本文選題：棉蚜　切入點(diǎn)：原生共生菌　出處：《南京農(nóng)業(yè)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：蚜蟲(chóng)體內(nèi)普遍存在共生菌,原生共生菌Buchnera aphidicola是蚜蟲(chóng)不可缺少的重要組成部分。缺失原生共生菌Buchnera后,蚜蟲(chóng)將不能繁殖后代。共生菌的種群密度與其宿主的生理與生態(tài)適應(yīng)能力等密切相關(guān)。但是,至今有關(guān)蚜蟲(chóng)體內(nèi)原生共生菌種群密度的調(diào)控機(jī)制還不清楚。因此,本文以存在寄主專(zhuān)化型的棉蚜為對(duì)象,研究了寄主植物、宿主棉蚜的基因型、棉蚜體內(nèi)的溶菌酶及次生菌等對(duì)Buchnera種群密度的影響,以期明確蚜蟲(chóng)體內(nèi)原生共生菌種群的調(diào)控機(jī)理。研究獲得了以下主要結(jié)果:(1)Buchner 原生共生菌對(duì)棉蚜種群的凈增殖率、平均壽命和內(nèi)稟增長(zhǎng)率有顯著影響。抗生素去除或降低Buchnera種群密度后,棉蚜的凈增殖率和內(nèi)稟增長(zhǎng)率顯著降低、世代平均壽命延長(zhǎng)。自然條件下的棉花型棉蚜在西葫蘆上的內(nèi)稟增長(zhǎng)率顯著高于在豇豆和棉花上的,但經(jīng)高濃度抗生素脫共生后,其在西葫蘆、豇豆和棉花上內(nèi)稟增長(zhǎng)率顯著下降,且寄主間不再有顯著差異。原生共生菌對(duì)棉蚜的繁殖和寄主利用有一定的影響。(2)瓜型棉蚜體內(nèi)原生共生茵B(yǎng)uchnera種群密度和菌胞數(shù)量都顯著高于棉花型棉蚜。南京地區(qū)西葫蘆和木槿上的棉蚜體內(nèi)原生共生菌Buchnera種群數(shù)量顯著高于黃瓜和棉花上的,并且黃瓜上的又顯著高于棉花上的。不同基因型棉蚜體內(nèi)共生菌的種群密度有一定的差異,但這種差異受棉蚜取食寄主的影響。瓜型和棉花型棉蚜轉(zhuǎn)移到新的植物上后,其體內(nèi)原生共生菌種群密度和棉蚜的存活率均會(huì)發(fā)生顯著波動(dòng),但生活多代后,兩者均會(huì)趨于穩(wěn)定;并且棉花型棉蚜在西葫蘆上生活后,其體內(nèi)的Buchnera種群密度會(huì)升高到瓜型棉蚜在黃瓜上生活的水平,而瓜型棉蚜在豇豆上生活后,其體內(nèi)Buchnera菌的密度會(huì)降低到棉花型棉蚜的水平。棉蚜的寄主植物和基因型顯著影響了原生共生菌Buchnera的種群密度。(3)利用PCR的方法檢測(cè)棉蚜種群中10種次生菌的感染率,發(fā)現(xiàn)棉蚜種群除有部分個(gè)體感染了殺雄菌Arsenophonus外,沒(méi)有發(fā)現(xiàn)有其他次生共生菌的感染,不同寄主植物上棉蚜感染Arsenophonus菌的比率差異顯著,棉花上的棉蚜感染率高,而瓜類(lèi)作物上的棉蚜種群感染率低或完全不感染。去除殺雄菌Arsenophonus后棉蚜體內(nèi)原生共生菌Buchnera的種群密度顯著提高。(4)通過(guò)RT-PCT和RACE技術(shù),克隆得到了棉蚜溶菌酶基因全長(zhǎng)序列(680bp )。序列分析表明,棉蚜體內(nèi)的溶菌酶屬于i型溶菌酶,并且失去了海洋動(dòng)物i型溶菌酶中存在的兩個(gè)重要活性位。棉蚜體內(nèi)的溶菌酶以可溶性蛋白和包涵體兩種形式存在。定量PCR測(cè)定發(fā)現(xiàn),瓜型棉蚜體內(nèi)溶菌酶基因的表達(dá)水平,以在西葫蘆上生活的棉蚜中最高,豇豆上生活的次之,而黃瓜上生活的最低。(5)植物提取物顯著影響了棉花型和瓜型棉蚜體內(nèi)原生共生菌Buchnera的種群密度。棉花型棉蚜取食含有黃瓜、西葫蘆、南瓜和豇豆葉片提取物的人工飼料后,原生共生菌種群密度顯著提高,但是取食含有棉花葉片提取物的人工飼料后,原生共生菌的密度沒(méi)有顯著變化。瓜型棉蚜取食含棉花葉片提取物的人工飼料后,其體內(nèi)原生共生菌Buchnera種群密度下降。取食添加棉酚和葫蘆素的人工飼料后,瓜型和棉花型棉蚜體內(nèi)原生共生菌Buchnera的種群密度都會(huì)發(fā)生顯著變化。高濃度棉酚(50ppm)顯著降低了兩專(zhuān)化型棉蚜體內(nèi)原生共生菌Buchnera的種群密度,但葫蘆素能引起原生共生菌種群密度的顯著升高。(6)對(duì)棉花型棉蚜(CO)、瓜型棉蚜(CU)和瓜型棉蚜轉(zhuǎn)移到豇豆上飼養(yǎng)五代時(shí)的品系(CU-cowpea)進(jìn)行轉(zhuǎn)錄組測(cè)序分析,結(jié)果表明,CO與CU棉蚜間基因表達(dá)水平顯著不同。與棉花型相比,瓜型棉蚜有1106個(gè)基因呈現(xiàn)顯著上調(diào)和2835個(gè)基因呈現(xiàn)顯著下調(diào);當(dāng)瓜型棉蚜在豇豆上飼養(yǎng)后,其有812個(gè)基因顯著下調(diào)和14492個(gè)基因顯著上調(diào)。棉花型和瓜型棉蚜間的差異表達(dá)基因顯著富集在與糖代謝、免疫進(jìn)程、病原體感染或共生、唾液分泌有關(guān)的KEGG途徑上。同時(shí),瓜型和棉花型棉蚜在解毒酶和唾液蛋白酶相關(guān)基因上出現(xiàn)了顯著的差異表達(dá),這些類(lèi)型的基因可能與棉蚜寄主專(zhuān)化型的調(diào)控有關(guān)。在棉蚜轉(zhuǎn)錄組中選取4個(gè)差異表達(dá)明顯的基因進(jìn)行RNA干擾,并檢測(cè)干擾成功后棉蚜體內(nèi)原生共生菌Buchnera的種群密度,結(jié)果表明,一個(gè)解毒酶基因U4155受干擾后,棉蚜體內(nèi)原生共生菌的種群密度顯著降低,而其他3個(gè)基因的干擾并沒(méi)有影響原生共生菌的數(shù)量�？傊�,棉蚜宿主的寄主植物、基因型和解毒酶基因表達(dá)水平和體內(nèi)的次生菌等參與了棉蚜體內(nèi)原生共生菌Buchnera種群密度的調(diào)控,并且寄主植物可通過(guò)植物次生代謝物來(lái)對(duì)棉蚜體內(nèi)的原生共生菌種群密度進(jìn)行調(diào)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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S433

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