| 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. |
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