| The transition from vegetative growth stage to vegetative reproduction stage plays a key role in the flowering and development of plants.The appropriate flowering time determines the development and reproduction of plants.The complex and diverse flowering regulatory factors in plants constitute six independent and interrelated genetic regulatory pathways: photoperiod pathway,vernalization pathway,temperature pathway,gibberellin pathway,autonomous pathway and age pathway.IQM6,coded by At3g58480,is a calmodulin-binding protein(Ca MBP)in Arabidopsis,which contains an IQ motif and may be a Ca MBP,and its mutant delays flowering under long days.In this paper,involvement of IQM6 in the regulation of flowering will be studied through physiological,and genetic experiments.The results are as follows:1)Identification of mutants.The homozygote of the mutant iqm6-1 with T-DNA insertion was obtained by PCR and RT-PCR analysis.Then iqm6-1 was identified as a single copy T-DNA insertion line through backcross method.2)Preliminary determination of genetic pathways involved in flowering regulation of IQM6.In our physiological experiments,iqm6-1 showed a late-flowering phenotype in long-day condition,and had more rosette leaves than wild type Col,and these phenotypes were more obvious in short-day condition,indicating that iqm6-1 do not participate in photoperiod pathway.After treated with vernalization,flowering time and the number of rosette leaves of iqm6-1 were similar to wild type,indicating that iqm6-1did not participate in vernalization pathway.The application of exogenous GA3 could promote the flowering of both iqm6-1 and wild type Col,but did not affect the late flowering phenotype and the number of rosette leaves of iqm6-1.At the same time,the flowering process of iqm6-1 treated with GA3 was consistent with that of wild type without GA3 treatment,which indicated that iqm6-1 normally responded to Gibberellin(GA).The germination rate of iqm6-1 and Col was inhibited after PAC treatment,but recovered after PAC+GA3 treatment,indicating that iqm6-1 was not involved in the synthesis of endogenous GA.Furthermore,RT-PCR and RT-q-PCR were used to analyzethe transcriptional level of key genes regulating the genetic pathway of flowering.Compared with wild type Col,the FLC transcription level of iqm6-1 was up-regulated in leaf and shoot meristem,while the transcription levels of SPL3,AP1 and LFY were down-regulated.There was no significant difference in the transcription level of other key genes in photoperiod,vernalization,gibberellin,temperature and autonomous pathway between iqm6-1 and Col.Based on the results of gene expression and physiological experiments,involvement of IQM6 in the pathway of photoperiod,vernalization,gibberellin,temperature and autonomy was excluded.It was speculated that IQM6 might be involved in regulation of age-pathway flowering.3)Construction and identification of iqm6-2.To further confirm the fuction of IQM6 found above,another mutant line of IQM6 was constructed by gene editing CRISPR/Cas9 technology and identified as iqm6-2.iqm6-2 showed similar phenotype to iqm6-1 when the physiological experiments were conducted.Moreover,like iqm6-1,iqm6-2 displayed significantly up-regulated transcript levels of the key flowering genes of age pathway,micro RNA156 and significantly down-regulated transcript levels of SPL3,AP1 and LFY.In conclusion,IQM6 is involved in the age-pathway flowering regulation.4)Analysis of protein interaction between IQM6 and Ca M.The yeast two-hybrid experiment confirmed that IQM6 could interact with Ca M5.When the first amino acid L in IQ motif of IQM6 mutated into S,however,two proteins cannot bind with each other,indicating that IQ motif and its first amino acid L were the key to the binding of IMQ6 and Ca M.5)In addition,to further validate the flowering regulation pathway of IQM6,complementary expression lines,over-expression lines and double mutants of iqm6/flc need to be constructed.To do so,corresponding vectors have been constructed. |
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