【作者】 周亦红；

【导师】 韩召军；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2003， 博士

【摘要】 褐飞虱Nilaparvata lugens(st(?)l)是我国和亚洲稻区的重要害虫。抗性水稻品种的选育和利用因其安全、经济、有效而成为褐飞虱防治措施中的首选。然而，随着水稻抗虫品种的推广应用，能够克服品种抗性的害虫新生物型却不断出现，这严重制约了褐飞虱综合治理和水稻抗虫育种工作的深入开展。 褐飞虱生物型问题自出现以来就引起了国内外学术界的高度重视。许多学者从水稻品种对褐飞虱的抗性机制、褐飞虱生物型的监测、褐飞虱生物型的生物学、形态学、细胞学、生理生化、遗传学及鉴定技术等方面进行了广泛研究，获得了大量的有价值的资料，大大加深了人们对生物型问题的认识和了解。然而，由于褐飞虱生物型问题的复杂性以及传统研究手段的限制，对一些根本性的问题，如褐飞虱生物型致害性的遗传基础及遗传规律，致害性发生和变异的机制，褐飞虱生物型与水稻品种之间的互作机理等，目前仍然缺乏深入了解。另外，虽然多种鉴定褐飞虱群体生物型的方法已经得到广泛应用，但如何准确、快速地对褐飞虱个体生物型进行鉴定，一直是亟待解决的技术难题。澄清和解决上述问题，对于褐飞虱生物型的有效治理以及抗虫育种工作的深入开展有着极其重要的意义。 从生化及分子水平研究害虫生物型之间的差异，探询害虫与抗性水稻品种之间的互作机制，是从根本上揭示褐飞虱生物型致害性形成机制的重要途径，而目前有关的研究还很少。近年来的研究表明，褐飞虱生物型的致害性并非由单一的基因控制，而是具有复杂的遗传背景。不同生物型致害性上的差异可能涉及到多种基因在质或量上的差异表达。本研究主要以此为切入点，在建立褐飞虱雌成虫对抗性水稻品种Mudgo的个体致害性指标的基础上，采用近年来新发展起来的分子生物学技术——抑制性差减杂交方法，研究生物型1和生物型2之间基因的差异表达情况，分离、鉴定生物型致害性相关基因。另外，由于代谢酶在昆虫对寄主的适应性方面起着重要作用，我们还初步考察了抗性水稻品种对褐飞虱体内几种代谢酶的影响。上述有关的研究结果总结如下： (1)用水稻敏感品种TN1和具有不同抗性基因的品种Mudgo，ASD7和Rathu Heenati饲养稻褐飞虱，研究不同品种对褐飞虱体内酯酶、谷胱甘肽转移酶和多功能氧化酶氧-脱甲基活性的影响。结果表明，褐飞虱在3种抗性品种上饲养一代后，其酯酶活性与敏感品种上的试虫相比没有显著差异。然而，取食Mudgo和取食ASD7的雌成虫之间酯酶活性达到显著性差异；取食Mudgo和Ruthu Heenati的雄成虫酯酶 褐飞虱生物型的研究的M值分别为取食TNI的2．78和2．58倍。褐飞虱体内酯酶活性还因其生物型的不同而异。对褐飞虱生物型1和生物型2个体酯酶活力分布的测定结果表明，生物型2雌、雄成虫酯酶活性均显著低于生物型1。另外，在ASD7与Rathu Heenati上饲养一代后，褐飞虱雌、雄成虫GSTS的活性显著高于取食W 和MSdgO的个体，但抗性品种对褐飞虱成虫的多功能氧化酶氧一脱甲基活性没有明显影响。因此，水稻品种对褐飞虱代谢酶的影响因品种的特性，代谢酶的类型，甚至褐飞虱的性别而异。 门）在 28 t IC下，观测了褐飞虱羽化 24h内短翅型雌成虫在敏感水稻品种 TNI及抗性品种MUdg。上的寿命及体重增量。结果表明，取食抗性品种时，试虫的平均增重与寿命之间表现出明显的正相关。寿命在7天内的个体，存活期间平均体重增量为负值，而存活7天以上的个体则平均增重明显。进一步研究了初羽化的褐飞虱长翅型雌成虫在W 及MUdg。上取食第1－4天的体重增量、寿命及产卵量，结果发现试虫在Mlldg。上的这3个生物学参数之间存在明显正相关。其中，寿命及体重增量较好地反映出害虫个体对抗性品种的致害能力，可作为个体致害性指标．根据研究结果，我们将羽化后在Mudgo上存活7天以上，或最初4天内体重增量大于0．ling的雌成虫定为能够致害该抗性品种的个体。 （3）以若虫存活率为致害性指标，采用家系纯化的方法，对室内已选育30多代的褐飞虱生物型1和生物型2实验种群分别进行了一代和2代的进一步纯化培养，并考察了两种生物型纯化过程中群体及个体家系致害性的变化情况。结果表明，纯化后的两种生物型群体基本上保持了原有的致害特性，表明生物型的致害性具有一定的遗传稳定性。通过纯化培养，我们获得了群体致害性差异明显、遗传背景相对一致的两个生物型实验群体。但研究中也发现，在纯化过程中，两种生物型的子代家系在致害性上均出现了较高比例的变异，表明生物型的致害性存在着复杂的遗传背景。 （4）以家系纯化后的褐飞虱生物型1和生物型2短翅型雌成虫为材料，采用抑制性差减杂交的方法分离两种生物型雌成虫差异表达的基因。以生物型1为对照群体 （driver），生物型2为目标群体（tester）进行正向差减杂交，同时以生物型2为对照群体，生物型1为目标群体，进行反向差减杂交，结果构建了分别含有480和299个CDNA克隆的正向和反向差减文库。从获得的正、反向差减文库中分别随机挑选150个CDNA克隆进行差示筛选。通过两轮差示筛选更多还原

【Abstract】 The brown planthopper (BPH), Nilaparvata lugens (Stal), is one of the most destructive pests of rice-growing areas in Asia. Resistant rice varieties have been used as the preferential method in the control of this pest for its safety, economy and efficiency. However, the BPH biotypes, which can "overcome" the resistance of rice varieties with different major resistance genes, occurs soon after the release of resistant rice varieties, and this has become a serious problem for resistant rice breeding and pest controlling.Extensive studies have been made on the biology, morphology, cytology, biochemistry and genetics of different BPH biotypes, the monitoring method of biotypes in the field, and the resistance mechanism of rice varieties to this pest, et al. Valuable data accumulated and it has greatly deepened our knowledge about the BPH biotypes. However, due to the extra complex of this problem and the limitation in the former research measures, some key problems, such as the genetic basis and hereditary characters of the biotype virulence, the occurrence and variation mechanism of biotypes, the interaction between BPH biotypes and rice varieties, are still waiting for clarification. In addition, although many methods are applicable for the identification of biotype populations, it has been a pressing task to establish some rapid, convenient and accurate means for the identification of individuals. Resolution of all these problems would be most important for the progress in resistant rice breeding and pest controlling.To clarify the key questions about the BPH biotypes, it would be most helpful to study the biochemical and molecular variation between different biotypes. Yet up to now, limited research has been done on this aspect. Anyway, some research results indicated from recent studies that the virulence of BPH biotypes seems to be controlled by multiple genes. Thus, the variation in different BPH biotypes is probably correlated to differential expression of many genes in quality or quantity. To identify the genes correlated with the virulence of BPH biotypes, we established the individual virulence indexes of BPH females to Mudgo (a rice variety with Bph1 resistance gene) in the first. According to the index, females with typical virulence were selected respectively from the purified biotype 1 and biotype 2family line. Then a newly-developed molecular technique -suppression subtract!ve hybridization (SSH), was used to separate differentially expressed genes between biotype 1 and biotype 2 females. Also, considering the importance of metabolizing enzymes in the adaptation of insects to their host plants, the influence of resistant rice varieties on some metabolizing enzymes in BPH was also investigated. The results are summed as follows:(1) Influence on Esterase (ESTs), glutathione S-transferases (GSTs) and mixed function oxidases (MFOs) in BPH was studied with one susceptible (TN1) and three resistant rice varieties (Mudgo, ASD7, and Ruthu Heenati). It was showed that one-generation feeding on resistant varieties did not make any significant difference in esterase activity. However, females feeding on Mudgo and ASD7 had significantly different esterase activity. Feeding on Mudgo made the Km of esterase in males increase as high as 2.78 times, and on Ruthu Heenati 2.58 times when compared with hoppers feeding on TN1. Esterase activity showed also some changes between different BPH biotypes. Study on distribution of individual esterase activity in biotype 1 and biotype 2 BPH populations indicated that esterase activity in BPH biotype 2, both male and females, was much lower than that of biotype 1. GSTs activity increased significantly after the hoppers feeding on ASD7 or Ruthu Heenati for one generation, but no changes was found in this enzyme when hpoppers were reared on Mudgo. For MFOs, no significant difference in the activity of microsomal O-demethylase was detected among various treatments. Finally, a conclusion was drown that the influence of host plant varieties on metabolizing enz更多还原