Preliminary Study On The Function Of Frigida-like Gene Family Members Related To Flowering In Chimonanthus Praecox

The flowering time of Calycanthaceae plants varies greatly,among which,wintersweet is delayed until winter to blossom,which makes wintersweet an ideal winter flower and woody plant,and is widely used in urban and rural landscape construction.However,there are few researches on the particularity of wintersweet in flowering time.According to the results of gene identification of the development pathway of wintersweet in our laboratory,it was found that FLC gene,a negative regulator of plant flowering,was absent in wintersweet,but there was a FRI gene closely related to the regulation of FLC.Therefore,in this study,Cp FRI gene was selected as a candidate gene to affect the flowering time of wintersweet.By further studying the function of Cp FRI gene,the role of this gene in the flowering process of wintersweet was preliminarily analyzed.The main research results are as follows:1.Based on the gene database of wintersweet H29 genome and transcriptome,a total of five FRI similar genes were selected.These five genes were selected as the representative genes of wintersweet FRI in this study,and they constituted members of the FRIGIDA-LIKE gene family,named Cp FRI1,Cp FRI2,Cp FRI3,Cp FRI4 and Cp FRI5 respectively.2.The evolutionary analysis of FRI gene showed that all the FRI genes of arabidopsis were clustered together with the FRI gene of wintersweet except the At FRL5 member of FRIGIDA-LIKE gene subfamily V in arabidopsis.Cp FRI1 and Cp FRI3 were clustered together with At FRL3,a member of the FRIGIDA-LIKE gene subfamily III in arabidopsis,and the homology was 100% and 83% respectively.Cp FRI2 was clustered together with At FRL1/2,a member of arabidopsis FRIGIDA-LIKE gene subfamily II,and the homology was 40%.Cp FRI4 was clustered together with At FRI,a member of the FRIGIDA-LIKE gene subfamily I in arabidopsis,and the homology was 77%.Cp FRI5 was clustered together with At FRL4a/4b,a member of the FRIGIDA-LIKE subfamily IV in arabidopsis,and the homology was 36%.3.The FRI gene was cloned using the c DNA of wintersweet H29 as the material.The ORFs of Cp FRI1,Cp FRI2,Cp FRI3,Cp FRI4,Cp FRI5 were 1626 bp,1689bp,1683 bp,1647bp,1632 bp respectively,and coding respectively 541,562,560,548,543 amino acids.The length of their FRIGIDA conserved domains is basically the same,consisting of 287,277,287,288,288 amino acids.4.The Cp FRI1,Cp FRI2,Cp FRI3,Cp FRI4 and Cp FRI5 gene p CAMBIA2300 overexpression vectors were constructed and expressed ectopic in model plant tobacco and arabidopsis respectively.It was found that there was no significant difference in flowering time between transgenic tobacco and empty vector tobacco,while the flowering time of each transgenic arabidopsis was significantly later than that of empty vector.Therefore,the functions of FRIGIDA-LIKE gene family members in wintersweet are not only highly similar,but also different in the regulation of flowering time between tobacco and arabidopsis.It is speculated that this difference may be related to whether plants need vernalization or not.The realization of late flowering function of the FRI gene in wintersweet requires to be supported by the demand of target plant for vernalization,so that the FRI gene can effectively act on the FLC gene and thus delay the flowering time of plants.5.The expression levels of different tissues and different flower development stages of wintersweet were analyzed by quantitative PCR.It was found that the expression levels of Cp FRI1,Cp FRI2,Cp FRI3,Cp FRI4 and Cp FRI5 genes in three different organs reached the maximum value in flowers,indicating that the FRIGIDA-LIKE gene family members of wintersweet have important biological significance in regulating plant flowering.The expression levels of Cp FRI1,Cp FRI2,Cp FRI3,Cp FRI4 and Cp FRI5 genes did not change significantly during the three flower development stages.The results showed that the low temperature experienced will not affect the expression of Cp FRI gene during nearly three months from bud to bloom of wintersweet,which also indirectly implies that wintersweet FRIGIDA-LIKE gene family members play a decisive role is not in the flower development stage,but is more likely to be in the flower induction stage before the flower development stage.