小RNA深度测序在柑橘新发病毒的鉴定及TuMV诱导的抗病毒新机制中的研究
发布时间：2019-07-10 08:19
【摘要】：我国果树资源丰富,各类水果总产量位居世界首位,因此其经济重要性不言而喻。病毒病害可影响果树植株的正常生长,甚至给相关产业造成严重损失。果树病毒病害种类较多,其中部分病害的病原至今未能确认,因此果树病毒病害的检测与鉴定对于生产及防治至关重要。果树病毒往往缺乏相应的草本寄主,在木本植物内的复制积累量低,分布不均且具有季节性差异,存在潜伏侵染的现象,且果树组织中多富含多糖多酚等复杂成分,这些因素限制了传统病毒检测方法在果树病毒上的鉴定工作。高通量测序技术(如小RNA深度测序技术)的发展大大加快了果树病毒检测和鉴定的效率,用于可鉴定包括柑橘在内的果树未知病毒,挖掘更多病毒种类。小RNA(small RNA,sRNA)深度测序技术具有快速、准确及高通量等优点,为植物病毒快速检测鉴定的新方法。本研究利用sRNA深度测序技术,鉴定两种不同种类的柑橘病毒,并以此探讨该技术在果树病毒鉴定中的应用;同时本文将对病毒侵染寄主植物(拟南芥及柠檬)后sRNA表达谱进行分析,以期为果树病毒的致病机理的研究及其防治提供新思路。通过sRNA深度测序技术,从采自重庆地区的表现黄化脉明症状的柠檬样品中鉴定到柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)。通过序列拼接最终获得了该病毒全长基因组序列,研究发现其分离物CQ与CYVCV土耳其分离物Y1、云南分离物YN及巴基斯坦分离物PK的序列一致性分别为97.3%、98.8%及97.6%,同时对病毒编码的开放阅读框(Open reading frame,ORF)也进行了分析。聚类分析表明,分离物CQ属于甲型线性病毒科(Alphaflexiviridae)柑橘病毒属(Mandarivirus)。除此之外,本文首次分析了CYVCV侵染柠檬后的病毒小RNA(virus-derived small interfering RNAs,vsiRNAs)表达谱特征,CYVCV vsiRNAs以21-及22-nts两种为主,它们在CYVCV基因组上连续且不均匀分布,源于病毒负链的vsiRNAs丰度比正链高。本研究对CYVCV vsiRNAs 5’-端碱基偏好性也做了分析,结果暗示了这些vsiRNAs可能与柑橘体内不同的Argonaute(AGO)蛋白结合进而行使各种生物学功能。该样品为CYVCV单独侵染。柑橘褪绿矮缩病(Citrus chlorotic dwarf disease,CCDD)也是柑橘上发现的一种病毒病害,最早发现于土耳其。本章继续利用sRNA深度测序技术对采自中国云南地区的表现褪绿畸形症状的柠檬样品进行病原鉴定。对sRNA表达谱分析后发现,类似于CYVCV,源自于病毒的contigs几乎覆盖了分离物CCDaV-TK4(Citrus chlorotic dwarf-associated virus,CCDaV,GenBank No.JQ920490)的全基因组。通过引物设计及实验成功获得该病毒基因组全长信息,全长为3642-nts。序列分析发现其与分离物CCDaV-TK4的基因组核苷酸序列一致性高达99.3%,其基因组与其它双生病毒结构类似,如单链环状dna、茎环结构及其内部的九核苷酸序列等。聚类分析表明该分离物ccdav-cn001属于双生病毒科(geminivididae)。本研究同样分析了ccdavvsirnas的表达谱,vsirnas以22-、21-及24-nts为主,病毒正义链vsirnas的丰度要高于反义链。结合vsirnas表达谱,预测并验证了该病毒基因组反义链存在一个内含子结构。分离物ccdavcn001是ccdav的第二条全长基因组序列,其发现丰富了ccdav的序列信息。通过上述两种不同种类柑橘病毒的鉴定,表明了srna深度测序技术在果树病毒快速鉴定方面的优势,同时对两种病毒vsirnas表达谱的分析,进一步加深和推动了我们对柑橘抗病毒分子机理的理解。基因沉默是真核生物体内广泛存在的一种基于srna活性的基因表达调节机制,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对较少。本研究将继续利用小rna深度测序技术对拟南芥的抗病毒机理进行分析研究,以期为包含柑橘在内的果树抗病毒分子机理研究提供思路。本章研究对象为芜菁花叶病毒(turnipmosaicvirus,tumv-gfp),它是侵染十字花科植物的一种重要病毒。本研究通过对tumv-gfp侵染拟南芥后诱导的小rna表达谱进行分析,发现与黄瓜花叶病毒突变体病毒(cucumbermosaicvirus-Δ2b,cmv-Δ2b)侵染拟南芥的结果类似,tumv-gfp侵染拟南芥后也能够诱导一类21-ntssrna的富集,此类srna由拟南芥1000多个编码基因产生,主要集中于基因的编码区域和及核糖体rrna的负链,由于此类srna是由病毒诱导所致,因此它们被命名为vasirnas(virus-activatedsirnas)。进一步数据分析及杂交实验验证了vasirnas的合成依赖于rdr1(rna-dependentrnapolymerase1)及dcl4(dicer-like4)。拟南芥突变体ein5(ethylene-insensitive5)健康植株本身可以产生一类21-ntssrna,而当接种tumv-gfp后ein5中vasirnas的积累量明显提高,表明ein5可以增强vasirnas通路;tumvmrna在ein5植株中的积累量降低和在rdr1中积累量升高,表明此通路可能具有抗病毒活性。cmv-Δ2b与tumv-gfp侵染拟南芥后诱导的rdr1共同靶标基因有369种,表明两种病毒侵染拟南芥均可诱导上百种内源基因产生vasirnas,该结果暗示了拟南芥的vasirnas合成通路针对不同病毒的侵染可能具有普遍性。vasirnas可导致寄主内源基因表达下调,基因功能分析发现这些内源基因多与植物基本的生命活动相关,推测这些编码基因的表达为病毒提供了其复制增殖需要的能量及原料,寄主关闭这些内源基因或对病毒在寄主细胞内的复制增殖造成不利影响,从而起到抗病毒的作用。本研究试图探究拟南芥vasirnas通路在柑橘上是否同样具有普遍性,以cyvcv侵染柠檬后的小rna表达谱为研究对象,以甜橙作为参考基因组,分析发现来源于柠檬编码基因及rrna区域的21-ntssrna,其丰度在cyvcv侵染前后基本不变,该结果说明CYVCV侵染柠檬后,并没有产生类似于拟南芥vasi RNAs的通路或柠檬体内vasiRNAs通路不明显,其背后原因尚待进一步研究。该结果为vsiRNAs机制在果树寄主上的首次尝试。
[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.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S436.66
，
本文编号：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.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S436.66
，
本文编号：2512473
本文链接：https://www.wllwen.com/shoufeilunwen/nykjbs/2512473.html
最近更新
教材专著
