Functional Characterization And Regulation Mechanism Of ShGT-1 Gene In Stress-responsiveness In Tomato

The abiotic stress is the main factor which threatens crop yield and quality at present.Therefore,future research of the response regulatory mechanisms under abiotic stress can provide people a theoretical basis to cultivate new varieties of stress tolerance.Trihelix transcription factors is a small family in plants.Trihelix transcription factors which could specifically bind GT element of genes,therefore also called GT transcription factor.They play an essential role in plant growth,development and environment stress resistance.Currently,the study of their function has been much reported,but the concrete regulation mechanism of trihelix members in the plant abiotic stress response is still unclear.Tomato plants are very vulnerable to adverse environment damage during development.Whereas the study that of GT transcription factors involved in tomato growth and abiotic stress is rare.So,exploring the molecular mechanisms of GT in tomato will contribute to increase our cognitive and create new germplasm.In this study,we selected a candidate gene which responses to low-temperature based on the results of hybridization in cold chip,then we isolated the ShGT-1 transcription factor from cold-resistant S.habrochaites LA1777,and used transgene technology to verify and analyze its function.The main studys in this paper are as follows:1.BLAST analysis indicates that the ShGT-1 encodes a protein containing 293 amino acids.Subcellular localization experiment proves that the nuclear localization of ShGT-1 protein.Comparing the gene sequence of cultivated tomato,there is only one amino acid change and one amino acid deletion in LA1777.2.The tissue expression pattern analysis reveales that ShGT-1 is widely expressed in organizations and is the constitutive expression,but it is highly expressed in root and flower,showing that ShGT-1 gene plays a significant role in regulating target genes in developmental growth process.ShGT-1 can express in response to the abiotic stress factors such as drought,low temperature and high salty,meanwhile,it also can be induced by plant hormone ABA and GA.The result reveals that ShGT-1 may be involved in environmental stress response positively.3.ShGT-1 was transformed into cultivated tomato A57 after its overexpression,altered individual phenotype of transgenic plants obviously,while the mutant plants displayed dwarfism with shorter nodes and the smallar size of leaves.Then we explore the reason of that dwarf phenotype,finding that transgenic tomato plants can recover the phenotype by GA-spraying.The result indicates that ShGT-1 has no influence in signal transduction pathway,so the dwarfism of transgenic lines is a kind of GA-sensitive.In order to future research the molecular mechanism of ShGT-1 causes dwaring of tomato plants,we analyzed the expression of GA biosynthesis genes in the tomato plants found that the key genes GA20ox4 and GA3 ox was significantly down-regulated in transgenic plants.Thus we conclude that the GAs synthesis of transgenic plants is restrained which may be the reason of dwarfing.4.In seeding stage,we did experiment to identificate the stress tolerance of tomato plants and found ShGT-1 transgenic lines with strong resistance under low temperature and drought stress than wild-type A57.Then we analyzed physiological indexes.Under low temperature,the proline contents rapidly accumulated when compared to wild-type,while the relative conductivity and MDA contents of transgenic plants are clearly lower.ShGT-1 over-expressed plants keep their stomata closed under drought stress than wild-types,meanwhile the relative water loss rate of excised-leaves is lower comparing to A57,therefore,the transgenic tomato plants show stronger moisture holding capacity.These results illustrate that ShGT-1 is a part of stress resistance regulation in tomato plants.5.We used yeast two-hybridization to filter the interacting proteins of ShGT-1 and found three proteins,which are Solyc02g067030(Sn RK1),Solyc02g067460(VDAC)and Solyc07g008070(Ca M),they are all related to abiotic stress,it shows that ShGT-1 may play a role in abiotic stress signal transduction.It has been reported that Sn RK1 involved in response to abiotic stress by the ABA-dependent pathway.After the study,we found that tomato transgenic plants expressed insensitivity to abscisic acid,but the expression of some genes in ABA synthesis and signal transduction pathway is up-regulated.Taken together,our date demonstrates that protein ShGT-1 responses to abiotic stress in tomato plants which by interaction with Sn RK1 in ABA-dependent signaling pathways.