次生内共生菌Rickettsia在烟粉虱MED隐种内的分布、传播以及对宿主生殖影响研究
发布时间：2018-09-18 15:55
【摘要】：烟粉虱Bemisia tabaci(Gennadius)是由35个遗传结构差别明显但是形态上无法区分的隐种所构成的复合种,其中烟粉虱地中海隐种(简称MED隐种,又称Q型)和小亚细亚隐种(简称MEAM1隐种,又称B型)是危害我国的主要入侵烟粉虱。2008年至今,MED隐种在中国大部分地区逐渐替代MEAM1隐种成为优势种,其中次生内共生菌(facultative symbiont)在MED隐种扩散和入侵方面发挥了重要作用,本论文以MED隐种的次生内共生菌Rickettsia为研究对象,从定性、定位、定量三个层面,垂直传播、水平传播、生物学影响三个方面,探究Rickettsia对MED隐种所产生的影响。首先利用标有荧光染料的特异性探针对MED隐种烟粉虱体内的Rickettsia进行荧光原位杂交,明确Rickettsia在MED隐种烟粉虱各发育阶段的分布。然后利用TaqMan探针法,以烟粉虱β-Actin基因为内源管家基因,Rickettsia 16srDNA(16s核糖体基因)为目的基因进行荧光定量PCR,明确MED隐种烟粉虱各发育阶段Rickettsia的拷贝数变化。同时进行三个独立生态实验:1、Rickettsia在MED隐种烟粉虱体内的垂直传播;2、Rickettsia在MED隐种烟粉虱体内的水平传播;3、Rickettsia对MED隐种烟粉虱生活史的影响。本论文主要研究结果如下:(1)Rickettsia在MED隐种烟粉虱体内各发育阶段都有分布。在MED隐种成虫体内的分布位置有两种:菌胞内部和菌胞外部。菌胞内部分布位置呈球形,是烟粉虱腹部特化的含菌细胞聚集而成的,菌胞外部位置主要是指烟粉虱腹部已成型的卵内。两种分布方式皆伴随有初生共生菌Portiera的分布;Rickettsia在MED隐种四龄若虫体内也存在两种分布位置,一种是主要分布在末端皿状孔和舌状突部位,另一种是随机分布在若虫胸部和头部;在MED隐种三龄、二龄、一龄若虫体内的分布均以虫体末端皿状孔和舌状突部位为主,其中,在二龄若虫体内还可分布于菌胞部位,在一龄若虫体内还可分布于触角、气管和口器等部位;Rickettsia在MED隐种卵内的分布位置主要在卵梗和卵柄部位。(2)Rickettsia在MED隐种烟粉虱成虫发育前期拷贝数变化差别较大,在成虫发育后期相对稳定。Rickettsia在整个发育初期含量是逐渐上升的,二龄时期同时也是生长发育最旺盛的时期达到了最高含量,三龄时期含量骤减达到最低点,随后出现回升,羽化5天达到第二个小高峰,随后保持在略高于初羽化时期的含菌量。从整个发育过程中来看,Rickettsia的含量整体趋势是先增加后减少。(3)Rickettsia在MED隐种内可随母代垂直传播,但无法水平传播。结果发现Rickettsia从雌亲代至后代的垂直传播率约为98%,从含有Rickettsia雄虫至不含Rickettsia雌虫的水平传播率约为0%。Rickettsia可以显著增加一龄到三龄若虫以及初羽化阶段烟粉虱的发育速率,其次含菌雌虫在卵、四龄若虫、初羽化时期发育速率均显著高于含菌雄虫。此外,Rickettsia可以显著提高烟粉虱后代性比,还能提高雌虫产卵量、后代羽化率以及成虫寿命,但影响并不显著。综上所述,本研究利用分子生物学技术,从分子水平上研究了Rickettsia在MED隐种不同发育阶段的分布位置以及拷贝数的变化,然后利用生态学方法,证明了Rickettsia在MED隐种内的传播效率及其对宿主生长发育的影响,所得结论为今后探究Rickettsia在MED隐种入侵方面所起的具体作用奠定基础,为揭示次生内共生菌在宿主体内可能所起的生物学功能提供依据。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433
本文编号：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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433
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