Construction Of Infectious Clone Of Citrus Tatter Leaf Virus

Citrus tatter leaf virus (CTLV) is one of the most important viruses. There’s usually latent period phenomenon of CTLV in many citrus varieties. In order to define the molecular properties and molecular variation characteristics of CTLV isolate in China, the complete genomic sequence of CTLV-STO isolate from Shatang orange (Citrus eticulate Blanco cv. Shatang) was obtained, and the infectious clone of the full-length cDNA of CTLV-STO was constructed to lay the foundation for considering the pathogenicity of CTLV and the interaction mechanism between CTLV and host in China. The obtained results are as followings:1. The complete genomic sequence (GenBank accession numbers JQ765412) of CTLV-STO isolate from China was determined by multisegment RT-PCR method (divided as six segments). It’s6,497nucleotides (nts) long, contained two overlapping open reading frames (ORFs), excluding the3’terminal poly (A) tail. The ORF1(37nt-6,354nt) encodes a putative242kDa polyprotein which containsreplication-associat-ed (Rep) region (methyl-transferase, papain-like protease, NTP-binding helicase and RNA-dependent RNA polymerase domains) and a coat protein (CP) region, CP (5,641nt-6,354nt) encodes27kDa coat protein, and the ORF2(4,788-5,750nts) encodes a36kDa putative movement protein (MP). The5’and3’terminal untranslational regions (UTRs) are compose of36nts and142nts, respectively. Sequence analysis showed that the amino acid identity of ORF1and ORF2, respectively, ranged from98.3%-78.6%and98.4%-82.7%, compared with reported nine ASGV (Apple stem grooving virus)/CTLV isolates, and are26.4%and28.4%with a Cherry virus A (CVA) isolate. The results suggested that there’s no obviously relevance to host source. Even though, interestingly, the amino acid sequence encoded by ORF1had98.3%-85.6%identities with reported ASGV, CTLV and CVA isolates, we found that the variable region I (532aa to570aa) and variable region Ⅱ(1,583aa to1,868aa) possessed90.0%-80.0%and96.9%-85.3%with the corresponding regions of CTLV-K and CTLV-Pk from citrus, and had only15.0%-20.0%and51.7%-64.0%with that of other ASGV/CTLV isolates. Multiple alignments suggested that many amino acid residues of the variable region Ⅱ of CTLV-STO, CTLV-K and CTLV-Pk were highly conserved, and CTLV-Li and ASGV-Li23also showed high similarity. In addition, those conserved amino acid residues of CTLV-STO, CTLV-K and CTLV-Pk from citrus isolates were inexistent in the other ASGV isolates, and the CTLV-Li and CTLV-Li23from lily isolates, as well. Therefore, we predicted that the variable region I and variable region II might be related to host selectivity of ASGV.2. Six sequence fragements (F1, F2, F3, F4, F5and F6) covering the whole genome of CTLV-STO were assembled using overlap extension PCR method. Then they were used to construct the recombinant plasmids pMD18-T M1and pMD18-T M2. The full-length cDNA clone (pCTLV-23) and the partial cDNA clone (pCTLV-13) of CTLV-STO was constructed with the recombinant plasmids pMD18-T M1and pMD18-T M2which were double digested using restriction enzyme Sph I/Hpa I and restriction enzyme Not I/Hpa I, respectively. The deleted region of recombinant plasmid pCTLV-13was1235nt-1811nt of the complete genomic sequence of CTLV-STO.3. The recombinant plasmids pCTLV-13and pCTLV-23were transcribed in vitro, and the transcripts were inoculated to Nicotiana benthamian by the mechanical inocu-lation method. The results showed that the transcript can infect Nicotiana benthamian, but still need further validation.