【作者】 周利飞；

【导师】 刘映红；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2007， 硕士

【摘要】 烟草是重庆市的重要经济支柱产业。但近年来烟草花叶病毒病和烟蚜Myzus persicae(Sulzer)发生普遍、危害严重，已成为各烟区威胁烟叶生产的主要病虫害，给烟叶生产造成巨大经济损失。本文对比研究了3种寄主及感染TuMV的相应寄主对烟蚜生长发育和繁殖的影响；成功克隆出烟草TuMV、TMV和CMV分离物的CP基因，进行了序列测定和分析。其研究结果为进一步探明植物寄主、烟草花叶病毒和烟蚜的关系，控制烟蚜的发生和烟草花叶病毒病的传播奠定了一定基础；为烟区TuMV、TMV和CMV病毒株系分化研究，病毒外壳蛋白表达，生产高效价的抗血清，建立烟草花叶病毒田间快速检测体系提供了技术支持。本文主要研究结果如下：1 3种寄主及感染TuMV的相应寄主对烟蚜生长发育的影响本文在供试温度25±0.5℃、相对湿度75±5％、光周期L：D=14：10条件下，研究了3种寄主及感染TuMV的相应寄主对烟蚜生长发育和繁殖的影响，结果表明，烟蚜具有一定的寄主适应性，寄主植物对烟蚜生长发育和繁殖存在显著影响。在其他条件相同的情况下，在3种健康寄主中，青菜上的烟蚜若虫历期最短(5.6929d)，产仔量(34.4286头)最大，产仔高峰日出现最早，死亡高峰日出现得最晚；运用生命表参数对烟蚜取食这3种寄主后的生长发育、繁殖情况进行综合评价，结果表明烟蚜在3种健康寄主植物上的r_m大小顺序排列为：青菜(0.2681)＞甘蓝(0.2572)＞烟草(0.2271)，说明烟蚜最嗜取食青菜，且在青菜上繁殖力最强，其次为甘蓝，最不嗜取食烟草。3种寄主病株对烟蚜生长发育和繁殖也有一定影响。烟蚜取食烟草病株和甘蓝病株的产仔量分别为21.4143头和21.4727头，显著高于取食青菜病株的产仔量(13.2714头)；3种感病寄主按产仔高峰大小排序为：甘蓝病株(4.0455头)＞烟草病株(2.7647头)＞青菜病株(1.6508头)；烟草病株和甘蓝病株上的产仔高峰日皆为第11天，青菜病株为第14天。说明青菜病株上烟蚜繁殖力最弱，而烟草病株和甘蓝病株上烟蚜繁殖力没有显著差异。烟蚜在3种寄主病株上的r_m大小顺序排列为：甘蓝病株(0.2556)＞烟草病株(0.2158)＞青菜病株(0.1729)，结果表明青菜病株对烟蚜影响最大，烟草病株次之，甘蓝病株对烟蚜的影响最小。与取食健康寄主相比，3种寄主病株均导致烟蚜若蚜的存活率和成蚜的繁殖力降低。在3种寄主健株和病株上，Weibull模型c值均大于1，烟蚜存活率曲线都属于Ⅰ型。在健康甘蓝上的c值最大(4.9265)，存活率下降最快，烟草病株上的c值最小(2.2708)，存活率下降最慢：烟草病株和甘蓝病株上的烟蚜死亡高峰日均比健康寄主上的提前，而青菜病株上的烟蚜死亡高峰日则比健康寄主上的晚；3种寄主病株和健株上烟蚜历期和繁殖力比较的结果显示，甘蓝病株导致烟蚜若虫龄期延长，而使成虫寿命缩短，产仔量减少；在青菜病株上，烟蚜若虫期显著长于在健康植株上的若虫期，在病株上的产仔量则显著降低；与健株相比，在烟草病株上的烟蚜除了1龄若虫历期和成蚜繁殖前期显著缩短外，若虫期、成虫寿命和产仔量都没有显著差异。以上结果表明，烟蚜虽然最嗜取食青菜，且在青菜上繁殖力最强，青菜有利于烟蚜生长发育和繁殖，但感染TuMV的青菜病株对烟蚜种群的影响也最大。与健株相比，烟草病株对烟蚜生长发育和繁殖没有显著影响。2武隆烟区TuMV、TMV和CMV外壳蛋白基因克隆及序列分析本研究从少量的病叶中提取到高质量的总RNA，利用RT-PCR分别扩增出重庆武隆烟区TuMV、TMV和CMV分离物的CP基因，并成功克隆了这3种病毒的CP基因，完成了序列测定，进行了同源性分析和系统发育树构建。这为烟区TuMV、TMV和CMV病毒株系分化研究和建立烟草花叶病毒田间快速检测体系提供了技术支持。同源性比较结果表明，本实验克隆的TuMV CP基因与GenBank上部分TuMV CP基因序列核苷酸同源性在86.1％～99.6％之间，氨基酸同源性在92.3％～99.0％之问。与TuMV-CO同源性最高的是AY090660(Tu7)，核苷酸同源性为99.6％，氨基酸同源性为99.0％。同源性比较和系统发生树表明，这些病毒分离物可分为4组，而重庆分离物与world-B组分离物同源性较高，由此可以确定TuMV重庆分离物属于Brassica(B)致病型中的world-B组。将本实验分离的TMV分离物与已知的TMV序列进行比较，核苷酸同源性在71.9％～99.8％之间，氨基酸同源性在78.5％～100.0％之间。其中与本实验分离的TMV分离物同源性最高的为Yunnan分离物(DQ352813)，核苷酸和氨基酸同源性分别高达99.8％和100.0％，表明它们可能为同一株系。经同源性比较，结果显示本实验分离的CMV分离物与已知序列的核苷酸同源性在75.1％～99.5％之间，氨基酸同源性在77.5％～98.6％之间，这些分离物总体可分为两大组，S70105、Z12818、D00463、U22822、AF198103、L15336和AJ131622为组Ⅱ，其余为组Ⅰ。除了D49496，亚组Ⅰ与CMV-CQ的核苷酸和氨基酸序列同源性均在90.0％以上；而与CMV亚组Ⅱ的核苷酸和氨基酸同源性仅在75.1％～76.7％和77.5％～80.2％之间，所以CMV-CQ分离物应该归属于CMV亚组Ⅰ。更多还原

【Abstract】 Recently, Myzus persicae (Sulzer) and tobacco mosaic diseases, the most main pest and diseases, appeared on tobacco and have caused tremendous economic losses on the production of tobacco. Influence of three hosts on development, survival and reproduction of Myzus persicae (Sulzer). RT-PCR was used to clone the coat protein (CP) genes of TuMV-CQ, TMV-CQ and CMV-CQ. Meanwhile, these six coat protein genes were sequenced and compared with other homologous sequences in GenBank. This thesis provides some reliable technique and theory for controlling Myzus persicae (Sulzer) and tobacco diseases spreading and harming. At the same time, the results will be helpful to construct faster molecular and serological methods and study on strains of TuMV, TMV and CMV.The main results of this thesis are as follows:1 Influence of different hosts on development, survival and reproduction of Myzus persicae (Sulzer)The effects of host plants and host infected TuMV on development and reproduction of Myzus persicae (Sulzer) were studied under the condition of 25±0.5℃, RH 75%±5%, L:D=14:10. The results showed that there were obvious difference in the development and reproduction of Myzus persicae (Sulzer). On Brassica chinensis (Linn.), the duration of nymph was shortest (5.6929d), fecundity was biggest (34.4386), and the date of maximum mortality was latest among these three hosts. The innate capacities of population increase (r_m) of three hosts were given in the order: Brassica chinensis (Linn.) (0.2681)＞cabbage (0.2572)＞tobacco (0.2271). According to the parameters of life table, we inferred that the most suitable host of Myzus persicae (Sulzer) is Brassica chinensis (Linn.), but the most unsuitable host is tobacco in the above three hosts.In these three hosts infected TuMV, the fecundity of Myzus persicae (Sulzer) on tobacco (21.4727) and cabbage (21.4143) were significantly higher than those on Brassica chinensis (Linn.). On the date of maximum fecundity, the order of fecundity of Myzus persicae (Sulzer) on three hosts infected TuMV is cabbage (4.0455)＞tobacco (2.7647)＞Brassica chinensis (Linn.) (1.6508). The date of maximum fecundity on tobacco and cabbage were 11, but on Brassica chinensis (Linn.) was 14. The innate capacities of population increase (r_m) of three hosts infected TuMV were given in the order: cabbage(0.2556)＞tobacco (0.2158)＞Brassica chinensis (Linn.)(0.1729). The result showed that the influence of Brassica chinensis (Linn.) infected by virus on the population was biggest, but the cabbage was smallest.By comparison of healthy tobacco, the survival rate of nymph and the reproduction of adult on three hosts infected TuMV were lower. The values C of Weibull model for the population survival rate of aphid were all greater than 1 on different healthy and infected hosts. So the survival rate of nymph all belonged toⅠtype. The c value of the survival rate on cabbage was biggest, but on tobacco was smallest. Dates of maximum mortality of aphid on tobacco and cabbage infected TuMV were earlier than on healthy hosts, but on infected Brassica chinensis (Linn.) was later. The result showed that the duration of nymph on infected cabbage was longer, duration of adult was shorter, and reproduction was reduced. On infected Brassica chinensis (Linn.), the duration of nymph was significantly higher than on healthy hosts, and the reproduction was also reduced. There was no significant difference on the duration of nymph and adult, and the reproduction between healthy and infected tobacco. These suggested that comparison of healthy hosts, the influence of Brassica chinensis (Linn.) infected by virus on the population was biggest, but the tobacco was smallest.2 Coat protein gene sequence analysis of TuMV, TMV and CMV in ChongqingThe integrated and pure RNA was obtained from a bit of infected tobacco leaves. The CP gene of these three viruses from Chongqing was amplified by reverse-transcriptase polymerase chain reaction (RT-PCR) and it was cloned and sequenced. Then sequence homology was analyzed and the phylogenetic tree was built.Comparison of the nucleotide and amino acid sequences of TuMV-CQ with the corresponding sequences of several TuMV isolates on Genebank showed that the homology are 86.1%～99.6% at nucleotide level, and 92.3%～99.0% at amino acid level. TuMV-CQ shares 99.6 % and 99.0% homologies at the nucleotide and deduced amino acid levels, respectively, with the strain TU7. The result showed that TuMV-CQ could belong to group world-B.Compared with the corresponding sequences of TMV isolates, the homology of TMV-CQ are 71.9%～99.8% at nucleotide level, and 78.5%～100.0% at amino acid level. TMV-CQ shares 99.8% and 100.0% at the nucleotide and deduced amino acid levels, with one of the Yunnan strain, indicating that they could belong to the same strain.Comparison of the nucleotide and amino acid sequences of CMV-CQ with the corresponding sequences of several CMVⅠisolates showed that the homology are 89.8%～99.5% at nucleotide level, and 91.9%～98.6% at amino acid level, on the other hand, it were 75.1%～76.7% at nucleotide level, and 77.5%～80.2% at amino acid level with the strains of CMVⅡ, which suggested that the CMV-CQ belongs to CMVⅠother than CMVⅡ.更多还原