次生內(nèi)共生菌Rickettsia在煙粉虱MED隱種內(nèi)的分布、傳播以及對宿主生殖影響研究
發(fā)布時間：2018-09-18 15:55
【摘要】：煙粉虱Bemisia tabaci(Gennadius)是由35個遺傳結(jié)構(gòu)差別明顯但是形態(tài)上無法區(qū)分的隱種所構(gòu)成的復(fù)合種,其中煙粉虱地中海隱種(簡稱MED隱種,又稱Q型)和小亞細(xì)亞隱種(簡稱MEAM1隱種,又稱B型)是危害我國的主要入侵煙粉虱。2008年至今,MED隱種在中國大部分地區(qū)逐漸替代MEAM1隱種成為優(yōu)勢種,其中次生內(nèi)共生菌(facultative symbiont)在MED隱種擴(kuò)散和入侵方面發(fā)揮了重要作用,本論文以MED隱種的次生內(nèi)共生菌Rickettsia為研究對象,從定性、定位、定量三個層面,垂直傳播、水平傳播、生物學(xué)影響三個方面,探究Rickettsia對MED隱種所產(chǎn)生的影響。首先利用標(biāo)有熒光染料的特異性探針對MED隱種煙粉虱體內(nèi)的Rickettsia進(jìn)行熒光原位雜交,明確Rickettsia在MED隱種煙粉虱各發(fā)育階段的分布。然后利用TaqMan探針法,以煙粉虱β-Actin基因為內(nèi)源管家基因,Rickettsia 16srDNA(16s核糖體基因)為目的基因進(jìn)行熒光定量PCR,明確MED隱種煙粉虱各發(fā)育階段Rickettsia的拷貝數(shù)變化。同時進(jìn)行三個獨立生態(tài)實驗:1、Rickettsia在MED隱種煙粉虱體內(nèi)的垂直傳播;2、Rickettsia在MED隱種煙粉虱體內(nèi)的水平傳播;3、Rickettsia對MED隱種煙粉虱生活史的影響。本論文主要研究結(jié)果如下:(1)Rickettsia在MED隱種煙粉虱體內(nèi)各發(fā)育階段都有分布。在MED隱種成蟲體內(nèi)的分布位置有兩種:菌胞內(nèi)部和菌胞外部。菌胞內(nèi)部分布位置呈球形,是煙粉虱腹部特化的含菌細(xì)胞聚集而成的,菌胞外部位置主要是指煙粉虱腹部已成型的卵內(nèi)。兩種分布方式皆伴隨有初生共生菌Portiera的分布;Rickettsia在MED隱種四齡若蟲體內(nèi)也存在兩種分布位置,一種是主要分布在末端皿狀孔和舌狀突部位,另一種是隨機(jī)分布在若蟲胸部和頭部;在MED隱種三齡、二齡、一齡若蟲體內(nèi)的分布均以蟲體末端皿狀孔和舌狀突部位為主,其中,在二齡若蟲體內(nèi)還可分布于菌胞部位,在一齡若蟲體內(nèi)還可分布于觸角、氣管和口器等部位;Rickettsia在MED隱種卵內(nèi)的分布位置主要在卵梗和卵柄部位。(2)Rickettsia在MED隱種煙粉虱成蟲發(fā)育前期拷貝數(shù)變化差別較大,在成蟲發(fā)育后期相對穩(wěn)定。Rickettsia在整個發(fā)育初期含量是逐漸上升的,二齡時期同時也是生長發(fā)育最旺盛的時期達(dá)到了最高含量,三齡時期含量驟減達(dá)到最低點,隨后出現(xiàn)回升,羽化5天達(dá)到第二個小高峰,隨后保持在略高于初羽化時期的含菌量。從整個發(fā)育過程中來看,Rickettsia的含量整體趨勢是先增加后減少。(3)Rickettsia在MED隱種內(nèi)可隨母代垂直傳播,但無法水平傳播。結(jié)果發(fā)現(xiàn)Rickettsia從雌親代至后代的垂直傳播率約為98%,從含有Rickettsia雄蟲至不含Rickettsia雌蟲的水平傳播率約為0%。Rickettsia可以顯著增加一齡到三齡若蟲以及初羽化階段煙粉虱的發(fā)育速率,其次含菌雌蟲在卵、四齡若蟲、初羽化時期發(fā)育速率均顯著高于含菌雄蟲。此外,Rickettsia可以顯著提高煙粉虱后代性比,還能提高雌蟲產(chǎn)卵量、后代羽化率以及成蟲壽命,但影響并不顯著。綜上所述,本研究利用分子生物學(xué)技術(shù),從分子水平上研究了Rickettsia在MED隱種不同發(fā)育階段的分布位置以及拷貝數(shù)的變化,然后利用生態(tài)學(xué)方法,證明了Rickettsia在MED隱種內(nèi)的傳播效率及其對宿主生長發(fā)育的影響,所得結(jié)論為今后探究Rickettsia在MED隱種入侵方面所起的具體作用奠定基礎(chǔ),為揭示次生內(nèi)共生菌在宿主體內(nèi)可能所起的生物學(xué)功能提供依據(jù)。
[Abstract]:Bemisia tabaci (Gennadius) is a complex of 35 cryptic species with distinct genetic structure but morphological indistinguishability. Mediterranean cryptic species of Bemisia tabaci (Mediterranean cryptic species, also known as Q-type) and subtropical cryptic species of Asia Minor (MEAM1 cryptic species, also known as B-type) are the main invasive species of Bemisia tabaci. In most areas of China, MEAM1 is gradually replaced as the dominant species. The facultative symbiont plays an important role in the diffusion and invasion of MED cryptophytes. In this paper, Rickettsia, a secondary endosymbiont of MED cryptophytes, is studied from three aspects: qualitative, localization, quantitative, vertical and horizontal transmission. The effects of Rickettsia on the development of MED cryptospecies were investigated in three aspects: first, the fluorescence in situ hybridization of Rickettsia in MED cryptospecies with a specific probe labeled with fluorescent dye was performed to determine the distribution of Rickettsia in the developmental stages of MED cryptospecies. Because of the endogenous housekeeping gene, Rickettsia 16srDNA (16s ribosomal gene) was used as the target gene for fluorescence quantitative PCR to determine the copies of Rickettsia at different developmental stages of the MED cryptorch whitefly. The main results of this study are as follows: (1) Rickettsia is distributed in all developmental stages of the Cryptococcus tabaci. There are two types of distribution in the adult of Cryptococcus MED: inside and outside the cell. The distribution of the cell is spherical in shape and is in the abdomen of the whitefly. Specialized bacteria-containing cells aggregated in the outer position of the cells mainly refer to the formed eggs in the abdomen of Bemisia tabaci.Both distribution patterns are accompanied by the distribution of the primary symbiotic bacteria Portiera.Rickettsia also has two distribution positions in the fourth instar nymphs of cryptospecies MED, one is mainly distributed in the terminal discoid foramen and the tongue process, the other is mainly distributed in the terminal discoid foramen and the tongue process. It is distributed randomly in the nymph's chest and head; in the third, second and first instar cryptozoa, dish-shaped foramen and tongue-shaped process are mainly distributed in the end of the nymph's body; in the second instar nymph, it can also be distributed in the cell parts; in the first instar nymph, it can also be distributed in the antennae, trachea and mouth organs; Rickettsia in the egg of cryptozoa in MED. The distribution of Rickettsia was mainly in the pedicel and stalk of eggs. (2) Rickettsia had a great difference in the number of copies in the early stage of development and was relatively stable in the late stage of adult development. The content of Rickettsia increased gradually in the early stage of development, and reached the highest level in the second and the most vigorous stage of growth and development. The content of Rickettsia increased first and then decreased during the whole development process. (3) Rickettsia could be transmitted vertically with the mother generation, but could not be transmitted horizontally. The results showed that the vertical transmission rate of Rickettsia from female parent to offspring was about 98%, and the horizontal transmission rate from male Rickettsia to female Rickettsia was about 0%. Rickettsia could significantly increase the development rate of Bemisia tabaci at the first to third instar nymphs and early emergence stages, followed by female bacteria in eggs, fourth instar nymphs and early emergence stages. In addition, Rickettsia could significantly increase the sex ratio of offspring of Bemisia tabaci, increase the egg production of female, the emergence rate of offspring and the longevity of adults, but the effect was not significant. In summary, molecular biology techniques were used to study the different developmental stages of Rickettsia in MED cryptospecies. Distribution and copy number changes, and then using ecological methods to prove the transmission efficiency of Rickettsia in MED cryptophyte and its impact on host growth and development, the conclusions for the future study of Rickettsia in MED cryptophyte invasion of the specific role laid the foundation for revealing the secondary endosymbiotic bacteria in the host may be. It provides a basis for biological functions.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S433
本文編號：2248400
[Abstract]:Bemisia tabaci (Gennadius) is a complex of 35 cryptic species with distinct genetic structure but morphological indistinguishability. Mediterranean cryptic species of Bemisia tabaci (Mediterranean cryptic species, also known as Q-type) and subtropical cryptic species of Asia Minor (MEAM1 cryptic species, also known as B-type) are the main invasive species of Bemisia tabaci. In most areas of China, MEAM1 is gradually replaced as the dominant species. The facultative symbiont plays an important role in the diffusion and invasion of MED cryptophytes. In this paper, Rickettsia, a secondary endosymbiont of MED cryptophytes, is studied from three aspects: qualitative, localization, quantitative, vertical and horizontal transmission. The effects of Rickettsia on the development of MED cryptospecies were investigated in three aspects: first, the fluorescence in situ hybridization of Rickettsia in MED cryptospecies with a specific probe labeled with fluorescent dye was performed to determine the distribution of Rickettsia in the developmental stages of MED cryptospecies. Because of the endogenous housekeeping gene, Rickettsia 16srDNA (16s ribosomal gene) was used as the target gene for fluorescence quantitative PCR to determine the copies of Rickettsia at different developmental stages of the MED cryptorch whitefly. The main results of this study are as follows: (1) Rickettsia is distributed in all developmental stages of the Cryptococcus tabaci. There are two types of distribution in the adult of Cryptococcus MED: inside and outside the cell. The distribution of the cell is spherical in shape and is in the abdomen of the whitefly. Specialized bacteria-containing cells aggregated in the outer position of the cells mainly refer to the formed eggs in the abdomen of Bemisia tabaci.Both distribution patterns are accompanied by the distribution of the primary symbiotic bacteria Portiera.Rickettsia also has two distribution positions in the fourth instar nymphs of cryptospecies MED, one is mainly distributed in the terminal discoid foramen and the tongue process, the other is mainly distributed in the terminal discoid foramen and the tongue process. It is distributed randomly in the nymph's chest and head; in the third, second and first instar cryptozoa, dish-shaped foramen and tongue-shaped process are mainly distributed in the end of the nymph's body; in the second instar nymph, it can also be distributed in the cell parts; in the first instar nymph, it can also be distributed in the antennae, trachea and mouth organs; Rickettsia in the egg of cryptozoa in MED. The distribution of Rickettsia was mainly in the pedicel and stalk of eggs. (2) Rickettsia had a great difference in the number of copies in the early stage of development and was relatively stable in the late stage of adult development. The content of Rickettsia increased gradually in the early stage of development, and reached the highest level in the second and the most vigorous stage of growth and development. The content of Rickettsia increased first and then decreased during the whole development process. (3) Rickettsia could be transmitted vertically with the mother generation, but could not be transmitted horizontally. The results showed that the vertical transmission rate of Rickettsia from female parent to offspring was about 98%, and the horizontal transmission rate from male Rickettsia to female Rickettsia was about 0%. Rickettsia could significantly increase the development rate of Bemisia tabaci at the first to third instar nymphs and early emergence stages, followed by female bacteria in eggs, fourth instar nymphs and early emergence stages. In addition, Rickettsia could significantly increase the sex ratio of offspring of Bemisia tabaci, increase the egg production of female, the emergence rate of offspring and the longevity of adults, but the effect was not significant. In summary, molecular biology techniques were used to study the different developmental stages of Rickettsia in MED cryptospecies. Distribution and copy number changes, and then using ecological methods to prove the transmission efficiency of Rickettsia in MED cryptophyte and its impact on host growth and development, the conclusions for the future study of Rickettsia in MED cryptophyte invasion of the specific role laid the foundation for revealing the secondary endosymbiotic bacteria in the host may be. It provides a basis for biological functions.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S433
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