两种韧皮部杆菌媒介木虱内生原核生物和线粒体基因组研究
发布时间：2018-07-26 10:01
【摘要】：亚洲柑橘木虱Diaphorina citri和马铃薯木虱Bactericera cockerelli是农业生产上的重要害虫。其中亚洲柑橘木虱为候选的韧皮部杆菌“Candidatus Liberibacter asiaticus(CLas)”和“Ca.L.americanus”的媒介昆虫,该细菌与柑橘黄龙病(Citrus Huanglongbing)密切相关,给全世界柑橘生产造成严重威胁。马铃薯木虱主要为害马铃薯、番茄、辣椒等茄科植物,不仅造成“木虱黄叶病”,还是马铃薯斑马病(Zebra chip disease)病原“Ca.L.solanacearum(CLso)”的媒介昆虫。这两种病原菌均为难培养细菌,目前没有有效的药剂进行控制,因此对着两种媒介昆虫的研究和从基因组中获取有用的生物信息对防控柑橘黄龙病和马铃薯斑马病有着重要的指导作用。本文首先探索亚洲柑橘木虱和马铃薯木虱的人工饲养方法和获取韧皮部杆菌的规律,得到带菌量高的木虱DNA样品并用于第二代测序(Next Generation Sequencing,NGS),组装两种媒介昆虫所携带的内生原核生物和线粒体基因组,并进一步分析和应用。具体研究内容及结果如下:(1)通过比较九里香Murraya exotica、酸橘Citrus sunki和马水橘Citrus reticulate三种寄主不同成熟度的梢对柑橘木虱各龄若虫和成虫存活率的影响,发现柑橘木虱若虫的生长发育依赖于嫩梢,而成虫在成熟梢上的存活率高于嫩梢,且酸橘上饲养效果较好。通过研究两个番茄Solanum lycopersicum品种(“Big boy”和“Yellow pearl”)对马铃薯木虱若虫和成虫生物学特性的影响,发现马铃薯木虱在“Yellow pearl”上的4龄若虫和产卵前期更短,且若虫总存活率和成虫存活率显著高于“Big boy”。(2)亚洲柑橘木虱和马铃薯木虱的若虫期获菌率和获菌量均高于成虫期,分别在饲菌后第6 d和第12 d开始达到显著性差异。马铃薯木虱成虫获菌效率远高于亚洲柑橘木虱,第18 d已达90%,且CT值显著下降,而亚洲柑橘木虱只达到33.33%,CT值仍没有显著变化,此时马铃薯木虱的韧皮部杆菌拷贝数约为亚洲柑橘木虱的3倍。(3)通过两种木虱的测序文库进行组装得到内生原核生物基因组,其中亚洲柑橘木虱样本中获得柑橘黄龙病病原细菌CLas YCPsy株系,原生共生菌“Ca.Carsonella ruddii”YCCR株系,次生共生菌“Ca.Profftella armature”YCPA株系的染色体和质粒基因组;另外在该样本组装序列中找到与Wolbachia sp.相关的291个序列。马铃薯木虱样本中获得马铃薯斑马病病原细菌CLso RSTM株系,并首次报导了原生共生菌“Ca.C.ruddii”BC株系和与次生共生菌Wolbachia sp.相关的605个序列。(4)首次获得亚洲柑橘木虱和马铃薯木虱完整的线粒体基因组。其中采自美国加利福尼亚州的马铃薯木虱线粒体全基因组大小为15220 bp,采自中国广东省和美国加利福尼亚州的亚洲柑橘木虱线粒体全基因组分别为14996和15013 bp。三个线粒体基因组均含有13个蛋白编码基因、2个核糖体RNA基因、22个转移RNA基因和1个控制区。基因排序均与昆虫原始线粒体的顺序相同。通过对马铃薯木虱与Gen Bank数据库现有三种木虱(枸杞木虱Paratrioza sinica,木通木虱Cacopsylla coccinea和朴树木虱Pachypsylla venusta)线粒体基因组的13个基因的核酸多态性进行分析,发现cox1基因最保守;对这4个基因组控制区进行比对,发现马铃薯木虱多出一段260 bp的特异序列,并能形成3个茎环二级结构。基于13个蛋白编码基因构建系统发育树,其结果与基于形态学的分类结果一致。采自中国广东省和美国加州的亚洲柑橘木虱的线粒体基因组存在52个变异碱基。通过3种方法(cox1基因、trn Asn基因和利用3个nad基因的常规PCR)验证采自中国和美国多个地区的木虱样品,结果表明加利福尼亚州的亚洲柑橘木虱样品可能来源于美国东部地区,为当地黄龙病防控起到理论的指导作用。此外,通过两个地区的线粒体基因组的保守区域设计了20对引物,用于扩增采自全球各地区的亚洲柑橘木虱线粒体全基因组,并发现同个样品线粒体的控制区可能存在多种形态。完整的木虱线粒体基因组可更好地应用到种群多态性和系统发育分析当中,具有巨大的应用价值。
[Abstract]:The Asian citrus Diaphorina citri and the potato lice Bactericera cockerelli are important pests in agricultural production. The Asian citrus lice are the candidate phloem "Candidatus Liberibacter asiaticus (CLas)" and "Ca.L.americanus" medium insects, which are closely related to the citrus yellow dragon disease (Citrus Huanglongbing). Related, causing serious threat to the production of Citrus all over the world. Potato lice mainly harm potato, tomato, pepper and other Solanaceae plants, not only cause the "yellow leaf disease of lice", but also the vector of "Ca.L.solanacearum (CLso)", the pathogen of Zebra chip disease. These two pathogens are all difficult to cultivate bacteria. Effective drug control, so the study of two media insects and the acquisition of useful biological information from the genome have an important guiding role in preventing and controlling Citrus Yellow Dragon disease and potato zebra disease. This paper first explores the artificial feeding methods and the rules of phloem bacilli in the Asian citrus and the potato lice. The DNA samples with high bacteria number and second generation sequencing (Next Generation Sequencing, NGS) were used to assemble the endogenous prokaryotes and mitochondrial genome carried by two vectors, and further analyzed and applied. The specific contents and results were as follows: (1) by comparing the Murraya exotica, Citrus Sunki, and horse water tangerine C. The effects of itrus reticulate on the survival rate of nymphs and adults of citrus plant lice at all ages were found. It was found that the growth and development of the nymphs were dependent on the shoots, and the survival rate of the adults on the mature shoots was higher than that of the shoots, and the effect was better on the citrus fruit. Two tomato Solanum lycopersicum varieties ("Big Bo" were studied. "Big Bo" ("Big Bo") The effects of Y "and" Yellow pearl ") on the biological characteristics of the nymphs and adults of the potato lice found that the 4 age nymphs and proooprop of the" Yellow pearl "were shorter in the" Yellow pearl ", and the total survival rate and adult survival rate of the nymphs were significantly higher than that of" Big boy ". (2) the nymph of the Asian citrus and the potato lice The amount was higher than that of the adult period, and the significant difference between the sixth D and twelfth d after the feeding was reached. The yield of the adult potato lice was much higher than that of the Asian citrus fruit lice, eighteenth d had reached 90%, and the CT value decreased significantly, while the Asian Citrus lice reached only 33.33%, and the CT value still did not change significantly. 3 times of the Asian citrus lice. (3) the endophytic genome was assembled through the sequencing Library of two species of lice, of which the citrus yellow dragon disease pathogen CLas YCPsy strain, the Ca.Carsonella ruddii YCCR strain, and the secondary symbiotic "Ca.Profftella armature" YCPA strain were obtained. In addition, 291 sequences related to Wolbachia sp. were found in the assembly sequence of the sample. The CLso RSTM strain of potato zebra disease pathogenic bacteria was obtained from the sample of potato lice, and 605 sequences related to the primary symbiont "Ca.C.ruddii" BC strain and the secondary symbiotic Wolbachia sp. were reported for the first time. 4) the complete mitochondrial genome of the Asian citrus lice and the potato lice was obtained for the first time. The total genome size of the mitochondrial genome of the potato lice collected from California, USA was 15220 BP, and the whole genome of the citrus tree lice collected from Guangdong and California, China, was 14996 and 15013 bp., respectively, and three lines. The body genome contains 13 protein coding genes, 2 ribosome RNA genes, 22 transfer RNA genes and 1 control areas. The sequence of gene sequencing is the same as that of the insect original mitochondria. There are three species of lice (lcifberry lice Paratrioza Sinica, lice Cacopsylla coccinea and Pu Shu lice) of the potato lice and the Gen Bank database. Pachypsylla venusta) the nucleotide polymorphism of the 13 genes in the mitochondrial genome was analyzed and the cox1 gene was most conserved. The comparison of the 4 genomic control regions showed that the potato lice had a 260 BP specific sequence and could form 3 stem ring two structures. The phylogenetic tree was constructed based on 13 protein coding genes. The results coincide with the morphological based classification results. The mitochondrial genome of citrus citrus from Guangdong, China and California, China has 52 variants in the mitochondrial genome. 3 methods (cox1, TRN Asn, and 3 nad genes) were used to verify the sample of lice collected from many regions in China and the United States. The results showed that Calif. The Asian citrus tree lice may be derived from the eastern region of the United States to guide the prevention and control of Rehmannia disease. In addition, 20 pairs of primers were designed through the conserved regions of the mitochondrial genome in two regions to amplify the whole genome of the Asian citrus lice from all regions of the world and find the same sample. There may be a variety of morphologies in the control areas of the mitochondria. The complete mitochondrial genome of lice can be better applied to population polymorphism and phylogenetic analysis, which has great application value.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433
本文编号：2145639
[Abstract]:The Asian citrus Diaphorina citri and the potato lice Bactericera cockerelli are important pests in agricultural production. The Asian citrus lice are the candidate phloem "Candidatus Liberibacter asiaticus (CLas)" and "Ca.L.americanus" medium insects, which are closely related to the citrus yellow dragon disease (Citrus Huanglongbing). Related, causing serious threat to the production of Citrus all over the world. Potato lice mainly harm potato, tomato, pepper and other Solanaceae plants, not only cause the "yellow leaf disease of lice", but also the vector of "Ca.L.solanacearum (CLso)", the pathogen of Zebra chip disease. These two pathogens are all difficult to cultivate bacteria. Effective drug control, so the study of two media insects and the acquisition of useful biological information from the genome have an important guiding role in preventing and controlling Citrus Yellow Dragon disease and potato zebra disease. This paper first explores the artificial feeding methods and the rules of phloem bacilli in the Asian citrus and the potato lice. The DNA samples with high bacteria number and second generation sequencing (Next Generation Sequencing, NGS) were used to assemble the endogenous prokaryotes and mitochondrial genome carried by two vectors, and further analyzed and applied. The specific contents and results were as follows: (1) by comparing the Murraya exotica, Citrus Sunki, and horse water tangerine C. The effects of itrus reticulate on the survival rate of nymphs and adults of citrus plant lice at all ages were found. It was found that the growth and development of the nymphs were dependent on the shoots, and the survival rate of the adults on the mature shoots was higher than that of the shoots, and the effect was better on the citrus fruit. Two tomato Solanum lycopersicum varieties ("Big Bo" were studied. "Big Bo" ("Big Bo") The effects of Y "and" Yellow pearl ") on the biological characteristics of the nymphs and adults of the potato lice found that the 4 age nymphs and proooprop of the" Yellow pearl "were shorter in the" Yellow pearl ", and the total survival rate and adult survival rate of the nymphs were significantly higher than that of" Big boy ". (2) the nymph of the Asian citrus and the potato lice The amount was higher than that of the adult period, and the significant difference between the sixth D and twelfth d after the feeding was reached. The yield of the adult potato lice was much higher than that of the Asian citrus fruit lice, eighteenth d had reached 90%, and the CT value decreased significantly, while the Asian Citrus lice reached only 33.33%, and the CT value still did not change significantly. 3 times of the Asian citrus lice. (3) the endophytic genome was assembled through the sequencing Library of two species of lice, of which the citrus yellow dragon disease pathogen CLas YCPsy strain, the Ca.Carsonella ruddii YCCR strain, and the secondary symbiotic "Ca.Profftella armature" YCPA strain were obtained. In addition, 291 sequences related to Wolbachia sp. were found in the assembly sequence of the sample. The CLso RSTM strain of potato zebra disease pathogenic bacteria was obtained from the sample of potato lice, and 605 sequences related to the primary symbiont "Ca.C.ruddii" BC strain and the secondary symbiotic Wolbachia sp. were reported for the first time. 4) the complete mitochondrial genome of the Asian citrus lice and the potato lice was obtained for the first time. The total genome size of the mitochondrial genome of the potato lice collected from California, USA was 15220 BP, and the whole genome of the citrus tree lice collected from Guangdong and California, China, was 14996 and 15013 bp., respectively, and three lines. The body genome contains 13 protein coding genes, 2 ribosome RNA genes, 22 transfer RNA genes and 1 control areas. The sequence of gene sequencing is the same as that of the insect original mitochondria. There are three species of lice (lcifberry lice Paratrioza Sinica, lice Cacopsylla coccinea and Pu Shu lice) of the potato lice and the Gen Bank database. Pachypsylla venusta) the nucleotide polymorphism of the 13 genes in the mitochondrial genome was analyzed and the cox1 gene was most conserved. The comparison of the 4 genomic control regions showed that the potato lice had a 260 BP specific sequence and could form 3 stem ring two structures. The phylogenetic tree was constructed based on 13 protein coding genes. The results coincide with the morphological based classification results. The mitochondrial genome of citrus citrus from Guangdong, China and California, China has 52 variants in the mitochondrial genome. 3 methods (cox1, TRN Asn, and 3 nad genes) were used to verify the sample of lice collected from many regions in China and the United States. The results showed that Calif. The Asian citrus tree lice may be derived from the eastern region of the United States to guide the prevention and control of Rehmannia disease. In addition, 20 pairs of primers were designed through the conserved regions of the mitochondrial genome in two regions to amplify the whole genome of the Asian citrus lice from all regions of the world and find the same sample. There may be a variety of morphologies in the control areas of the mitochondria. The complete mitochondrial genome of lice can be better applied to population polymorphism and phylogenetic analysis, which has great application value.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433
【参考文献】
相关期刊论文 前1条
1 范国成;刘波;吴如健;李韬;蔡子坚;柯冲;;中国柑橘黄龙病研究30年[J];福建农业学报;2009年02期
，本文编号：2145639
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