小RNA深度測序在柑橘新發(fā)病毒的鑒定及TuMV誘導(dǎo)的抗病毒新機制中的研究
發(fā)布時間：2019-07-10 08:19
【摘要】：我國果樹資源豐富,各類水果總產(chǎn)量位居世界首位,因此其經(jīng)濟重要性不言而喻。病毒病害可影響果樹植株的正常生長,甚至給相關(guān)產(chǎn)業(yè)造成嚴(yán)重損失。果樹病毒病害種類較多,其中部分病害的病原至今未能確認,因此果樹病毒病害的檢測與鑒定對于生產(chǎn)及防治至關(guān)重要。果樹病毒往往缺乏相應(yīng)的草本寄主,在木本植物內(nèi)的復(fù)制積累量低,分布不均且具有季節(jié)性差異,存在潛伏侵染的現(xiàn)象,且果樹組織中多富含多糖多酚等復(fù)雜成分,這些因素限制了傳統(tǒng)病毒檢測方法在果樹病毒上的鑒定工作。高通量測序技術(shù)(如小RNA深度測序技術(shù))的發(fā)展大大加快了果樹病毒檢測和鑒定的效率,用于可鑒定包括柑橘在內(nèi)的果樹未知病毒,挖掘更多病毒種類。小RNA(small RNA,sRNA)深度測序技術(shù)具有快速、準(zhǔn)確及高通量等優(yōu)點,為植物病毒快速檢測鑒定的新方法。本研究利用sRNA深度測序技術(shù),鑒定兩種不同種類的柑橘病毒,并以此探討該技術(shù)在果樹病毒鑒定中的應(yīng)用;同時本文將對病毒侵染寄主植物(擬南芥及檸檬)后sRNA表達譜進行分析,以期為果樹病毒的致病機理的研究及其防治提供新思路。通過sRNA深度測序技術(shù),從采自重慶地區(qū)的表現(xiàn)黃化脈明癥狀的檸檬樣品中鑒定到柑橘黃化脈明病毒(Citrus yellow vein clearing virus,CYVCV)。通過序列拼接最終獲得了該病毒全長基因組序列,研究發(fā)現(xiàn)其分離物CQ與CYVCV土耳其分離物Y1、云南分離物YN及巴基斯坦分離物PK的序列一致性分別為97.3%、98.8%及97.6%,同時對病毒編碼的開放閱讀框(Open reading frame,ORF)也進行了分析。聚類分析表明,分離物CQ屬于甲型線性病毒科(Alphaflexiviridae)柑橘病毒屬(Mandarivirus)。除此之外,本文首次分析了CYVCV侵染檸檬后的病毒小RNA(virus-derived small interfering RNAs,vsiRNAs)表達譜特征,CYVCV vsiRNAs以21-及22-nts兩種為主,它們在CYVCV基因組上連續(xù)且不均勻分布,源于病毒負鏈的vsiRNAs豐度比正鏈高。本研究對CYVCV vsiRNAs 5’-端堿基偏好性也做了分析,結(jié)果暗示了這些vsiRNAs可能與柑橘體內(nèi)不同的Argonaute(AGO)蛋白結(jié)合進而行使各種生物學(xué)功能。該樣品為CYVCV單獨侵染。柑橘褪綠矮縮病(Citrus chlorotic dwarf disease,CCDD)也是柑橘上發(fā)現(xiàn)的一種病毒病害,最早發(fā)現(xiàn)于土耳其。本章繼續(xù)利用sRNA深度測序技術(shù)對采自中國云南地區(qū)的表現(xiàn)褪綠畸形癥狀的檸檬樣品進行病原鑒定。對sRNA表達譜分析后發(fā)現(xiàn),類似于CYVCV,源自于病毒的contigs幾乎覆蓋了分離物CCDaV-TK4(Citrus chlorotic dwarf-associated virus,CCDaV,GenBank No.JQ920490)的全基因組。通過引物設(shè)計及實驗成功獲得該病毒基因組全長信息,全長為3642-nts。序列分析發(fā)現(xiàn)其與分離物CCDaV-TK4的基因組核苷酸序列一致性高達99.3%,其基因組與其它雙生病毒結(jié)構(gòu)類似,如單鏈環(huán)狀dna、莖環(huán)結(jié)構(gòu)及其內(nèi)部的九核苷酸序列等。聚類分析表明該分離物ccdav-cn001屬于雙生病毒科(geminivididae)。本研究同樣分析了ccdavvsirnas的表達譜,vsirnas以22-、21-及24-nts為主,病毒正義鏈vsirnas的豐度要高于反義鏈。結(jié)合vsirnas表達譜,預(yù)測并驗證了該病毒基因組反義鏈存在一個內(nèi)含子結(jié)構(gòu)。分離物ccdavcn001是ccdav的第二條全長基因組序列,其發(fā)現(xiàn)豐富了ccdav的序列信息。通過上述兩種不同種類柑橘病毒的鑒定,表明了srna深度測序技術(shù)在果樹病毒快速鑒定方面的優(yōu)勢,同時對兩種病毒vsirnas表達譜的分析,進一步加深和推動了我們對柑橘抗病毒分子機理的理解�；虺聊钦婧松矬w內(nèi)廣泛存在的一種基于srna活性的基因表達調(diào)節(jié)機制,目前抗病毒基因沉默的研究多集中在草本植物上,木本植物上的研究相對較少。本研究將繼續(xù)利用小rna深度測序技術(shù)對擬南芥的抗病毒機理進行分析研究,以期為包含柑橘在內(nèi)的果樹抗病毒分子機理研究提供思路。本章研究對象為蕪菁花葉病毒(turnipmosaicvirus,tumv-gfp),它是侵染十字花科植物的一種重要病毒。本研究通過對tumv-gfp侵染擬南芥后誘導(dǎo)的小rna表達譜進行分析,發(fā)現(xiàn)與黃瓜花葉病毒突變體病毒(cucumbermosaicvirus-Δ2b,cmv-Δ2b)侵染擬南芥的結(jié)果類似,tumv-gfp侵染擬南芥后也能夠誘導(dǎo)一類21-ntssrna的富集,此類srna由擬南芥1000多個編碼基因產(chǎn)生,主要集中于基因的編碼區(qū)域和及核糖體rrna的負鏈,由于此類srna是由病毒誘導(dǎo)所致,因此它們被命名為vasirnas(virus-activatedsirnas)。進一步數(shù)據(jù)分析及雜交實驗驗證了vasirnas的合成依賴于rdr1(rna-dependentrnapolymerase1)及dcl4(dicer-like4)。擬南芥突變體ein5(ethylene-insensitive5)健康植株本身可以產(chǎn)生一類21-ntssrna,而當(dāng)接種tumv-gfp后ein5中vasirnas的積累量明顯提高,表明ein5可以增強vasirnas通路;tumvmrna在ein5植株中的積累量降低和在rdr1中積累量升高,表明此通路可能具有抗病毒活性。cmv-Δ2b與tumv-gfp侵染擬南芥后誘導(dǎo)的rdr1共同靶標(biāo)基因有369種,表明兩種病毒侵染擬南芥均可誘導(dǎo)上百種內(nèi)源基因產(chǎn)生vasirnas,該結(jié)果暗示了擬南芥的vasirnas合成通路針對不同病毒的侵染可能具有普遍性。vasirnas可導(dǎo)致寄主內(nèi)源基因表達下調(diào),基因功能分析發(fā)現(xiàn)這些內(nèi)源基因多與植物基本的生命活動相關(guān),推測這些編碼基因的表達為病毒提供了其復(fù)制增殖需要的能量及原料,寄主關(guān)閉這些內(nèi)源基因或?qū)Σ《驹诩闹骷毎麅?nèi)的復(fù)制增殖造成不利影響,從而起到抗病毒的作用。本研究試圖探究擬南芥vasirnas通路在柑橘上是否同樣具有普遍性,以cyvcv侵染檸檬后的小rna表達譜為研究對象,以甜橙作為參考基因組,分析發(fā)現(xiàn)來源于檸檬編碼基因及rrna區(qū)域的21-ntssrna,其豐度在cyvcv侵染前后基本不變,該結(jié)果說明CYVCV侵染檸檬后,并沒有產(chǎn)生類似于擬南芥vasi RNAs的通路或檸檬體內(nèi)vasiRNAs通路不明顯,其背后原因尚待進一步研究。該結(jié)果為vsiRNAs機制在果樹寄主上的首次嘗試。
[Abstract]:The fruit tree resources of our country are rich, the total output of all kinds of fruit is the first place in the world, so its economic importance is self-evident. The virus disease can affect the normal growth of the fruit tree plant, and even cause serious loss to the related industry. The disease of fruit tree virus is more and more, and the pathogen of some diseases has not been confirmed so far, so the detection and identification of the disease of fruit tree virus is of great importance to the production and control. The fruit tree virus often lacks a corresponding herbal host, the replication accumulation amount in the woody plant is low, the distribution is uneven, and the fruit tree virus has the seasonal difference, the latent infection phenomenon exists, and the fruit tree tissue is rich in complex components such as polysaccharide polyphenol and the like, These factors limit the identification of traditional virus detection methods on fruit trees. The development of high-throughput sequencing technology, such as small-RNA deep sequencing technology, has greatly accelerated the efficiency of fruit tree virus detection and identification, and is used for identifying the unknown viruses of fruit trees, including citrus, and digging more virus types. Small RNA (sRNA) deep sequencing technology has the advantages of fast, accurate and high throughput, and is a new method for rapid detection and identification of plant virus. In this study, two different kinds of citrus viruses were identified by the technique of deep sequencing of sRNA, and the application of the technology in the identification of fruit trees was discussed, and the expression profiles of sRNA in the host plants (Arabidopsis and lemon) infected by the virus were analyzed. So as to provide a new thought for the research and prevention of the pathogenic mechanism of the fruit tree virus. Citrus yelow vein clear virus (CYVCV) was identified by sRNA deep sequencing technology from a lemon sample with a yellow-and-dark condition in the area of Chongqing. The whole-length genome sequence of the virus was obtained by sequence splicing, and the sequence consistency of the isolate CQ and CYVCV Turkey isolate Y1, Yunnan isolate YN and the isolate PK of Pakistan was 97.3%, 98.8% and 97.6%, respectively. The open reading frame (ORF) of the virus code was also analyzed. The cluster analysis shows that the isolates CQ belong to the genus Alphafexiiridae. In addition, for the first time, the expression profile of the virus-derived small RNA (vsiRNAs) infected by CYVCV was analyzed, and the CYVCV vsiRNAs were mainly composed of 21-and 22-nts, and they were not uniformly distributed on the CYVCV genome, and the vsiRNAs derived from the negative chain of the virus were higher than that of the positive chain. This study also made an analysis of the 5 '-terminal base preference of CYVCV vsiRNAs, and the results suggested that these vsiRNAs might be combined with different Argonaute (AGO) proteins in the citrus to exercise various biological functions. The sample was a single infection of CYVCV. Citrus chlorotic dwarf disease, CCDD is also a virus disease found on the citrus, and is the first to be found in Turkey. This chapter continues to use the sRNA deep sequencing technology to identify the lemon sample of the symptoms of chlorosis in Yunnan, China. It is found that similar to CYVCV, The whole genome of the isolated CCDaV-TK4 (CCDaV-TK4-associated virus, CCDaV, GenBank No. JQ920490) was almost covered with the virus-derived constigs. The full-length information of the viral genome was obtained by the primer design and experiment, and the total length was 3642-nts. In this study, the expression profile of ccdavvsirnas, vsirnas is mainly 22-,21-and 24-nts. The vsirnas abundance of the viral justice chain is higher than that of the antisense strand, and an intron structure of the antisense strand of the viral genome is predicted and verified in combination with the vsirnas expression profile. by the identification of the two different kinds of citrus viruses, the advantages of the srna deep sequencing technology in the rapid identification of the fruit tree virus are demonstrated, and the analysis of the expression profiles of the two viruses vsirnas, It further deepens and promotes our understanding of the mechanism of the anti-viral molecule of citrus. The gene silencing is a kind of gene expression regulation mechanism based on the srna activity, which is widely present in the eukaryotes, and the research on the anti-virus gene silencing is mainly on the herbaceous plants, In this study, the anti-viral mechanism of Arabidopsis thaliana was studied by using the small rna depth sequencing technology, in order to provide a train of thought for the study of the anti-viral molecular mechanism of fruit trees, including citrus. Tuumv-gfp), which is an important virus that infects the cruciferae, has been found to be similar to that of the cucumber mosaic virus mutant virus (cuckumbermosaicvirus-v2b, cmv-v2b), by analyzing the expression profile of the small rna induced by tuumv-gfp in the presence of arabidopsis thaliana. tuumv-gfp can also induce the enrichment of a class of 21-ntssrna, which is produced by more than 1000 coding genes in arabidopsis, which is mainly concentrated in the coding region of the gene and the negative chain of the ribosomal rna, as such srna is caused by the virus induction, So they were named vasirnas (virus-activated sirnas). Further data analysis and hybridization experiments demonstrated that the synthesis of vasirnas was dependent on rna-dependent and dcl4 (dicer-like4). The accumulation of tuumvmrna in the ein5 plants and the increase in the amount of accumulation in rdr1 indicate that this pathway may have antiviral activity. the results suggest that the viasirnas synthesis pathway of arabidopsis may be universal for the infection of different viruses. vasirnas may result in down-regulation of endogenous gene expression of the host, It is presumed that the expression of these coding genes provides the energy and the raw materials required for the replication and proliferation of the virus, the host closes these endogenous genes or has an adverse effect on the replication and proliferation of the virus within the host cell, so as to play an anti-virus role, and the research attempts to explore whether the vasirnas pathway of arabidopsis has the same universality on the citrus, The results show that the vsiRNAs mechanism is the first attempt of the vsiRNAs mechanism on the host of fruit trees.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S436.66
，
本文編號：2512473
[Abstract]:The fruit tree resources of our country are rich, the total output of all kinds of fruit is the first place in the world, so its economic importance is self-evident. The virus disease can affect the normal growth of the fruit tree plant, and even cause serious loss to the related industry. The disease of fruit tree virus is more and more, and the pathogen of some diseases has not been confirmed so far, so the detection and identification of the disease of fruit tree virus is of great importance to the production and control. The fruit tree virus often lacks a corresponding herbal host, the replication accumulation amount in the woody plant is low, the distribution is uneven, and the fruit tree virus has the seasonal difference, the latent infection phenomenon exists, and the fruit tree tissue is rich in complex components such as polysaccharide polyphenol and the like, These factors limit the identification of traditional virus detection methods on fruit trees. The development of high-throughput sequencing technology, such as small-RNA deep sequencing technology, has greatly accelerated the efficiency of fruit tree virus detection and identification, and is used for identifying the unknown viruses of fruit trees, including citrus, and digging more virus types. Small RNA (sRNA) deep sequencing technology has the advantages of fast, accurate and high throughput, and is a new method for rapid detection and identification of plant virus. In this study, two different kinds of citrus viruses were identified by the technique of deep sequencing of sRNA, and the application of the technology in the identification of fruit trees was discussed, and the expression profiles of sRNA in the host plants (Arabidopsis and lemon) infected by the virus were analyzed. So as to provide a new thought for the research and prevention of the pathogenic mechanism of the fruit tree virus. Citrus yelow vein clear virus (CYVCV) was identified by sRNA deep sequencing technology from a lemon sample with a yellow-and-dark condition in the area of Chongqing. The whole-length genome sequence of the virus was obtained by sequence splicing, and the sequence consistency of the isolate CQ and CYVCV Turkey isolate Y1, Yunnan isolate YN and the isolate PK of Pakistan was 97.3%, 98.8% and 97.6%, respectively. The open reading frame (ORF) of the virus code was also analyzed. The cluster analysis shows that the isolates CQ belong to the genus Alphafexiiridae. In addition, for the first time, the expression profile of the virus-derived small RNA (vsiRNAs) infected by CYVCV was analyzed, and the CYVCV vsiRNAs were mainly composed of 21-and 22-nts, and they were not uniformly distributed on the CYVCV genome, and the vsiRNAs derived from the negative chain of the virus were higher than that of the positive chain. This study also made an analysis of the 5 '-terminal base preference of CYVCV vsiRNAs, and the results suggested that these vsiRNAs might be combined with different Argonaute (AGO) proteins in the citrus to exercise various biological functions. The sample was a single infection of CYVCV. Citrus chlorotic dwarf disease, CCDD is also a virus disease found on the citrus, and is the first to be found in Turkey. This chapter continues to use the sRNA deep sequencing technology to identify the lemon sample of the symptoms of chlorosis in Yunnan, China. It is found that similar to CYVCV, The whole genome of the isolated CCDaV-TK4 (CCDaV-TK4-associated virus, CCDaV, GenBank No. JQ920490) was almost covered with the virus-derived constigs. The full-length information of the viral genome was obtained by the primer design and experiment, and the total length was 3642-nts. In this study, the expression profile of ccdavvsirnas, vsirnas is mainly 22-,21-and 24-nts. The vsirnas abundance of the viral justice chain is higher than that of the antisense strand, and an intron structure of the antisense strand of the viral genome is predicted and verified in combination with the vsirnas expression profile. by the identification of the two different kinds of citrus viruses, the advantages of the srna deep sequencing technology in the rapid identification of the fruit tree virus are demonstrated, and the analysis of the expression profiles of the two viruses vsirnas, It further deepens and promotes our understanding of the mechanism of the anti-viral molecule of citrus. The gene silencing is a kind of gene expression regulation mechanism based on the srna activity, which is widely present in the eukaryotes, and the research on the anti-virus gene silencing is mainly on the herbaceous plants, In this study, the anti-viral mechanism of Arabidopsis thaliana was studied by using the small rna depth sequencing technology, in order to provide a train of thought for the study of the anti-viral molecular mechanism of fruit trees, including citrus. Tuumv-gfp), which is an important virus that infects the cruciferae, has been found to be similar to that of the cucumber mosaic virus mutant virus (cuckumbermosaicvirus-v2b, cmv-v2b), by analyzing the expression profile of the small rna induced by tuumv-gfp in the presence of arabidopsis thaliana. tuumv-gfp can also induce the enrichment of a class of 21-ntssrna, which is produced by more than 1000 coding genes in arabidopsis, which is mainly concentrated in the coding region of the gene and the negative chain of the ribosomal rna, as such srna is caused by the virus induction, So they were named vasirnas (virus-activated sirnas). Further data analysis and hybridization experiments demonstrated that the synthesis of vasirnas was dependent on rna-dependent and dcl4 (dicer-like4). The accumulation of tuumvmrna in the ein5 plants and the increase in the amount of accumulation in rdr1 indicate that this pathway may have antiviral activity. the results suggest that the viasirnas synthesis pathway of arabidopsis may be universal for the infection of different viruses. vasirnas may result in down-regulation of endogenous gene expression of the host, It is presumed that the expression of these coding genes provides the energy and the raw materials required for the replication and proliferation of the virus, the host closes these endogenous genes or has an adverse effect on the replication and proliferation of the virus within the host cell, so as to play an anti-virus role, and the research attempts to explore whether the vasirnas pathway of arabidopsis has the same universality on the citrus, The results show that the vsiRNAs mechanism is the first attempt of the vsiRNAs mechanism on the host of fruit trees.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S436.66
，
本文編號：2512473
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