Molecular Genetics Study On The Involvement Of IQM5 In Floral Regulation In Arabidopsis

Flowering at the right time is important for plants’ reproduction and survival.The flowering transition is influenced by the complex external environment factors and endogenous genetic factors.It has been identified that there are 6 genetic regulatory pathways: photoperiod-,vernalization-,gibberellin-,autonomous-,temperature-and age-related,which are interrelated but independent of each other.AtIQM5 is encoded by AT5G57010,and IQM5 is a calmodulin binding protein(protein calmodulin-binding,CaMBP),containing 1 IQ-motif.Its deletion mutants iqm5-1 and iqm5-2 present a late flowering phenotype under both long day(LD)condition and short day(SD)condition.In this paper,RNAi,yeast two-hybrid,RT-PCR and RT-qPCR,and other experiments were conducted to analyze the function of IQM5 gene and its regulatory site in flowering regulation pathway.The results are as follows:1.Homozygous lines of iqm5-1 and iqm5-2 were screened by the resistant plate of Kan~+ and the molecular identification of DNA and RNA.Seeds were collected to reserve.2.We use physiological experiment to research the flowering pathway that IQM5 may be involved.After a vernalization treatment,iqm5-1 and iqm5-2 showed the same flowering ratio as wild type.Meanwhile,iqm5-1 and iqm5-2 displayed similar flowering phenotype or PAC sensitivity to wild type after gibberellic acid or PAC(paclobutrazol)treatments.Thus,the GA response and vernalization of iqm5 is intact,also,IQM5 is independent of the GA3-related pathway and vernalization pathway.iqm5 dispayed a moderate late-flowering under both LD condition and SD condition,indicating that IQM5 is not involved in photoperiod and autonomous pathway.In addition,the number of abaxial trichomes in both iqm5-1 and iqm5-2 is lower than wild type,illustrating that iqm5 delays the transition of juvenile-to-adult.The above characteristics of iqm5 were similar to that of the age-related pathwaymutants,suggesting that IQM5 might be involved in the regulation of aging flowering pathway.3.In addition,the disruption of IQM5 increases the transcript level of FLC(FLOWERING LOCUS C)in both shoot apical meristems(SAMs)and leaves,and decreases that of SPL15(SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15)in leaves and AP1 and LFY in SAMs,but it does not change the expression of other key genes in the flowering time-related pathway.These results are consistent with the physiological experimental results,which further indicate that IQM5 may be involved in the regulation of age-related pathway in Arabidopsis thaliana.4.IQM5 RNAi plant expression vector and transgenic IQM5 RNAi plants were constructed,and homozygous line were screened.RT-PCR and qRT-PCR were used to analyze the transcript expression of IQM5 and FLC between iqm5 and Ler.The results showed that,due to the endogenous FLC transcript level of Col was higher than Ler,and RNAi partly was not sufficient to affect the flowering phenotype,so IQM5 RNAi plants showed no late-flowering phenotype.5.Yeast two-hybrid experiment showed that when the L in the Q-motif was changed into S,IQM5 can not combine with CaM5,indicating that L is a key amino acid in IQM5 for CaM binding.6.The complementary expression vectors of IQM5 were constructed and delivered into iqm5-1,and the transgenic plants have been screened.7.The double mutants of iqm5 and flc have been conducted and the homozygous transgenic plants have been achieved to phenotype.