【作者】 宋震；

【导师】 周常勇； 李中安；

【作者基本信息】 西南大学 ， 植物病理学， 2013， 博士

【摘要】 柑橘是世界第一大水果。我国柑橘栽培面积超过290万公顷,产量达3000万吨,均居世界第一位。随着克里曼丁红橘(Citrus clementina Hort. ex Tan)和甜橙(C. sinensis cv. Valencia)全基因组序列的公布,以及柑橘抗病、抗逆等相关基因序列表达标签(Expressed sequence tags, EST)的大量累积,柑橘基因功能解析已成为目前的研究热点和挑战之一。病毒诱导的基因沉默(Virus-induced gene silencing, VIGS)是近年来发展的一种基因功能研究工具,与转基因、基因敲除、反义抑制等常用生物技术相比,试验周期短,不需要遗传转化,具有操作简便、成本低、高通量等优势。利用VIGS技术有望克服柑橘这种多年生木本作物童期长、遗传转化困难等瓶颈,加速其基因功能研究的进展。为此,本研究在柑橘碎叶病毒(Citrus tatter leaf virus, CTLV)检测方法及多态性研究的基础上,筛选CTLV分离株进行全基因组cDNA扩增、侵染性克隆构建,并进一步开展了在模式植物本生烟(Nicotiana benthamiana)上建立VIGS体系的研究,以期为在柑橘上实施VIGS进而研究其基因功能奠定基础。主要研究结果如下：1. CTLV一步法及巢式RT-PCR检测体系建立通过引物比对,建立了CTLV的一步法及巢式RT-PCR检测体系。其中,一步法RT-PCR产物889bp,包含整个CP及3’UTR序列,比目前的常规RT-PCR操作简便,污染几率小；巢式RT-PCR检测灵敏度高,比一步法RT-PCR至少提高100倍,检测模板总核酸的最低浓度约1.27pg/μL。2. CTLV多态性分析建立了CTLV分子变异的Hinf Ⅰ/RFLP检测体系,定义RFLP组群9个,发现CTLV以单一RFLP组群侵染为主,其中RFLP Ⅰ和RFLP Ⅱ组群可能是优势流行组群,但也存在混合侵染。该体系简便、快速、重复性好,可用于大规模样品的分子变异检测。对收集保存的18份CTLV分离株进行了指示植物鉴定、3’端序列分析及RFLP检测。结果表明,在指示植物腊斯克枳橙(C. sinensis×Poncirus trifoliata cv. Rusk)上表现弱到中等强度症状的6个分离株均属于或携带有RFLP Ⅱ或RFLPⅢ组群,而其它RFLP组群均表现出强毒株特征。在根据3’端序列(889bp)构建的系统进化树上,CTLV分离株聚为了2个同源性组群,同源性组群A包含的8个序列均为RFLP Ⅱ或RFLP Ⅲ组群；同源性组群B则包含了其它RFLP组群的所有序列。同时,在指示植物上表现较弱症状的多数(4/6)CTLV分离物划分在A组群,多数(10/12)CTLV强毒分离物则划分在B组群。可见,指示植物上的弱(中)毒分离株侵染症状-RFLPⅡ或RFLPⅢ组群-同源性A组群三者之间可能存在某种内在联系。RFLP组群检测可用于研究CTLV的分子变异,对于快速鉴定CTLV致病性强弱可能具有参考价值。3. CTLV基因组全长cDNA扩增及序列分析在通过RACE技术获得末端精确序列的基础上,建立了CTLV全长的RT-PCR体系,进而扩增并克隆了CTLV新会橙分离株及满头红分离株基因组全长cDNA。序列分析表明,CTLV-XHC(登录号KC588947)和CTLV-MTH(登录号KC588948)基因组全长均为6497nt[不包括3’poly(A)尾巴],均包含2个开放读码框,在推定的CP及MP上游均具有保守的亚基因组启动子核心序列‘’UUAGGU",与目前已报道的CTLV及苹果茎沟病毒(Apple stem grooving virus, ASGV)的基因组结构一致。同登录GenBank的所有CTLV及ASGV全基因组序列比对显示,CTLV-XHC与我国砂糖橘分离株CTLV-S序列一致性最高,为98%,在依据全基因组核苷酸序列构建的系统进化树上,CTLV-XHC与柑橘来源的CTLV分离株聚为一簇；有趣的是,CTLV-MTH与日本ASGV分离物序列一致性最高,为91%,而与台湾甜橙分离株CTLV-LC一致性最低,为82.0%,在系统进化树上CTLV-MTH与多数ASGV分离株聚为一簇。这一结果对于CTLV与ASGV的起源进化关系具有启示意义。4. CTLV侵染性克隆构建对6个CTLV基因组全长cDNA克隆进行体外转录并摩擦接种昆诺藜(Chenopodium quinoa),症状观察及RT-PCR检测结果表明,CTLV-XHC和CTLV-MTH的体外转录物具有侵染性。为获得适于农杆菌介导接种的CTLV侵染性克隆,以pCAMBIA1301为骨架构建了双元植物表达载体pCSSN:LB-2x35S-MCS-NOS-RB,通过瞬时表达GFP验证其有效性后,采用分步克隆策略将CTLV-XHC和CTLV-MTH分别插入到pCSSN载体2x35S启动子与NOS终止子之间。经筛选、鉴定获得了CTLV侵染性克隆pCSSN-XHC和pCSSN-MTH。农杆菌介导的本生烟(N. benthamiana)注射接种实验表明,二者的侵染症状与野生型病毒对照无明显差异,接种时共表达沉默抑制子p19提高了pCSSN-XHC和pCSSN-MTH的侵染率。5. CTLV诱导的VIGS体系建立以CTLV侵染性克隆pCSSN-XHC为基础,通过重组克隆在CP终止子之后引入NruⅠ识别序列,构建了VIGS载体pCTLV-00:LB-2x35S-XHC(Nru Ⅰ)-NOS-RB。反向插入402bp源于本生烟的八氢番茄红素脱氢酶(Phytoene desaturase, PDS)基因片段后,重组载体pCTLV-PDS402R通过农杆菌浸润接种本生烟,在22℃光照16h和20℃黑暗8h周期条件下,诱导产生了PDS基因沉默的白化表型。Real-time RT-PCR检测结果表明,pCTLV-PDS402R侵染植株PDS基因的相对表达量比空载体pCTLV-00对照下降约25%。说明基于CTLV上的VIGS体系初步建立。为提高VIGS沉默效率,引入具有自我剪切功能的核酶序列到pCTLV-00,构建了VIGS载体pCTLV1:LB-2x35S-XHC(Nru Ⅰ)-RZ-NOS-RB;进一步分别引入135bp、105bp、90bp和61bp的CP亚基因组启动子片段构建了VIGS载体pCTLV2、pCTLV3、 pCTLV4、pCTLV5。上述系列VIGS载体均反向插入了PDS基因片段,基因沉默效应正在评估当中。更多还原

【Abstract】 Citrus is the first important fruit crop by total output in the world. China ranks No.1with a total acreage of over2.9million hectares and an annual citrus production of over30million tons. At present, the genome sequence of clemantine tangor(Citrus clementina Hort. ex Tan and valencia sweet orange(C. sinensis cv. Valencia) have been published, and a large number of expressed sequence tags (ESTs) has been released to GenBank. In contrast to the enormous progress in obtaining sequence data, the identification of citrus genes functions has been being focused with challenge by its long juvenile period and genetic transformation difficulty. A tool to bridge the gap between sequence information and gene functions for citrus is in demand. Virus-induced gene silencing (VIGS) is a recently developed tool for gene function studies, which is particularly attractive for such woody species as citrus. Compared to commonly used biotechnological tools such as transgenic mean, gene deletion method and inhibition technique by antisense nucleotides, VIGS is rapid with low cost, no need for stable plant transformation, and allows a large-scale screening of genes for functional analysis. The aim of this paper was to develop viral vectors for gene silencing that might be useful to identify gene functions and for citrus genetic improvement. For this purpose, Citrus tatter leaf virus (CTLV) isolates were collected and detected by newly established One-step or Nested RT-PCR, and then were addressed to biological indexing and genetic polymorphism assay. Selected isolates were adopted to construct full-length cDNA clone of CTLV genome, followed by sequencing and engineering to a modified binary vector. Based on the clone shown to be infectious, a series VIGS vectors were developed and evaluated by silencing phytoene desaturase gene (PDS) upon agroinoculation into model plant leaves of tabacco Nicotiana benthamiana. The outputs are as follows.1. One-step and Nested RT-PCR for CTLV detectionA one-step RT-PCR and a nested RT-PCR were established for CTLV detection by comparing primer pairs. With a product of889bp in length, the One-step RT-PCR was more convenient and lower risk of contamination than that of conventional RT-PCR. The Nested RT-PCR was at least hundred times more sensitive than that of the One-step RT-PCR, and the lowest detection limit of the method reached1.27pg/μL, which could be very useful for CTLV diagnostic and certification programs.2. Genetic diversity of CTLVA method for detecting molecular variation of CTLV was developed based on restriction fragment length polymorphisms (RFLP) of the3’sequence amplified by RT-PCR, and nine Hinf I RFLP patterns were defined. The method was rapid, reliable, reproducible, and could be used for a large scale of samples. Detection data indicated that the majority of CTLV isolates presented as single well-defined patterns, among which RFLP I and RFLP Ⅱ were dominant. In contrast, a few samples presented as a mixture of two RFLP patterns, suggesting a mixed infection of CTLV variants.Eighteen isolates of CTLV were characterized by biological indexing, sequencing and RFLP assay. Six isolates induced mild to moderate symptoms on indexing plants (C. sinensis×Poncirus trifoliata cv. Rusk), and all of them were conformed to carry with RFLP II or RFLP Ⅲ. In contrast, four isolates carrying with RFLP I and six other samples exhibited RFLPⅣ-Ⅸ induced severe symptoms. In the phylogenetic tree constructed according to3’ nucleotide sequence, CTLV isolates tested were divided into two clusters clearly, of which phylogenetic groups A, included all sequence were conformed to carry with RFLP II or RFLP III patterns, while phylogenetic groups B included other RFLP restrictotypes. Also, phylogenetic groups A included the majority mild (moderate) strain (4/6), while phylogenetic groups B included the majority severe strain (10/12).The results above revealed somewhat correlation among mild (moderate) symptoms, phylogenetic groups A and RFLP Ⅱ or RFLP Ⅲ restrictotypes, suggesting RFLP analysis may be useful in quick identification of mild strains of CTLV.3. RT-PCR amplification and Sequence analyses of CTLVA single step RT-PCR for detecting the entire CTLV genomic RNA were established with a pair of primers specific to its exact5’and3’ends sequences, which were determined by RACE system (Clontech). Full-length cDNA of CTLV isolates XHC and MTH were successfully amplified according to the method, and then were cloned and sequenced. The complete genome sequence of CTLV-XHC and CTLV-MTH were determined to be6497nucleotides in length, excluding the3’-terminal poly(A) tract, and contained two putative overlapping open reading frames (ORFs). For the genome of CTLV-XHC and CTLV-MTH, the core sequences of the sgRNA promoter, UUAGGU, were also found upstream from the transcription start sites of both the putative CP and MP sgRNAs, which is broadly conserved among viruses in the Flexiviridae.Homology matrix of nucleotide sequences of CTLV-XHC, CTLV-MTH and other available Capilloviruses in GenBank was conducted, and then the phylogenetic tree derived from those sequences was constructed. CTLV-XHC has the maximum identity (98%) with CTLV-S, a strain isolated from Shatangju(C. reticulata) in China, and clustered together with isolates from citrus in the phylogenetic tree. Interestingly, CTLV-MTH showed the highest identity (91%) with an ASGV isolate from Japan, and the lowest identity (82.0%) with a CTLV isolate from sweet orange located at Taiwan. In the phylogenetic tree, CTLV-MTH was clustered together with most of ASGV isolates. These results may have enlightenment significance for CTLV and ASGV evolutionary relationships.4. Construction of full-length infectious clones of CTLVCapped in vitro transcripts of six full-length clones of CTLV were generated, and inoculated mechanically onto leaves of Chenopodium quinoa plants, and evaluated periodically for symptom development. In vitro transcripts from CTLV-XHC and CTLV-MTH elicited systemic infections in some of the inoculated plants, which were confirmed both by RT-PCR detection and symptom observation.In order to generate the agrobacterium-mediated infectious cDNA clones of CTLV, a binary vector pCSSN (LB-2x35S-MCS-NOS-RB) was firstly constructed based on pCAMBIA1301backbone, and evaluated for validity by GFP expression. The full-length cDNAs of CTLV-XHC and CTLV-MTH were inserted into pCSSN between2x35S promoter and NOS terminator respectively, and then were introduced into agrobacterium EHA105and inoculated N. benthamiana by agro-infiltration with or without co-expressing of the p19silencing suppressor. As a result, pCSSN-XHC and pCSSN-MTH were obtained with the corresponding full-length cDNA of CTLV via duplicated CaMV35S promoter, which were confirmed by sequencing. Part of plants agro-infiltrated with pCSSN-XHC or pCSSN-MTH were infected successfully according to RT-PCR results, and developed symptoms similar to those induced by the wild-type CTLV, but with a3-5d delay. When the p19silencing suppressor involved in the process of agrobacterium-mediated inoculation, higher infectivity of pCSSN-XHC or pCSSN-MTH were observed.5. Developing VIGS vector based on CTLVA VIGS vector pCTLV-00[LB-2x35S-XHC(Nru I)-NOS-RB] was constructed by introducing a unique Nru I restriction site to pCSSN-XHC at closely downstream of the CP. A402bp fragment of PDS from N. benthamiana was RT-PCR amplified and inserted into the restriction site in the antisense orientation. The recombinant (pCTLV-PDS402R) was inoculated onto leaves of N. benthamiana by agro-infiltration with co-expressing the p19silencing suppressor. Grown in a plant growth chamber at22/20℃day/night, and16/8h light/dark regime, some of those plants initiated white spots on the upper leaves25-30d post inoculation, indicating the PDS had been silenced. Quantification of PDS-mRNA by Real-time RT-PCR showed that tobacco leaves with a silencing phenotype had25%lower mRNA accumulation than that of leaves from plants infected with pCTLV-00. These results suggest that a VIGS system based on CTLV was preliminarily established.In order to improve effciency of the CTLV-mediated VIGS system, pCTLV1[LB-2x35S-XHC(Nru I)-RZ-NOS-RB] was developed by adding a sequence of Hepatitis delta virus ribozyme to pCTLV-00before its NOS terminator. Further modifications were done based on pCTLV1to generate pCTLV2, pCTLV3, pCTLV4, and pCTLV5, which contained a duplicated fragment of CP sgRNA promoter in different lengths(135bp,105bp,90bp, and61bp respectively). These vectors had been inserted with PDS fragment, and their efficiency were evaluating.更多还原