Cloning Of The Capsicum Annuum CTOM1 Gene And Construction Of The Expression Constructs For The Gene Silencing

The full-length cDNA of Capsicum annuum CTOM1, a gene with high similarity to AtTOMl of Arabidopsis thaliana, has been cloned by the methods of 5'RACE and 3'RACE, based on the partial cDNA sequence information of pepper. The length of the cDNA was 1244 base pairs (bp) long, encoding a 282 amino acids putative protein with six predicted motifs of trans-membrane structure. CTOM1 was 65.2% and 74.9% identical to AtTOMl, an A. thaliana host factor gene supporting the replication of tabacco mosaic tobamovirus (TMV) at the level of the nucleotide acids and the deduced amino acids respectively, and was 81.7% and 91.7% identical to TTOM1, a tomato host factor gene involved in the replication of cucumber mosaic cucumovirus (CMV), at the level of the nucleotide acids and the deduced amino acids respectively. Three types of expression constructs that could be used to study the function of CTOM1 were developed: an antisense plasmid that employed a 2.1kbp ofCTOMl genomic DNA at 5' end; a virus-based construct, pTVOO:CTOM1 that contained the full-length sequence of CTOMl cDNA in sense or antisense orientation; and RNAi constructs that were based on the CTOMl cDNA. These constructs were introduced into Agrobacterium tumefaciens via triparental conjugations and used to transform explants of pepper varietis "Tiandeng No. 1" and "Guiyan 99" . Twenty two transgenic plants of Tiandeng No. 1 that were transformed with the RNAi construct were obtainted. Twelve plants of Tiandeng No. 1 and 19 plants of Guiyan 99 that were resistance to Kanamycin and derived from transformation by antisense CTOMl construct were obtained. Within the observation period of 15 days, after being inoculated with TMV, the plants with the integrated RNAi sequence showed no sign of symptom, whereas the non-transformed showed severe symptom on day 9. These data suggest that CTOMl is a host factor gene involved in the replication of TMV.