Construction of Infectious Clones and Detection Improvement of Citrus tatter leaf virus
Date Issued
2012
Date
2012
Author(s)
Chang, Li
Abstract
Citrus tatter leaf virus (CTLV) belongs to Capillovirus, Betaflexiviridae with the genome of (+) ssRNA packaged by a filamentous virion. CTLV has been considered to be one of iportant citrus virus diseases, but the scientific data associated with CTLV are still rare so far. This thesis was dedicated to construct the infectious clones of full-length cDNA of CTLV to obtain more molecular and pathological information about CTLV. A CTLV isolate named CTLV-Pk collected from the diseased Ponkan mandarin was used for the cloning. The sequencing data reveals that the genome of CTLV-Pk consists of 6496 nucleotides (nt). The size is larger than most other genome sequences of CTLV isolates (6495 nt). The genome organization is similar to other capilloviruses, with two overlapping open reading frames (ORFs). ORF1 encodes a polyprotein p242 containing replication-associated domains and coat protein (CP). There are two variable regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868); ORF2 encodes a putative movement protein (MP). The nucleotide sequences of full-length genome of CTLV-Pk are similar (79.4-94%) to those of the other CTLV and Apple stem grooving virus (ASGV) isolates. The similarity of amino acid sequences is 85.3-95.8% for ORF1, 92-95.8% for CP and 93.4-99.1% for MP among those isolates. The phylogenetic analyses showed that the geographic separation might lead to the molecular differences among various capilloviruses. The complete genomic RNA of CTLV can be synthesized from infectious clones through in vitro transcription. In the inoculation tests, the selected infectious clone (pCTLV-Pk-8) could successfully infect Chenopodium quinoa at 22°C and incite the symptoms similar to the original CTLV-Pk isolate. The symptoms induced by pCTLV-Pk-8 were slightly later than those by CTLV-Pk. Approximately 73.7% of tested plants were positive for CTLV-infection in the inoculation tests, and they were able to maintain stability through serial passages on C. quinoa. Meanwhile, the results also indicated that the symptoms caused by in vitro transcripts from pCTLV-Pk-8 were not evenly distributed on C. quinoa. The trial using point mutation at translational start codon of CTLV-Pk coat protein (5642 nt) demonstrated that the mutant did not cause systemic symptoms on C. quinoa. For more sensitive detection of CTLV, this study also attempted to improve the RT-PCR and develop Real-time RT-PCR assays. The RT-PCR detection of CTLV with the newly devised primer pair CTLV-501 could obtain more specific and sensitive results even using only 1 pg of RNA template. The Real-time RT-PCR with the devised “CTLV-RT-TP TaqMan primers / probes” could better results even using only 10 fg of RNA template. The results presented in this thesis were expected to provide important references for the academic research and quarantine application in the future.
Subjects
Citrus tatter leaf virus
Apple stem grooving virus
infectious clone
Type
thesis
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