Arsenophonus菌在褐飞虱抗病免疫防御中的作用及机制

We use the brown planthopper (BPH), which is one of the most important rice pest in China, and its endosymbiont bacteria Arsenophonus (ARS) to study the function and mechanism of ARS in the immune response of BPH against the pathogenic fungi Metarhizium anisopliae. First, fluorescent in situ hybridization (FISH) will be used to study the tissue distribution of ARS in BPH. Second, immune-related enzyme activity, the number of haemocytes, the survival rate of haemocytes, haemocytes spreading rates and the encapsulation reaction in ARS-BPH haemolymph will be compared with those in non-ARS-BPH haemolymph to understand the physiological and biochemical function of ARS in the BPH imume response. Third, we test the transcriptome of ARS-BPH and non-ARS-BPH either infected or uninfected with pathogenic fungi M. anisopliae to find out the specific differential expression genes related to the BPH immune response to the pathogenic fungi, and use real-time quantitative polymerase chain reaction to verify the genes expression. Lastly, RNAi technology will be used to silence the specific differential expression genes in ARS-BPH to confirm the function of the genes in the BPH immune response against pathogenic fungi. The aim of this study is to find out the function and mechanism of ARS in the immune response of BPH against pathogenic fungi by using immunology, physiology and molecular biology methods, and this study will provide new ideas and a theoretical basis for how to enhance the fungi control function to BPH through impairing the BPH immune system by regulating its symbiotic bacteria ARS.

本研究以我国水稻生产的重要害虫褐飞虱及其共生细菌Arsenophonus(ARS)为研究对象，采用荧光原位杂交技术对褐飞虱体内ARS菌进行组织定位；通过测定含和不含ARS菌褐飞虱血淋巴中液体免疫相关酶活以及比较细胞免疫相关参数的异同，探明ARS在褐飞虱免疫应答反应的免疫生理生化作用；对含和不含ARS褐飞虱感染和未感染黄绿绿僵菌四个种群进行转录组测序及分析，筛选出ARS介导的褐飞虱抗病免疫应答的特异性差异表达基因，以期明确ARS在褐飞虱抗病免疫防御中的作用及相关免疫调控机理。并利用实时荧光定量PCR方法，对特异性差异表达基因进行验证，尔后利用RNAi技术对其生物功能进行验证。本研究从免疫学、生理生化与分子生物学角度探明ARS介导的褐飞虱抗病免疫防御的作用及机理，为今后如何通过调控ARS来削弱褐飞虱的免疫能力，以提高病原真菌对褐飞虱的自然控制作用提供防治新思路与理论依据。

褐飞虱Nilaparvata lugens (Stål)是我国水稻上的主要害虫，黄绿绿僵菌是其重要的病原真菌,褐飞虱内共生菌Arsenophonus可抑制黄绿绿僵菌对褐飞虱的毒力。本研究在筛选黄绿绿僵菌菌株和探索其保存方法的基础上，就共生菌Arsenophonus对黄绿绿僵菌毒力的影响及其与环境温度的关系进行了研究，并通过褐飞虱体内血细胞类型与数量的观察及对含和不含Arsenophonus褐飞虱、感染和未感染黄绿绿僵菌四个种群进行转录组测序及分析及相关解毒酶活性测定，筛选出Arsenophonus介导的褐飞虱抗病免疫应答的特异性差异表达基因，初步探讨了共生菌Arsenophonus在褐飞虱抗病免疫防御中的作用及相关免疫调控机理。结果如下：优化了黄绿绿僵菌的保存方法，采用滤纸片保存法操作简单，菌株活力不减退；黄绿绿僵菌的最适侵染温度为27度；褐飞虱的卵、1龄若虫、2龄若虫、3龄若虫、4龄若虫、5龄若虫及初羽化雌成虫、初羽化雄成虫体内均含有Arsenophonus，并且随着褐飞虱的发育，Arsenophonus在体内呈现积累的趋势；过氧化氢酶（CAT）、谷胱甘肽转移酶（GSH）、碱性磷酸酯酶（ALP）、胆碱酯酶（AchE）、羧酸酯酶（CES）和烯醇式酶（hEC）在含和不含Arsenophonus种群褐飞虱的不同发育阶段的含量有显著差异，喷施黄绿绿僵菌后发现， CES和hEC与黄绿绿僵菌侵染有关；褐飞虱血细胞构成简单，为浆血细胞和粒血细胞，还有非细胞形态的脂血胞，含有Arsenophonus褐飞虱和不含有Arsenophonus褐飞虱种群之间血细胞的类型不存在差异，但粒血细胞数量存在显著差异，含有Arsenophonus褐飞虱的粒血细胞数量显著高于不含有Arsenophonus的褐飞虱；黄绿绿僵菌处理后1~5 d，浆细胞数量在喷菌处理的褐飞虱体内显著升高，且含ARS的BPH高于不含ARS的BPH，并在第2 d两者之间差异极显著，表明浆细胞在褐飞虱应对黄绿绿僵菌感染过程中有重要作用，而Arsenophonus能进一步激发该作用。黄绿绿僵菌诱导与未诱导的ARS褐飞虱种群和非ARS褐飞虱种群的血淋巴的转录组测序结果表明：含有ARS褐飞虱种群和不含有ARS褐飞虱种群之间差异基因951个，感染黄绿绿僵菌之后，两种群之间的差异基因有2653个，差异基因的验证还有待于进一步研究。