The Function Analysis Of CIB3 Gene In Arabidopsis

Cryptochromes are photolyase-like blue light receptors that mediate various light responses in plants including photomorphogenesis and floral initiation.CRY2(Cryptochrome 2)can interact with CIB1(cryptochrome-interacting basic-helix-loop-helix 1)in a blue light-specific manner to promote FT(Flowering Locus T)transcription and floral initiation in Arabidopsis.However,the cibl loss-of-funnction mutant dids not show later flowering phenotype,which indicated that the function of CIB1 may be redundant to that of other bHLH proteins.Through phylogenetic analysis,additional members of the bHLH family related to CIB1 were found and named as CIB2,CIB3,CIB4,CIB5,CIL1 and CIL2 respectively.Transgenic Arabidopsis thaliana plants overexpressing CIB2,CIB4,CIB5 and CIL1 all flowered earlier than wild type except for CIB3(At3g07340).In this study,the bivalent vector pDT7G-ACT2::CIB3-MYC-UBQ10::cGR-CRY2 were constructed and transfered into rdr6 and cry2 mutant respectively.The flowering time of the transgenic plants was investigated in long-day(LD)photoperiods(16 hours light,8 hours dark).We aim to make the function of CIB3 clear in the floral initiation regulating,and whether this regulatory mechanism depends on the nucleus-located CRY2.The results of this study are as follows:1.Construction of bivalent vector and identification of transgenic Arabidopsis thaliana.The bivalent vector pDT7G-ACT2::CIB3-MYC-UBQ10::cGR-CRY2 was constructed using Infusion technology and was transferred into rdr6 and cry2 mutant respectively through inflorescence-dipping.We confirmed the transgenic plants co-expressing CIB3-MYC(human cellular homolog of avian myelocytomatosis virus)and cGR-CRY2 stably.The transgenic lines with CIB3-MYC and cGR-CRY2 co-expressing were symbolized by(CIB3-CRY2)/rdr6 and(CIB3-CRY2)/cry2 respectively.2.The phenotype identification of T3 or T4 lines of transgenic Arabidopsis thaliana.Transgenic plants were grown in long-day(LD)photoperiods and were treated with 30uM Dex(Dexamethasone)and Mock(without Dexmethasone)solution respectively.We investigated the time to flowering and the number of rosette leaves at the time of flowering.The results showed there was no significant flowering time difference between Dex and Mock treatment to cGR-CRY2/rdr6 plants.It indicated that the dose increase of CRY2 in the nucleus does not promote the initiation of flowering.(CIB3-CRY2)/rdr6 plants flowered slightly earlier than control(cGR-CRY2/rdr6)and there was no significant flowering time difference between Dex and Mock treatment.Interestingly,the(CIB3-CRY2)/cry2 plants flowered obviously earlier than the control of cGR-CRY2/cry2 and cry2,and there was no apparent flowering time difference between Dex and Mock treatment.It indicated that CIB3 has the CRY2-independent function of promoting flowering.3.The mRNA expression of the flowering time genes in transgenic Arabidopsis thaliana overexpressing CIB3.Utilizing the technology of QPCR(real-time quantitative polymerase chain reaction),we analyzed the mRNA expression of flowering genes including FT(Flowering Locus T)and FLC(FLOWERING LOCUS C)in transgenic plants(CIB3-CRY2)/rdr6 and(CIB3-CRY2)/cry2 respectively,comparing with the mRNA expression in each control plants.The results showed that the FT mRNA expression was promoted by CIB3 and the expression peak reached twice times than that in the control.But,the circadian rhythm of the FT mRNA expression was not affected in(CIB3-CRY2)/rdr6 plants during a 24-hour period At the same time,the mRNA expression of FLC was inhibited to some extent in(CIB3-CRY2)/rdr6 comparing to that in the control.The circadian rhythm of the FT mRNA expression was different than that in the control and Col4.The expression of FT mRNA reached a peak in the light period which was 6 hour-earliered than that in the control and reached another peak in the dark period..Meanwhile,the FLC mRNA expression was inhibited slightly in(CIB3-CRY2)/cry2 comparing to that in the control.4.The verification of the protein interaction between CIB3 and CRY2.The yeast two-hybrid results showed that CIB3 and CRY2 did not interact with each other in no matter in blue light or dark condition.CRY2 and CIB3 were expressed in human embryonic kidney(HEK293T)cells by Co-IP(Co-Immunoprecipitation)technology.The results showed that no protein interaction between CRY2 and CIB3 existed.It is inconsistent with the result reported previously that CIB3 can interact with CRY2 verified by vitro Pull-down assay.