关键词: 酵母同源重组, 柑橘叶斑驳病毒, 三元穿梭载体, 侵染性克隆

Abstract: 【Objective】 The objective of this study is to construct infectious cDNA clone of Citrus leaf blotch virus (CLBV) isolated from China and lay a foundation for studies on the pathogenesis of CLBV at the molecular level.【Method】Using GeneArt® pYES1L Vector as a template, a 2.1-kb fragment containing the yeast replication origin and Trp-1 gene were amplified by primer pair PYES2117F/PYES2117R. Plasmid of a binary vector DK1317-2 was digested with Sac II. The amplified and digested productions containing expected fragments were then purified with the Gel Extraction Kit and subjected to In-Fusion cloning. In this way a ternary shuttle vector pCY was obtained. Using Potato virus X (PVX) infectious clones as a template, pCY-PVX-F and pCY-PVX-R as primers, the full length cDNA of PVX genome was amplified. Plasmid pCY was digested with restriction endonuclease Stu I and Sma I. The fragment and the linearized vector obtained above were co-transformed into yeast YPH501 by lithium acetate conversion method, and the full-length cDNA clone of PVX genome was obtained by homologous recombination. The infectivity of the resulting constructs was verified by Agrobacterium tumefaciens mediated inoculation on N. benthamiana, and a rapid cloning system based on homologous recombination in yeast cell was established for construction of viral infectious clone. On this basis, two fragments covering the full-length genome of CLBV-HBYD were amplified, and CLBV-1 and CLBV-2 were obtained. The homologous recombination was then performed to assemble CLBV-1, CLBV-2 and pCY vector fragments using the cloning system established. Then, N. benthamiana and Jincheng (C. sinensis) was inoculated with A. tumefaciens carrying the recombinant plasmid pCY-CLBV. RT-PCR and Northern blot were used to detect the inoculated seedlings.【Result】A ternary yeast-E. coli-A. tumefaciens shuttle vector, pCY, was constructed based on DK1317-2 and pYES1L. The vector is 10 347 bp in length and contains the replication elements of three bacteria, and can be stably replicated in yeast, A. tumefaciens, and E. coli. Using this vector, 16 full-length cDNA clones of CLBV-HBYD genome were obtained through homologous recombination in yeast cell. One clone was randomly selected and sequenced (GenBank: MG572236). Sequence analysis showed that CLBV-HBYD is 8 747 nt in length and encodes 3 open reading frames (ORF). ORF1, ORF2 and ORF3 is 5 889, 1 089 and 1 092 nt, respectively. The nucleotide sequence identity between CLBV-HBYD and 9 isolates of CLBV ranged 79%-98%, in which the highest identity was 98% with EU857540 isolate from citrus, while only 79% was seen when compared with kiwifruit isolates JN983454, JN983455, JN983456 and JN900477. In the phylogenetic tree generated by MEGA 6 software, CLBV-HBYD clustered with other citrus isolates into the same cluster. Sixteen CLBV full-length cDNA clones were inoculated on N. benthamiana and Jincheng by A. tumefaciens mediated inoculation, and pCY empty vector was used as a negative control. After 20 days of inoculation, RNA was extracted for RT-PCR detection. The results showed that CLBV specific genes were detected in plants inoculated with 11 full-length cDNA clones of CLBV-HBYD, respectively. Five set of RT-PCR positive plants were randomly selected for further Northern blot. The results showed that specific bands of CLBV were detected by Northern blot in plants inoculated with pCY-CLBV 1, 2, 3, 14, 15, whereas no gene was detected in the control samples, indicating that these clones were infectious clones.【Conclusion】A rapid cloning system based on a ternary yeast-E. coli-A. tumefaciens shuttle vector pCY and homologous recombination in yeast was constructed, and an infectious cDNA clone of Chinese isolate of CLBV was developed for the first time.

Key words: yeast homologous recombination, Citrus leaf blotch virus (CLBV), ternary shuttle vector, infectious clone