【作者】 陈艳；

【导师】 刘芳；

【作者基本信息】 扬州大学 ， 植物保护， 2015， 硕士

【摘要】 灰飞虱I aodelphax striatellus (半翅目飞虱科)是亚洲地区水稻上的重要害虫。它不仅刺吸水稻汁液造成危害,更重要的是传播水稻条纹叶枯病毒(Rice sripe virus, RSV)造成水稻条纹叶枯病的流行。RSV是纤细病毒属代表成员,由灰飞虱以循回增殖型方式经卵传播。介体灰飞虱从感染植株吸食获毒后,RSV从肠壁内腔扩散到血淋巴及其它组织,最后到达唾液腺,在介体昆虫取食时又被传至植株。唾液腺是RSV传播需要克服的最后一道屏障,而RSV克服唾液腺屏障的机制缺乏研究。前期的研究发现,带毒灰飞虱唾液腺中α-亚基-ATP合酶(ATP synthase subunit alpha)、微管蛋白-α-2 (Tubulin alpha-2)、苹果酸脱氢酶(NADP-dependent malic enzyme)、线粒体内膜移位酶(Mitochondrial import inner membrane translocase)及泛素-60s核糖体L40前体(Ubiquitin-60S ribosomal protein L40 precursor)等5个蛋白的表达水平显著高于无毒灰飞虱唾液腺。本论文研究了苹果酸脱氢酶、线粒体内膜移位酶及泛素-60s核糖体L40前体等3个唾液腺差异蛋白的氨基酸全长序列及基因表达,并用RNA干扰研究了α-亚基-ATP合酶、微管蛋白-α-2、线粒体内膜移位酶基因对RSV传播的影响。主要结果如下：(1)利用克隆与RACE技术,从带RSV的灰飞虱体内成功扩增出2060 bp的苹果酸脱氢酶、661 bp线粒体内膜移位酶及1167bp的泛素-60s核糖体L40前体的的核苷酸全长序列。将全长核苷酸序列提交到NCBI的GenBank数据库中,分别命名为LsNADP、LsMIT及LsUBI。采用实时定量PCR的方法对LsNADP、LsMIT及LsUBI基因表达进行相对定量,结果发现带毒灰飞虱唾液腺和全身中这3个基因的表达均显著上调。与不带毒灰飞相比,带毒灰飞虱唾液腺中L sNADP基因、LsMIT基因及LsUBI的相对表达量分别提高了17.4%、123.6%和62.2%。LsNADP、LsMIT和LsUBI基因在带RSV灰飞虱虫体中的相对表达量较无毒灰飞虱分别提高了25.0%、43.4%和16.7%。(2)采用RNAi研究了α-亚基-ATP合酶(LsATP)、线粒体内膜移位酶基因(LsMIT)、微管蛋白-α-2 (LsTUB)对灰飞虱传播RSV的影响。取食了含dsRNA的人工饲料后,灰飞虱中LsATP、LsMIT、LsTUB的相对表达分别较对照下降了63%、82%、76%,表明RNA干扰效果明显。吸食了dsATP和dsMIT后,灰飞虱的传毒与对照并无差异,未发现这两个基因对灰飞虱传毒有明显影响。取食含dsTUB人工饲料的带毒灰飞虱的传毒率为12%,仅为取食纯人工饲料的对照的30%。表明LsTUB在介体传播RSV中具重要作用。LsTUB协助RSV克服介体唾液腺屏障的机制待进一步详细的研究。更多还原

【Abstract】 The small brown planthopper (SBPH) Laodelphax striatellus (Fallen) (Homoptera:Delphacidae) is one of the most important pest insects of rice in Asia. In addition to injuring rice plants Oryza sativa by sap-sucking via its piercing-sucking mouthparts, L. striatellus also acts as the most important vector of rice stripe virus (RSV), which causes rice stripe disease. RSV is a typical member of the genus Tenuivirus and is transmitted only in a persistent, circulative-propagative manner by L. striatellus. After being acquired, the virus viruses move through the insect vector, from the gut lumen into the hemolymph or other tissues and finally into the salivary glands from which these viruses are introduced back into the plant during insect feeding. Our previous study indicated that the expressions of some proteins, such as ATP synthase subunit alpha, tubulin alpha-2, NADP-dependent malic enzyme, mitochondrial import inner membrane translocase and ubiquitin-60S ribosomal protein L40 precursor in the salivary gland of viruliferous L. striatellus were significantly higher than those of naive vectors. The complete cDNA sequences and relative expressions of genes encoding NADP-dependent malic enzyme, mitochondrial import inner membrane translocase and ubiquitin-60S ribosomal protein L40 precursor were examined. The functions of genes encoding ATP synthase subunit alpha, tubulin alpha-2 and mitochondrial import inner membrane translocase in RSV transmission were investigated. The results were as follows.(1) The complete cDNA sequences genes encoding NADP-dependent malic enzyme, mitochondrial import inner membrane translocase and ubiquitin-60S ribosomal protein L40 precursor of viruliferous L. striatellus was obtained by reverse transcription polymerase chain reaction (RT-PCR) with rapid amplification of cDNA ends (RACE). The full-length sequences of NADP-dependent malic enzyme gene (Gene name:LsNADP), mitochondrial import inner membrane translocase (Gene name:LsMIT) and ubiquitin-60S ribosomal protein L40 precursor (Gene name:LsUBI) were 2060 bp,661bp and 1167 bp, respectively. The relative expressions of these three genes were significantly higher in viruliferous L. striatellus than those in naive vectors. In the salivary glands, the relative expressions of LsNADP, LsMIT and LsUBI increased by 17.4%,123.6% and 62.2%, respectively, compared to the control. In the vector body, the LsNADP, LsMIT and LsUBI were up-expressed increasing by 25.0%,43.4% and 16.7%, respectively, compared to the control.(2) RNAi silencing of LsATP, LsMIT and LsTUB genes were used to examine their functions in RSV transmission by viruliferous L. striatellus. The significance of RNA interference was obtained. The dsRNA decreased target gene expression by 63% for LsATP, by 82% for LsMIT, and by 76% for LsTUB, respectively, compared to controls. No obvious difference was observed in virus transmission rate by viruliferous L. striatellus treated with dsATP (dsMIT) compared to control, indicating that LsATP and LsTUB had less significance in RSV transmission. Virus inoculation rate of viruliferous L. striatellus treated with dsTUB was 12%, decreasing by 70% compared to the control. It indicated that LsTUB played an important role in RSV transmission. The underlying molecular mechanism of interaction between LsTUB and RSV needs further investigation.更多还原