棉蚜体内原生共生菌Buchnera aphidicola种群密度的调控机制

发布时间：2018-03-18 09:32

本文选题：棉蚜　切入点：原生共生菌　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

【摘要】：蚜虫体内普遍存在共生菌,原生共生菌Buchnera aphidicola是蚜虫不可缺少的重要组成部分。缺失原生共生菌Buchnera后,蚜虫将不能繁殖后代。共生菌的种群密度与其宿主的生理与生态适应能力等密切相关。但是,至今有关蚜虫体内原生共生菌种群密度的调控机制还不清楚。因此,本文以存在寄主专化型的棉蚜为对象,研究了寄主植物、宿主棉蚜的基因型、棉蚜体内的溶菌酶及次生菌等对Buchnera种群密度的影响,以期明确蚜虫体内原生共生菌种群的调控机理。研究获得了以下主要结果:(1)Buchner 原生共生菌对棉蚜种群的净增殖率、平均寿命和内禀增长率有显著影响。抗生素去除或降低Buchnera种群密度后,棉蚜的净增殖率和内禀增长率显著降低、世代平均寿命延长。自然条件下的棉花型棉蚜在西葫芦上的内禀增长率显著高于在豇豆和棉花上的,但经高浓度抗生素脱共生后,其在西葫芦、豇豆和棉花上内禀增长率显著下降,且寄主间不再有显著差异。原生共生菌对棉蚜的繁殖和寄主利用有一定的影响。(2)瓜型棉蚜体内原生共生茵Buchnera种群密度和菌胞数量都显著高于棉花型棉蚜。南京地区西葫芦和木槿上的棉蚜体内原生共生菌Buchnera种群数量显著高于黄瓜和棉花上的,并且黄瓜上的又显著高于棉花上的。不同基因型棉蚜体内共生菌的种群密度有一定的差异,但这种差异受棉蚜取食寄主的影响。瓜型和棉花型棉蚜转移到新的植物上后,其体内原生共生菌种群密度和棉蚜的存活率均会发生显著波动,但生活多代后,两者均会趋于稳定;并且棉花型棉蚜在西葫芦上生活后,其体内的Buchnera种群密度会升高到瓜型棉蚜在黄瓜上生活的水平,而瓜型棉蚜在豇豆上生活后,其体内Buchnera菌的密度会降低到棉花型棉蚜的水平。棉蚜的寄主植物和基因型显著影响了原生共生菌Buchnera的种群密度。(3)利用PCR的方法检测棉蚜种群中10种次生菌的感染率,发现棉蚜种群除有部分个体感染了杀雄菌Arsenophonus外,没有发现有其他次生共生菌的感染,不同寄主植物上棉蚜感染Arsenophonus菌的比率差异显著,棉花上的棉蚜感染率高,而瓜类作物上的棉蚜种群感染率低或完全不感染。去除杀雄菌Arsenophonus后棉蚜体内原生共生菌Buchnera的种群密度显著提高。(4)通过RT-PCT和RACE技术,克隆得到了棉蚜溶菌酶基因全长序列(680bp )。序列分析表明,棉蚜体内的溶菌酶属于i型溶菌酶,并且失去了海洋动物i型溶菌酶中存在的两个重要活性位。棉蚜体内的溶菌酶以可溶性蛋白和包涵体两种形式存在。定量PCR测定发现,瓜型棉蚜体内溶菌酶基因的表达水平,以在西葫芦上生活的棉蚜中最高,豇豆上生活的次之,而黄瓜上生活的最低。(5)植物提取物显著影响了棉花型和瓜型棉蚜体内原生共生菌Buchnera的种群密度。棉花型棉蚜取食含有黄瓜、西葫芦、南瓜和豇豆叶片提取物的人工饲料后,原生共生菌种群密度显著提高,但是取食含有棉花叶片提取物的人工饲料后,原生共生菌的密度没有显著变化。瓜型棉蚜取食含棉花叶片提取物的人工饲料后,其体内原生共生菌Buchnera种群密度下降。取食添加棉酚和葫芦素的人工饲料后,瓜型和棉花型棉蚜体内原生共生菌Buchnera的种群密度都会发生显著变化。高浓度棉酚(50ppm)显著降低了两专化型棉蚜体内原生共生菌Buchnera的种群密度,但葫芦素能引起原生共生菌种群密度的显著升高。(6)对棉花型棉蚜(CO)、瓜型棉蚜(CU)和瓜型棉蚜转移到豇豆上饲养五代时的品系(CU-cowpea)进行转录组测序分析,结果表明,CO与CU棉蚜间基因表达水平显著不同。与棉花型相比,瓜型棉蚜有1106个基因呈现显著上调和2835个基因呈现显著下调;当瓜型棉蚜在豇豆上饲养后,其有812个基因显著下调和14492个基因显著上调。棉花型和瓜型棉蚜间的差异表达基因显著富集在与糖代谢、免疫进程、病原体感染或共生、唾液分泌有关的KEGG途径上。同时,瓜型和棉花型棉蚜在解毒酶和唾液蛋白酶相关基因上出现了显著的差异表达,这些类型的基因可能与棉蚜寄主专化型的调控有关。在棉蚜转录组中选取4个差异表达明显的基因进行RNA干扰,并检测干扰成功后棉蚜体内原生共生菌Buchnera的种群密度,结果表明,一个解毒酶基因U4155受干扰后,棉蚜体内原生共生菌的种群密度显著降低,而其他3个基因的干扰并没有影响原生共生菌的数量。总之,棉蚜宿主的寄主植物、基因型和解毒酶基因表达水平和体内的次生菌等参与了棉蚜体内原生共生菌Buchnera种群密度的调控,并且寄主植物可通过植物次生代谢物来对棉蚜体内的原生共生菌种群密度进行调控。
[Abstract]:In aphids widespread symbiotic bacteria, native symbiotic bacteria Buchnera aphidicola is an important part of aphids indispensable. Lack of native symbiotic bacteria Buchnera, aphids will not breed. The physiological and ecological population density of the symbiotic bacteria and its host adaptation ability closely related. However, so far the aphid body native symbiotic bacteria density regulation mechanism is not clear. Therefore, based on the existing host biotype aphids as object, studied the host plant genotype, host aphid, Aphis gossypii in vivo effects of lysozyme and secondary bacteria on Buchnera population density, in order to clear the primary regulation mechanism of symbiotic bacteria in aphids population. Main results are as follows: (1) Buchner primary symbiotic bacteria on cotton aphid net reproductive rate, life expectancy and intrinsic growth rate has a significant effect on removing or reducing Buchnera antibiotics. The population density of cotton aphid, the net reproductive rate and intrinsic growth rate decreased significantly, prolong the life expectancy. The cotton aphid generations under natural conditions, the intrinsic in pumpkin growth rate was significantly higher than that in cowpea and cotton, but the high concentration of antibiotics in the symbionts, zucchini, cowpea and cotton on the intrinsic growth rate decreased significantly, and no significant difference. Between the host primary symbionts on cotton aphid reproduction and host utilization have certain effect. (2) in Cucurbit specialized primary symbiotic bacteria Buchnera population density and number of bacteria were significantly higher than that of the cotton cotton aphid. The symbiosis of Zucchini and hibiscus aphid in the original Nanjing area number of bacteria Buchnera population was significantly higher than that of cucumber and cotton, and the cucumber was significantly higher than that of cotton. The population density of different genotypes of cotton aphid in symbiotic bacteria have some differences, but the difference by cotton Effect of aphid feeding on the host. The transfer of melon type and cotton biotypes to new plants, the survival rate of the symbiotic bacteria in the native density of cotton aphid and will significantly fluctuate, but after so many generations of living, both will tend to be stable; and the cotton aphid in summer squash life, its body Buchnera population density will rise to cucurbit specialized living in cucumber on the level, and cucurbit specialized in cowpea life, the in vivo Buchnera bacteria density will be reduced to the level of the cotton aphid. The population density of cotton aphid host plant and genotype significantly affected total native bacteria Buchnera (3) detection. 10 secondary bacteria infection rate in cotton aphid population using the PCR method, in addition to some individual aphid populations found infected with male killing bacteria Arsenophonus, found no other secondary bacteria infection of different host plants of cotton aphid infected with Arsenoph The percentage of onus was significant, high rate of cotton aphid infection, and melon crops the cotton aphid population on the low infection rate or no infection. The removal of population density of male killing bacteria Arsenophonus in vivo after aphid primary symbionts of Buchnera increased significantly. (4) by RT-PCT and RACE technology, we cloned the full-length sequence of lysozyme of cotton aphid gene (680bp). Sequence analysis showed that the aphid body belongs to type I lysozyme and lysozyme activity has lost two important marine animal type I lysozyme bits. There lysozyme as a soluble protein in cotton aphid and inclusion bodies in two forms. The quantitative determination of PCR expression levels found in Cucurbit specialized lysozyme gene the highest life in summer squash aphids, cowpea time in life, and life. The lowest on Cucumber (5) plant extracts significantly affected the cotton and cucumber biotypes in native The population density of symbiotic bacteria Buchnera. The cotton aphids fed with cucumber, squash, pumpkin and artificial diet of cowpea leaves extract, native symbiotic bacteria density increased, but feeding on artificial diet containing extract of cotton leaves after primary symbiotic bacteria density did not change significantly. The artificial diet containing extracts of melon aphid feeding type cotton leaves after the in vivo primary symbiont Buchnera population density decreased. Adding gossypol and cucurbitacin feeding on artificial diet, population density and cotton aphid in melon type primary symbionts Buchnera will change significantly. The high concentration of gossypol (50ppm) significantly reduced the population density of two biotypes of cotton aphid in primary the symbiotic bacteria Buchnera, but cucurbitacin can cause primary endosymbionts bacteria density increased significantly. (6) of the cotton aphid, Aphis gossypii (CO) melon type (CU) and melon aphid transfer type To the five generation of cowpea breeding lines (CU-cowpea) of transcriptome sequencing analysis results showed that CO and CU gene expression levels were significantly different between cotton. Compared with cotton, melon biotypes of 1106 genes was significantly up-regulated and 2835 genes showed significantly reduced; when the melon biotypes in cowpea breeding after the 812 genes were down regulated and 14492 genes were up-regulated. Differences between cotton and cucumber biotypes of gene expression is significantly enriched in carbohydrate metabolism, immune process, pathogen infection or symbiosis, salivary KEGG pathways related. At the same time, melon type and cotton biotypes of detoxifying enzymes and protease in saliva related genes showed significantly different expression, regulation of genes may be associated with these types of specialized aphid host type. Select 4 differences of gene expression were RNA interference in cotton aphid transcriptome, and detection of interference The population density of cotton aphid in, after reactive primary symbionts Buchnera showed that a detoxification enzyme gene U4155 interference in vivo after aphid native symbiotic bacteria population density decreased significantly, and the interference of the other 3 genes did not affect the number of primary endosymbionts. In short, the host plant aphid host genotype, and detoxification the enzyme gene expression in vivo and the secondary bacteria in vivo primary symbiotic bacteria Buchnera control cotton aphid population density, and host plants by plant secondary metabolites to native symbiotic bacteria in vivo on cotton aphid density for regulation.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433

【相似文献】