兩種韌皮部桿菌媒介木虱內生原核生物和線粒體基因組研究
發(fā)布時間：2018-07-26 10:01
【摘要】：亞洲柑橘木虱Diaphorina citri和馬鈴薯木虱Bactericera cockerelli是農業(yè)生產上的重要害蟲。其中亞洲柑橘木虱為候選的韌皮部桿菌“Candidatus Liberibacter asiaticus(CLas)”和“Ca.L.americanus”的媒介昆蟲,該細菌與柑橘黃龍病(Citrus Huanglongbing)密切相關,給全世界柑橘生產造成嚴重威脅。馬鈴薯木虱主要為害馬鈴薯、番茄、辣椒等茄科植物,不僅造成“木虱黃葉病”,還是馬鈴薯斑馬病(Zebra chip disease)病原“Ca.L.solanacearum(CLso)”的媒介昆蟲。這兩種病原菌均為難培養(yǎng)細菌,目前沒有有效的藥劑進行控制,因此對著兩種媒介昆蟲的研究和從基因組中獲取有用的生物信息對防控柑橘黃龍病和馬鈴薯斑馬病有著重要的指導作用。本文首先探索亞洲柑橘木虱和馬鈴薯木虱的人工飼養(yǎng)方法和獲取韌皮部桿菌的規(guī)律,得到帶菌量高的木虱DNA樣品并用于第二代測序(Next Generation Sequencing,NGS),組裝兩種媒介昆蟲所攜帶的內生原核生物和線粒體基因組,并進一步分析和應用。具體研究內容及結果如下:(1)通過比較九里香Murraya exotica、酸橘Citrus sunki和馬水橘Citrus reticulate三種寄主不同成熟度的梢對柑橘木虱各齡若蟲和成蟲存活率的影響,發(fā)現(xiàn)柑橘木虱若蟲的生長發(fā)育依賴于嫩梢,而成蟲在成熟梢上的存活率高于嫩梢,且酸橘上飼養(yǎng)效果較好。通過研究兩個番茄Solanum lycopersicum品種(“Big boy”和“Yellow pearl”)對馬鈴薯木虱若蟲和成蟲生物學特性的影響,發(fā)現(xiàn)馬鈴薯木虱在“Yellow pearl”上的4齡若蟲和產卵前期更短,且若蟲總存活率和成蟲存活率顯著高于“Big boy”。(2)亞洲柑橘木虱和馬鈴薯木虱的若蟲期獲菌率和獲菌量均高于成蟲期,分別在飼菌后第6 d和第12 d開始達到顯著性差異。馬鈴薯木虱成蟲獲菌效率遠高于亞洲柑橘木虱,第18 d已達90%,且CT值顯著下降,而亞洲柑橘木虱只達到33.33%,CT值仍沒有顯著變化,此時馬鈴薯木虱的韌皮部桿菌拷貝數(shù)約為亞洲柑橘木虱的3倍。(3)通過兩種木虱的測序文庫進行組裝得到內生原核生物基因組,其中亞洲柑橘木虱樣本中獲得柑橘黃龍病病原細菌CLas YCPsy株系,原生共生菌“Ca.Carsonella ruddii”YCCR株系,次生共生菌“Ca.Profftella armature”YCPA株系的染色體和質�；蚪M;另外在該樣本組裝序列中找到與Wolbachia sp.相關的291個序列。馬鈴薯木虱樣本中獲得馬鈴薯斑馬病病原細菌CLso RSTM株系,并首次報導了原生共生菌“Ca.C.ruddii”BC株系和與次生共生菌Wolbachia sp.相關的605個序列。(4)首次獲得亞洲柑橘木虱和馬鈴薯木虱完整的線粒體基因組。其中采自美國加利福尼亞州的馬鈴薯木虱線粒體全基因組大小為15220 bp,采自中國廣東省和美國加利福尼亞州的亞洲柑橘木虱線粒體全基因組分別為14996和15013 bp。三個線粒體基因組均含有13個蛋白編碼基因、2個核糖體RNA基因、22個轉移RNA基因和1個控制區(qū)�；蚺判蚓c昆蟲原始線粒體的順序相同。通過對馬鈴薯木虱與Gen Bank數(shù)據(jù)庫現(xiàn)有三種木虱(枸杞木虱Paratrioza sinica,木通木虱Cacopsylla coccinea和樸樹木虱Pachypsylla venusta)線粒體基因組的13個基因的核酸多態(tài)性進行分析,發(fā)現(xiàn)cox1基因最保守;對這4個基因組控制區(qū)進行比對,發(fā)現(xiàn)馬鈴薯木虱多出一段260 bp的特異序列,并能形成3個莖環(huán)二級結構。基于13個蛋白編碼基因構建系統(tǒng)發(fā)育樹,其結果與基于形態(tài)學的分類結果一致。采自中國廣東省和美國加州的亞洲柑橘木虱的線粒體基因組存在52個變異堿基。通過3種方法(cox1基因、trn Asn基因和利用3個nad基因的常規(guī)PCR)驗證采自中國和美國多個地區(qū)的木虱樣品,結果表明加利福尼亞州的亞洲柑橘木虱樣品可能來源于美國東部地區(qū),為當?shù)攸S龍病防控起到理論的指導作用。此外,通過兩個地區(qū)的線粒體基因組的保守區(qū)域設計了20對引物,用于擴增采自全球各地區(qū)的亞洲柑橘木虱線粒體全基因組,并發(fā)現(xiàn)同個樣品線粒體的控制區(qū)可能存在多種形態(tài)。完整的木虱線粒體基因組可更好地應用到種群多態(tài)性和系統(tǒng)發(fā)育分析當中,具有巨大的應用價值。
[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.
【學位授予單位】：華南農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S433
本文編號：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.
【學位授予單位】：華南農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S433
【參考文獻】
相關期刊論文 前1條
1 范國成;劉波;吳如健;李韜;蔡子堅;柯沖;;中國柑橘黃龍病研究30年[J];福建農業(yè)學報;2009年02期
，本文編號：2145639
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