Function Of The VERNALIZATION1Gene And The Analysis Of Its Direct Targets In Brachypodium Distachyon L

VRN1is the decisive gene for the transition of temperate gramineous crops from vegetative growth to reproductive. It responses to vernalization and plays an important role in heading and the development of floral organ in wheat. Dues to its large genome, long growth cycle and difficult genetic transformation, At present, the functional analysis of VRN1for temperate gramineous mainly based on the mutants of VRN1. However, as the key vernalization response genes in temperate gramineae, it really needs to be further studied for the specific function of VRN1in the course of the entire flowering regulation network and the upstream and downstream relationship of the three key flowering rugulation genes VRN1, VRN2and FT.This study mainly based on tne material of Brachypodium distachyon ecotype Bd21. In order to explore the function of the BdVRN1, cDNA of VRN1was cloned and the two over-expression vectors were built with the promoters of35SCaMV and ZmUbi respectively. Both the two over-expression vectors were used for the genetic transformation experiment of the wild ecotype Bd21. The ZmUbi promoter, stronger in temperate gramineous, could lead to early flowering transgenic plants and more than95%of the tissue culture seedlings flowered in its differentiation stage and even some leaves of the early flowering plants trended to spike differentiation. Compared with the wild type, the transgenic plants promoted by35SCaMV flowered earlier for one or two leaves. This indicated that the degree of early flowering promoted by VRN1was closely related with the expression of VRN1. The higher expression of exogenous VRN1gene, the better effect promoted by VRN1and the earlier flowering time.In order to further explore the function of VRN1, this study also made an overexpression experiment of BdVRN1promoted by ZmUbi under short days. Over-expression of BdVRN1could lead to early flowering and the early flowering transgenic plants could be seen in the differential medium as well under short days. Owing to the low level expression of endogenous flowering promoting factors in short days, such as FT, VRN1and so on, the early flowering phenotype of transgenic plants was a bit weaker than LD’s. Only about5%of the plants flowered in the stage of differentiation in SD. It showed that VRN1is the key flowering regulation gene both in SD and LD.The wheat mvp mutant displayed never blossom. In order to further prove the function of the BdVRNl, the BdVRN1RNAi vector was constructed and genetic transformation experiment was done. Corresponding to its different effect of silence, the transgenic plants of BdVRN1RNAi showed different degrees of the later flowering phenotype and it flowered with9to15leaf numbers. This further proved that VRN1indeed could promote flowering and it played a key role in plant flowering regulation.Over-expression of BdVRNl in Brachypodium distachyon could lead to early flowering both in SD and LD. However, there was no no obvious abnormalities in its early flower organ, indicating that there were some direct downstream target genes of BdVRN1which played a key role in floral organ development. To further explore the molecular mechanism of flowering regulation, the expression of some key flowering related genes were tested in the transgenic plants. All the genes we tested in the study, especially the two FT homologous genes, did not displayed any changes both in the over-expression and RNAi transgenic plants. This indicated that FT may locate in the upstream of VRN1and increase the expression of VRN1to promote flowering in temperate gramineous.To explore the direct downstream target genes of VRN1, the over-expression vectors of VRN1with fused tag were constructed. The over-expression experiment with fusion protein showed that only the vector of BdVRNl-3xHA had no effect on the function of VRN1and the transgenic plants appeared early flowering phenotype. The vectors with tags in the front end and both ends colud not be used in our following experiments. They destroyed the function of VRN1and the transgenic plants displayed no early flowering phenotype. In addition, this research also made an exploration for the estrogen induced experiment. The results of the induced experiment showed that the effect of induced resistance was the best and the target gene was the brightest of the plants after8hours disposed with estrogen.