Functional Analysis Of FCA And FLC-Like Gene In Brachypodium Distachyon L.

The growth of plants is divided into vegetative growth and reproductive growth.The latter is particularly critical,and the flowering transformation from vegetative growth to reproductive growth which is mainly regulated by external environmental signals and genetic factors of endogenous development.Appropriate flowering time has a decisive effect on the level of plant yield and quality.There are six major pathways regulating flowering time in Arabidopsis:vernalization pathway,autonomous pathway,photoperiod pathway,gibberellin pathway,age pathway and temperature pathway.The FCA gene is a key gene in the autonomous pathway and is a flowering promoting gene.Compared with AtFCA,OsFCA cloned from rice has high homology but whether OsFCA gene regulates flowering time is still not clear.However,in the major temperate grass plants such as wheat,barley and Brachypodium distachyon,whether or not the FCA gene functions,if there is a function,which gene is downstream of the FLC gene,there are few reports on these issues.Brachypodium distachyon is a new type of temperate grass model plant,closely related to major grass crops such as wheat and barely,and has similar advantages to the dicotyledonous Arabidopsis thaliana:short growth cycle,small genome size,simple growing conditions and self-pollination,tissue culture technology is quite mature.Therefore,the study of the function of Brachypodium distachyon genes helps us to better understand the gene functions of wheat and other food crops.It has been predicted in the literature that the three genes,BdMADS37,BdODDSOC1,and BdODDOSC2,may be the FLC-Like gene,and it was recently determined that BdODDOSC2 is the FLC gene in Brachypodium distachyon.Therefore,in this study,we mainly used reverse genetics to construct BdFCA RNAi vector and overexpression vector to study the function of BdFCA gene and preliminary study of the function of BdMADS37 and BdODDSOC1.The main results are as follows:?1?The FCA genes in Brachypodium distachyon were relatively homologous to barley,wheat and Aegilops tauschii.BdFCA can encode two RRM RNA binding proteins and one WW protein binding domain with very high conservation.?2?BdFCA RNAi transgenic lines showed late flowering,indicating that the FCA gene in Brachypodium distachyon is functional.After 8 weeks of vernalization of the BdFCA RNAi transgenic line,the late flowering phenotype could be restored,indicating that there is an autonomous pathway regulated by the FCA gene in Brachypodium distachyon.?3?AtFCA overexpression was slightly early flowering,but Ubi::BdFCA T₁ transgenic plants had no obvious phenotype compared to wild type.We need to obtain BdFCA-transgenic homozygous plants and further investigate whether overexpression of the gene affects plant flowering by observing the phenotype and related gene quantification.?4?Phylogenetic tree analysis revealed that the BdMADS37 gene is closely related to AetMADS51-like gene.Compared to Arabidopsis FLC,the encoded protein of BdMADS37contains a highly conserved MADS-box domain.?5?BdMADS37 gene was expressed in various tissues and organs of plants at different growth stages,but the expression level was relatively low in stem apical.The expression level of BdMADS37 gene was higher in the vegetative growth period than in the heading stage.These results suggest that the BdMADS37 gene may play a role in the period of vegetative growth.?6?BdMADS37 mutant obtained using the CRISPR-Cas9 technique has no apparent phenotype compared to the wild type.This indicates that the BdMADS37 gene does not affect flowering time and is not an inhibitor of flowering.?7?The phenotype of Ubi::BdMADS37 transgenic line under long day conditions compared with the wild-type shows:slightly early flowers,dwarf,the number of branches increased number of main branch,the total number of spikelets increased,the seeds became smaller.?8?Under long-day conditions,the Ubi::BdODDSOC1-OXT₀ transgenic plants showed late flowering compared to the wild type plants.Since we obtained too few transgenic lines,we needed to obtain more transgenic lines and related data to prove the reliability of results.