Functional Characterization Of SlCR1 Gene And Regulatory Mechanism To The Cold Resistance In Tomato

Tomato(Solanum lycopersicum)is a kind of vegetable crop that widely naturalized in our country,and mainly use the greenhouse.When cultivatied in open air,the ability of tomato to resist cold environment in its germination and seedling stage is very important to save funding and reduce agricultural economic loss.The original center of the tomato is the Andes,and now,the ancestor of cultivated tomatoes is cherry tomato.Tomato is a kind of thermophilic crops,with continual domestication,tomato's ability to fight against the cold environment in different growth stage in the life has been improved.This research has an account for the germination index of the 252 tomato re-sequencing materials at 15?and 25?,and by using association analysis,we finally candidate a gene Sl CR1(Cold Resistance,Sl CR1),which associated with the cold resistance in germination stage.The main experimental results are as follows: 1.In the coding region of the cold resistence gene Sl CR1,there is an SNP site closely associated with the phenotype of the re-sequencing materials.This SNP is located in the coding region of Sl CR1,in cold resistant materials,the base of the SNP tends to be G,in the cold sensitive re-sequencing materials,the base of the SNP tends to be A,and the change of single base could lead to the change of amino acid.Differences of Sl CR1 promoter sequence are not correspond to the phenotype of the extreme materials,at the same time,the expressive level of Sl CR1 in extreme materials has no significant differences between 15? and 25?,and the gene expression among extreme materials are not correspond to its phenotype,this means that the sequence change of coding region was the cause of extreme character differences among materials.2.Transgenic functional verification showed,when overexpressing the Sl CR1,germination rate index(GRI)of the T2 generation is reduced.The sooner seeds can be able to germinate at the low temperature,the lower germination rate index is,shows that Sl CR1 can help improving tomato's ability to fight against the cold environment,and promoting its germination.When treated the seedlings of overexpression T2 lines in low temperature(3?),its wilting level is lower than control WT(TS-9).Overexpressing the Sl CR1 can improve the cold resistance oftomato in the seedling stage,suggests that Sl CR1 can positive regulate the cold resistance in tomato's seedling and germination stage.3.Yeast two-hybrid(Y2H)and bimolecular fluorescent complementary(Bi FC)showed that Sl CR1 can interact with a transcription factor protein Sl PP2-B1,Sl PP2-B1 is a gene in the family which coding F-box transcription factor protein,F-box protein is mumber of ubiquitin ligase E3.Sl PP2-B1 can be expressed in multiple tissues,and the expression level is higher in stem epidermis,flowers and immature green fruits,while the expression level is increased remarkably at low temperature stress.The expressive level of Sl PP2-B1 transgenic lines have no difference in germination stage compared with WT(TS-9),therefore,the gene has no relationship with tomato's cold resistance in germination stage,but when overexpress the gene,the cold resistance of OE(over expression)transgenic lines in seedling stage are lower than the WT,thus,the gene regulates the cold resistance in tomato's seedling stage in a negative pathway.4.The expression level of Sl CR1 was decreased in the T1 generation Sl PP2-B1 overexpressed transgenic lines,and the expression level of Sl PP2-B1 was increased in the T1 OE transgenic plants of Sl CR1,indicating that there might be a feedback regulation mechanism between the two genes.5.Sl PP2-B1 can interact with a voltage-gated anion channel protein VDAC1,and VDAC1 can also interact with Sh CIGT,the VDAC proteins have very close connection with the plant resistance to abiotic stress.Sl PP2-B1 cannot interact with Sh CIGT or Sl PP2-B1.