Abstract: 【Objective】The objective of this study is to clone tomato resistance-related gene SlHin1, to analyze its bioinformatics characters, tissue expression and subcellular localization, evaluate its anti-role in the process of the infection and movement of Tobacco mosaic virus (TMV), and to provide a theoretical basis for tomato resistance breeding. 【Method】 Gene cloning and RT-PCR were combined to isolate a gene which might encode SlHIN1. Bioinformatics tools were applied to analyze both the DNA sequence and the characteristics of encoded protein sequence, MEGA6.0 was used to make multiple sequence alignments between the SlHIN1 amino acid sequences and their homologous sequences, and the phylogenetic tree of homologous species was constructed. The subcellular localization test of SlHIN1 was analyzed by using fusion expression vector pCV-mGFP-C1, then the gene was inserted into the expression vector by Sal I and Bam H I digestion. The real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to analyze to the expression levels of SlHin1 in different tomato tissues. The gene fragment was inserted into the plant expression vector pFGC5941 by Nco I and Bam HI digestion. The SlHIN1 was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the SlHIN1 expressed leaves were inoculated with TMV-GFP. The accumulation of the virions was detected by indirect ELISA to investigate the antiviral function of the gene. Western blot was used to evaluate the expression of SlHIN1 protein.【Result】Using RT-PCR method, the tomato Hin1 was obtained from tomato (Solanum lycopersicon cv. Ailsa Craig) leaves and was designated as SlHin1 (GenBank number: KU195820). SlHin1 was 675 bp in length. It was predicted to encode a protein with 225 amino acid residues, a molecular weight of 26.1 kD and a theoretical isoelectric point of 9.35. SlHin1 contains the LEA-14 domains structure, doesn’t have transmembrane segments and locates on chromosome 10 (Solyc10g081980). Sequence analysis and phylogenetic tree analysis showed that SlHIN1 shared approximately 80% similarity with HIN1 from other solanaceae plants and was close to that of rice and sorghum monocotyledons. Subcellular localization was showed that SlHIN1 distributed on the plasma membrane of the leaf epidermis cell of N. benthamiana, which is consistent with the predicted results. The results of qRT-PCR showed that the SlHin1 was of tissue-specificity, whose expression decreased from tomato roots, leaves to stems. The SlHIN1 was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the SlHIN1 expressed leaves were inoculated with TMV-GFP. After 4 days, there was no green fluorescence observed in the SlHIN1 expressed leaves under UV light, but the green fluorescence could be observed in the control group. With the passage of the inoculation, the sporadic fluorescence on the leaves of the treated group was slightly enlarged after 7 days post inoculation, and the green fluorescence of the control group had spread to the leaf. The spread of virus was inhibited and the time that the virus needed to reach the lobus cardiacus was longer, the content of the virions was less than that of control group through indirect ELISA test result. Western blot analysis showed that there was a specific band at about 26 kD in the nitrocellulose membrane, and no corresponding control was observed in the empty vector, indicating that the SlHIN1 protein was successfully expressed at the injection site.【Conclusion】Hin1 genes are present in all tested species, and the SlHIN1 is localized on the plasma membrane of the leaf epidermis cell of N. benthamiana. Transiently expressing SlHIN1 could inhibit the accumulation of the virions and spread of the virus, which can reflect that it may be involved in the resistance reaction of Solanaceae plants to TMV causing them to have resistance.

Key words: tomato, SlHin1, Tobacco mosaic virus (TMV), expression, anti-virus