Functional Analyses Of Vernalization Related Gene TaVRN2

Floral transformation plays an important role in sexual reproduction of plants.Wheat as an inportant food crop,vernalization pathways and photoperiod pathways are very important ways to affect flowering.In order to further study the role of wheat VRN2 gene in wheat flowering,the wheat VRN2 gene expression pattern,circadian rhythm,and vernalization response of wheat were studied using Yannong 15 wheat cultivar as materials,meanwhile,Agrobacterium-mediated transgene technology was used for the second transformation.Brachypodium distachyon preliminarily verifies the function of wheat VRN2 gene.The main results are as follows:?1?We cloned and isolated homologous genes from the wheat VRN2 gene locus from wheat cultivars.We named them ZCCT1-A1,ZCCT1-B1,ZCCT1-D1 and ZCCT2-A2,ZCCT2-B2,ZCCT2-D2.The homologous amino acid sequences of these homologs are as high as 95-96%.By homology analysis,we found that the TaVRN2 protein isolated from wheat was highly homologous to homologous proteins TmZCCT1 and TmZCCT2 in wheat and Hv ZCCT-Ha and Hv ZCCT-Hb in barley.Between 76-86%.Further amino acid sequence alignments revealed that they all contained a typical CCT-domain region and a zinc finger structure.However,the difference is that specific site-specific point mutations occur in the CCT-domain of the wheat VRN2 protein.?2?Analysis of the expression pattern of VRN2 gene in Yannong 15 wheat variety.The overall expression patterns of ZCCT1 and ZCCT2 were found to be similar,with high expression levels during the vegetative stage and low levels at the heading stage,with almost no signal detected.Expression at different sites revealed that the expression was highest in young leaves and low in old leaves.However,almost no expression was detected in flag leaves,stems,panicles,and roots.At the same time,real-time fluorescence quantitative analysis of ZCCT1-D1,ZCCT2-B2 and ZCCT2-D2 revealed that the expression levels in different sites were similar to the overall expression patterns of ZCCT1 and ZCCT2.?3?The wheat TaVRN2 gene has a typical CCT-domain,which participates in the photoperiodic pathway and participates in flowering.Therefore,we detected the circadian rhythm of wheat TaVRN2 gene.It was found that the expression patterns of ZCCT1 and ZCCT2 are similar in both long-day and short-day conditions and have a certain rhythm.And ZCCT1 reaches the peak 2 hours earlier than ZCCT2.Real-time fluorescence quantitative analysis of ZCCT1-D1,ZCCT2-B2 and ZCCT2-D2 showed that their expression trends were consistent with the overall expression trends of ZCCT1 and ZCCT2.?4?Since the TaVRN2 gene is involved in the vernalization pathway of wheat flowering,we tested the vernalization response of wheat TaVRN2 gene.Studies have shown that both ZCCT1 and ZCCT2 respond to vernalization.At the same time,real-time fluorescence quantitative analysis of ZCCT1-D1,ZCCT2-B2 and ZCCT2-D2 revealed that the vernalization response pattern was similar to the overall expression pattern of ZCCT1 and ZCCT2.?5?Overexpression of ZCCT1-A1,ZCCT1-B1,ZCCT1-D1 and ZCCT2-A2,ZCCT2-B2,and ZCCT2-D2,found TaZCCT1-A1,TaZCCT1-B1 and TaZCCT1-D1 overexpression plants in long daylight Under the condition,there is obvious late flower phenotype,and the late flower phenotype can be reversed by vernalization.The TaZCCT2-A2,TaZCCT2-B2 and TaZCCT2-D2 overexpressed plants showed no flowering phenotype under long-day conditions and could not flower under saturated vernalization conditions?8 weeks?,prolonged vernalization time to 14 weeks,TaZCCT2-B2 still can't flower.?6?Quantitative analysis of expression levels of other flowering genes in over-expressed plants.qRT detection results of over-expression of wheat TaZCCT1-D1 and TaZCCT2-D2gene Brassica napus plants in T₀ generation after vernalization related flowering genes showed TaZCCT1-D1 overexpression of VRN1 and FT almost restored WT expression,while TaZCCT2-D2 overexpressed VRN1 and FT had very low expression.