Transcriptional Analysis Of Tomato Under High Temperature Stress Via Solexa Sequencing And Cloning And Functional Analysis Of LeCOR413-TM1Gene

Temperature stress is one of the major factors limiting the yield and geographical distribution of tomato. To develop a variety of tomatoes with resistance to high temperature or low temperature is an important way to ease the temperature stress effectively.In this study, we analyzed the transcriptome of tomato under high temperature stress via high-throughput sequencing of Solexa. The analysis of up-and down-regulated genes before and after high temperature treatment reveals the complicated regulatory networks of tomato. Moreover, a down-regulated tomato gene which shows higher homology with cold-regulated gene (COR) was selected, isolated from the leaves of tomato variety Zhongshu6, and named LeCOR413-TMl. Its sequence characteristics, expression patterns and the physiological function of resistance in transgenic tobacco were investigated. The main results were as follows:(1) Sequencing depths of4661314and4981634tags were achieved in the control and high temperature libraries, and each contains186280and226513specific tags.4563284and4862014clear labels were achieved, respectively, through the further eliminating the false positive labels. Overall,3302958(72.38%) and3211809(66.06%) matching labels were found by comparing the tomato gene database website. We analyzed the most differentially regulated tags with a log2ratio>2or<-2using a greater statistically significant value (P<0.001) as well as false discovery rates (FDR<0.01), founding284up-and154down-regulated genes,36up-and28down-regulated genes were involved in the environmental response,108up-and36down-regulated genes were involved in the transcriptional regulation, and53up-and58down-regulated genes were involved in the metabolism. With unknown genes account for about18.9%.(2) Using homologous primers sequences, we cloned the full-length cDNA by RT-PCR from tomato leaves, and named it LeCOR413-TM1(AW034114). The full length of LeCOR413-TM1is726bp and the ORF is663bp, encoding221amino residues. Transcripts of LeCOR413-TM1accumulated to the highest level in leaves; the expression of LeCOR413-TM1was induce by4℃, PEG, ABA and high light. (3) p35S-LeCOR413-TMl-GFP fusion protein was constructed and transiently expressed in tobacco. It was observed with confocal microscopy that the green fluorescence was clearly associated with chloroplasts and colocalized with the red autofluorescence of chloroplasts, demonstrating the LeCOR413-TMl subcellular localization on chloroplast.(4) The full-length LeCOR413-TMl cDNA was recombined into the expression vector pBI121downstream of the35S-CaMV promoter to form sense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404by the freezing transformation method and the transgenic tobacco plants were verified by PCR and qRT-PCR. The results indicated that the LeCOR413-TM1gene had been into tobacco genome.(5) Compared with WT, the transgenic tobacco showed higher seed germination and better seedling growth under chilling stress (4℃). These results indicated that the overexpression of LeCOR413-TMl improved the low temperature resistance of transgenic tobacco.(6) Under chilling stress (4℃), the transgenic tobacco showed lower superoxide radical (O2-) and hydrogen peroxide (H2O2) levels and less membrane damage by NBT and DAB staining and the determination of relative conductivity. The polyphasic fluorescence induction kinetics analysis of WT and transgenic tobacco indicated that under normal condition, there is no difference in WT and transgenic tobacco. However, the rise range of transgenic tobacco K was significantly lower than that WT, indicating that overexpression of LeCOR413-TM1alleviated the photoinhibition of PSII.