, caused environmental pollutions but also resulted in pesticide residue. Thus, the control of aphid had become a major problem on industrial production of chrysanthemum. Development of the genetic resistance to aphid infestation has become a priority in chrysanthemum breeding programmes. However, there was a few reports of studies on the aphid resistance of chrysanthemum. So, the purpures of this study was to explore the identification of the method of aphid-resistance in chrysanthemum cultivars, describe the aphid resistance mechanisms and develope the new aphid-resist germplasm of chrysanthemum via genetic engineering. We carried a series studies on artificial aphid inoculation test, compared different aphid resistant varieties of leaf epidermal surface morphology, antioxidant and defense enzyme activity in response to aphid infestation and try to transformation of chrysanthemum with LLA gene. The main results are follows:1. Chrysanthemum aphid resistance grade and different aphid-resistance to different cultivars.In order to discover aphid resistant germplasm, aphid number and aphid number ratio were investigated in 35 cut chrysanthemum cultivars via artificial aphid inoculation test. The results indicated that different chrysanthemum cultivars had different levels of aphid resistance. By 21 days after inoculation, the number of aphid varied upon to cultivars ranging from 0 to 267. The aphid resistance of chrysanthemum can be divided into five grades in terms of aphid number ratio, i.e., high resistance with an aphid number ratio of 0-0.25, moderate resistance (0.26-0.50), resistant grade (0.51-0.75), low resistance (0.76-1.25) and non-resistant grade (>1.25), respectively. Among 35 tested cultivars,15 cultivars represented by’Jinba’,’Fengcheju 1’, ’Nannonggongfen’ are non-resistant cultivars with aphid number ratio above 1.25. Four cultivars represented by’Xiaoli’,’Han 1’, ’Nannongchaxiang’ are cultivars of low resistance to aphid with aphid number ratio of 0.76-1.25. Three cultivars, ’Yangguang’ , ’Mantouju’ and ’Youxiang’ , are aphid resistant cultivars (aphid number ratio,0.51-0.75). Two cultivars,’Han 5’ and’ Han 6’, are moderately resistant to aphid (aphid number ratio,0.26-0.50), while 11 cultivars represented by’Han 4’, ’Nannongzixuanzhang’,’Keiun’ are highly resistant to aphid (aphid number ratio,0-0.25).2. Study on leaf epidermal micro-morphology of different aphid resistance cultivars, activities of antioxidant and defense enzymes in chrysanthemum cultivars of different aphid resistance in response to aphid inoculation.Using high-resistant cultivar’Keiun’, morderate-resistant cultivar’Han 6’and susceptible cultivar ’Jinba’ as material, a comparative analysis of structural characteristics of leaf surface (trichomes, glands, wax, etc.) and antioxidant and defense enzyme activity in response to aphid infestation, we found that high resistant cultivar’Keiun’ had thicker, higher and denser trichomes, bigger and full glands, and more waxiness on the lower leaf epidermis than that on moderately resistant’Han 6’and susceptible’Jinba’. Preliminary inferred that aphid resistance in chrysanthemum had related to leaf trichomes, glands, wax content. After aphid inoculation, the activity of antioxidant and defense enzymes levels were significantly different. In resistant cultivars, superoxide dismutase (SOD), peroxidase (POD), ascorbic acid enzyme (APX) activity levels rising faster than susceptible cultivar and peaking earlier. APX activity has been significantly changed in resistant cultivars. Preliminary inferred that aphid resistance in chrysanthemum had related to leaf trichomes, glands, wax content. After aphid inoculation, the activity of antioxidant enzymes and defense levels were significantly different. In resistant cultivars, superoxide dismutase (SOD), peroxidase (POD), ascorbic acid enzyme (APX) activity levels rising faster than susceptible cultivar and peaking earlier. APX activity has been significantly changed in resistant cultivars and the activity of SOD, POD during 3 d to 7 d were observed in resistant cultivars of ’Keiun’ and ’Han6’ than in ’Jinba’. Polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) activity were enhanced in aphid inoculated plants compared with the control. It responsed increased fastly and peak earlier, and maintain high activity levels post-inoculation. We suggest that antioxidant and defense enzymes both contribute to aphid resistance of these chrysanthemum cultivars, it can be used as one of biochemical marker for aphid resistance of seedlings of cut flower in chrysanthemum.3. Transgenic Chrysanthemum Plants Expressing a LLA Gene via Agrobacterium-mediated.Using leaf disks of cut-flower chrysanthemum ’Jinba’ as explants, the concentration of the appropriate antibiotic selection has been studied, The results showed that the leaf was very sensitive to hygromycin (Hyg) and carbenicillin (Carb),10 mg·L-1 of Hyg concentration had been completely inhibited differentiation and rooting of adventitious buds could be completely restricted with 20 mg·L-1. In actual research, concentration of Hyg for selecting was a little lower than critical concentration, so we chose 8-10 mg·L-1 as the optimal concentration of Hyg for Leaf disc regeneration and 20 mg·L-1 for root selecting. Also, the leaf was very also sensitive to Carb. The Carb concentration had a certain degree inhibition of chrysanthemum. As the concentration elevated, the effect of inhibition increased, but the rate of bud differentiation had declined. Carb concentration 500 mg·L-1 could completely control the growth of bacterium but inhibit the shoot regeneration severely. While the concentration declined with 300 mg·L-1, it could be restrain the bacterium completely but keep higher regeneration rate of 50%. So we carried with gradual-reducing bacterium controlling method, using 500 mg·L-1 of Carb concentration in the early stage, then declined to 250-300 mg·L-1.The LLA gene was successfully transferred into chrysanthemum via Agrobacterium-mediated leaf transformation, One hundred and five resistance bud were obtained. Fifty-four putative transformants of chrysanthemum were obtain after root screening. Polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) analysis showed that the LLA gene was successfully integrated into the chrysanthemum genome and expressed in seven transgenic lines. The artificial aphid inoculation test showed that the aphid resistance of different transgenic plants was different, and the rate of aphid population inhibition ranged from 12.2% to 76.8% with an average inhibition rate of 41.8% in transformed chrysanthemum.更多还原