Functional Characterization Of Cold-response Genes SmICE1s And SmCBFs In Solanum Melongena L.

Freezing-sensitive eggplant as a main kind of solanaceae vegetable,is vulnerable to cold injury at all stages of growth and development,which caused serious disruption to the growth and development and serious decline in quality and yield.Eggplant fruit is rich in anthocyanin,and low temperature is an important environmental factor affecting anthocyanin biosynthesis.The cold-response pathway and the molecular mechanism of anthocyanin biosynthesis regulated by low temperature are largely unknown.In order to reveal the above scientific problems,we studied the function of ICE1 s and CBFs on cold-response and anthocyanin biosynthesis.The main results were achieved as follows.1,The molecular identification and functional characterization of SmCBF1,SmCBF2 and SmCBF3 from eggplant were studied.SmCBFs belonging to the AP2/EREBP transcription factors could specifically bind to the DRE/CRT cis-element to positively regulate cold-response.SmCBFs regulated the anthocyanin biosynthesis via interaction with SmMYB113.In this study,full length of SmCBF1,SmCBF2 and SmCBF3 were cloned from eggplant.SmCBF proteins belong to the CBF/DREB1 subfamily of AP2/ERF family.Alignment of the sequences obtained from c DNA and genomic DNA indicated that the SmCBF genes are intronless.Based on the quantitative RT-PCR,the expression of three SmCBFs were all induced by cold stress,drought,high-salinity and abscisic acid(ABA)treatments.Subcellular localization suggested that SmCBF1 and SmCBF2 were all located in nucleus,while SmCBF3 was located in cytosol and nucleus.SmCBFs could specifically bind to the DRE/CRT cis-element and induce the expression of COR genes on the basis of yeast one hybrid(Y1H)assay and Dual-Luciferase(Dual-LUC)assay.Overexpression of SmCBF1,SmCBF2 and SmCBF3 in Arabidopsis could increase the freezing-tolerance,but SmCBF2 and SmCBF3 have stronger effect on cold-response than SmCBF1.Previous study suggested that SmMYB113 is a key regulator of anthocyanin biosynthesis in eggplant.The interaction between SmCBFs and SmMYB113 were confirmed by yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(Bi FC).The R3 domain of SmMYB113 and the C-terminal of SmCBFs were responsible for the interaction.So SmCBFs may participate in regulating the accumulation of anthocyanin.Dual-LUC assay indicated that the expression of SmCHS and SmDFR were significantly improved by SmMYB113.When SmCBFs were co-expressed with SmMYB113,the activity of SmMYB113 in activating SmCHS and SmDFR was enhanced.SmCBFs and SmTT8,a b HLH partner of SmMYB113,could synergistically enhance the transcriptional activation of SmMYB113-mediated SmCHS and SmDFR.When SmCBFs and SmMYB113 were co-infiltrated,the anthocyanin content and the expression level of anthocyanin biosynthesis related genes were significantly higher than infiltrated SmMYB113 alone in Nicotiana tabacum leaves.Then,it was observed that overexpression of SmCBFs resulted in more visible anthocyanin pigments in young seedlings within 7 days than WT Arabidopsis.The anthocyanin contents and the expression of anthocyanin biosynthetic genes were significantly increased in transgenic lines compared with WT after the cold treatment.This research revealed that SmCBFs as positive regulatory factors were involved in cold-response pathway,and put forwarded a regulation pathway describing the possible mechanism of SmCBFs participate in the anthocyanin biosynthesis of eggplant.2,The molecular identification and functional characterization of SmICE1 and SmICE1 a encoding MYC-type b HLH transcription factor were studied.SmICE1 s positively regulated cold-response by promoting the expression of SmCBFs.In this study,we cloned two ICE1-like genes from eggplant(SmICE1 and SmICE1a).The proteins encoded by SmICE1 and SmICE1 a were localized to the nucleus.The transcription level of SmICE1 and SmICE1 a were induced by low temperature,although the increase was only about 2-3 times.Dual-LUC assay revealed that SmICE1 s can enhance the expression of SmCBFs.Overexpression of the SmICE1 s in Arabidopsis enhanced the freezing tolerance,increased the expression level of cold-resposive genes and caused multiple biochemical changes,including that transgenic lines have higher proline contents and lower levels of electrolyte leakage and malondialdehyde than wild type in cold condition.Moreover,the freezing-tolerence of SmICE1 transgenic plants was weaker than SmICE1 a overepression plants.These results indicated that SmICE1 s were crucial transcription regulatory factors in the cold-response pathway.Bioinformatics analysis,Y2 H and Bi FC suggested that SmICEIs interact with SmYABBY.Nevertheless,the function of SmICEIs and SmYABBY on anthocyanin biosynthesis pathway needs to be studied further.