Isolation And Functional Analysis Of TETRASPANIN3 Gene Relative To Tomato Yellow Curl Virus(TYLCV)Resistance In Solanum Lycopersicum

The tomato plants infected by tomato yellow leaf curl virus are dwarf.and their leaves are small,yellow,and curl,and their growth,and their bossom and fruit been seriously affected,and the yield reducted.Recently,tomato yellow leaf curl disease has ocured and has different degree effect around the world.and has become one of the most serious diseases of tomato.To improve the resistance to diseases of tomato through gene engineering in tomato breeding became more and more important.Through cloning anti TYLCV gene into plants,or inhibit the expression of gene which promote TYLCV infecting plants can improve the resistance of plants toTYLCV.Based on the transcriptome data of tomato.the expression of TETRASPANIN3 significantly up-regulated after beening induced by TYLCV,suggesting that TETRASPANIN3 may be associated with the resistance of tomato to TYLCV,and TETRASPANIN3 may be a target gene of resistance to TYLCV breeding.TETRASPANIN protein is a transmembrane protein with the highly conserved amino acid sequence and special structure.and formed by four transmembrane domains and three loops which made the foour transmembrane domain connect,two loops out of cell membrane.a loop at the inner of cell membrane.The highly conservation of TETRASPANIN protein,showed that the TETRASPANIAN gene family have similar function.The function of TETRASPANIN3 have never been reported in tomato.The research was conducted to study the effect of TETRASPANIN3 on the resisitance to tomato yellow leaf curl virus(TYLCV)and the development of tomato plants.The phylogenetic tree of TETRASAPANIN3 was constructed by using NCB1 datebase and the MEG5.0 software to analysis the evolution of TETRASPANIN3.The results show that the TETRASPANIANIN3 and VITIS VINIFERA gene of grape were in a distinct branch and TETRASPAANIN3 has further relationship with other genes.The expression pattern of TETRASPANIN3 in different organs were determined by qRT-PCR.The results show that the TETRASPANIN3 was expressed in all organs,the higher expression in stem,the old leaf,flower and the lower expression in root,fruit.and the expression in the new leaf was in between.The response of TETASPANIN3 during TYLCV infection were determined by qRT-PCR,The results show that the TETRASPANIN3 was up-regulated during TYLCV infection and consist with the data of tomato RNA-seq after TYLCV inducing.To further study the function of TETRASPANIN3 in tomato,the TETRASPANIN3 of tomato was silenced by VIGS technology,then observed phenotype of TETRASPANIN3 gene silenced plants and detected resistance to TYLCY of gene silenced plants.The tomato PDS gene was used as a positive control experiment.After injecting PDS gene to tomato plants 2-3 weeks,some tomato leaves become white,suggesting that the PDS gene has been slienced.At the same time,detecting the expression of TETRASPANIN3.The results show that the expression of TETRASPANIN3 in gene silenced plants was 35%of the control group(TRV2 vector),then the gene silenced significantly.The TETRASPANIN3 silenced plant has a significantly difference phenotype with the control group,and the TETRASPANIN3 silenced plant has heavly distorted leaves.The results show that TETRASPANIN3 gene has an effect on the growth and development of tomato,and this consist with the effect of TORNADO1 and TORANAD02 which were TETRASPANIN3 homologous genes in Arabidopsis on root radial and periclinal division.The TETRASPANIN3 silenced plant has distorted leaves,and has an over 50%decline in the quantity of TYLCV,suggesting that TETRASPAANIN3 act as a positive regulator of the infection of TYLCV to tomato plants.TETRASPANIN3 gene was cloned by RT-PCR in tomato,and the plant expressive vector of TETRASPANIN3 gene was constructed and through agrobacterium mediated the transformation of tobacco.T1 generation was obtained by T0 transgenic tobacco plants self-fertilize.Tl transgenic tobacco was inoculated with TYLCV and the resistance to virus was detected by qRT-PCR detecting the quantity of virus in tobacco leaves.The results showed that TETRASPANIN3 transgenic tobacco leaves contained more virus than non transgenic tobacco leaves.That suggesting that the TETRASPANIN3 gene act as a positive regulator of the infection of TYLCV to tobacco plants.Through the above results.concluded that TETRASPANIN3 have an effect on the development of tomato leaves and act as a positive regulator of the infection of TYLCV to tomato plants.