Research On The Regulation Of Transcription Factors Related To Sugar Beet Bolting Based On WGCNA Screening

Vernalization is one of the important factors that determine the growth period and planting distribution in sugar beet.It is of great significance to cultivate anti-vernalization sugar beet varieties for early sowing in the north and winter sowing in the south,and to improve the yield of sugar beet and sugar.Bioinformatics,RNA-Seq and molecular biology experiments were combined to explore the transcription factors that regulate bolting and flowering in sugar beet under vernalization in this study.After vernalization in sugar beet KWS9147,the key genes regulating bolting and flowering were screened through determination of agronomic trait,phylogenetic analysis of flowering homologous genes and qRT-PCR analysis.RNA-Seq was further performed to identify the DEGs after vernalization,and WGCNA and plant TFDB were used to determine the transcription factors that bind to the key genes of bolting and flowering,and molecularly verified them.The results showed that:（1）Through the agronomic traits BI,BD and expression patterns of flowering-related genes in sugar beet with different degrees of vernalization,the vernalization process was divided into three stages,namely,before vernalization（0 w）,insufficient vernalization（0 w-10 w）and sufficient vernalization（after 10 w）.Among them,the expression patterns of BvGI and BvFVE1 had a certain correlation with the change trends of agronomic traits BI and BD,and there were significant changes between insufficient and sufficient vernalization stages,and the gene expression level did not change significantly with the recovery of room temperature culture.Therefore,we believed that BvGI and BvFVE1 were involved in the regulating of vernalization response in sugar beet.（2）The transcriptomes of sugar beet before vernalization（0 w）,insufficient vernalization（2 w and 6 w）and sufficient vernalization（10 w and 14 w）were analyzed.A total of 7586 DEGss were identified and divided into 18 co-expressed modules by WGCNA.The Co-expression modules highly correlated with BvGI and BvFVE1 were further analyzed using plant TFDB,and three transcription factors with binding sites for BvGI were screened,namely BvLHY,BvCRF3 and BvTCP4,and BvNLP7 with binding sites for BvFVE1.（3）Using transient transformation systems in tobacco cells,it was proved that BvLHY,BvCRF3,BvTCP4 and BvNLP7 were all nuclearlocalized transcription factors.Yeast one-hybrid assay showed that the HIS3 reporter gene of pHIS2-pro GI and pHIS2-pro FVE1 recombinant vector could be expressed in yeast cells.After co-transformation of transcription factors and adding a certain amount of 3-AT inhibitor,HIS3 reporter gene could be expressed in the control group,but could not be expressed in the experimental group.This indicated that BvLHY,BvCRF3 and BvTCP4 inhibited the expression of BvGI,and BvNLP7 inhibited the expression of BvFVE1.At the same time,we also proved that BvCRF3 played an inhibitory role by binding to the WRE3 motif in the BvGI promoter region through the Yeast one-hybrid promoter motif experiment These results provide an important theoretical basis and technological reserve for breeding vernalization-insensitive sugar beet and benefit for early spring sowing and late autumn harvesting in sugar beet production.