Genome-wide Identification Of BES1 Transcription Factor Family In Potato(Solanum Tuberosum.l)and The Construction Of StBES1-2 Transgenic Potato Lines

BES1(bril EMS SUPPRESSOR 1)is a class of important transcription factors in the downstream of brassinosteroid(BR)signaling pathway,which play an important role in promoting plant growth and response to stresses in Arabidopsis thaliana and rice.At present,the BES1 transcription factor family has been identified in many plants such as Arabidopsis and rice.Potato(Solanum tuberosum L.)is the fourth largest crop after maize,rice and wheat in china,which is rich in nutrients.Many studies have been reported on the regulation of BR on potato growth and development,but BES1 family in potato has not been systematically studied.In this study,BES1 gene family was identified in potato at the genome-wide level by bioinformatics method and the sequence characteristics and phylogenetic relations of StBES1 transcription factor family proteins,as well as the structure,conserved motif and reaction elements of StBES1 transcription factor family genes were analyzed.The expression patterns of StBES1 family genes were analyzed by qRT-PCR,and StBES1 transcription factors were verified by subcellular localization and transcriptional activation essay.Three overexpressed StBES1-2 potato lines were obtained by genetic transformation of potato,the study laid a foundation for further understanding the role of BES1 in potato tuber formation and resistance to adversity.Main research results are as follows:1.A total of 9 BES1 protein sequences,named StBES1-1 to StBES1-9,were obtained by searching the whole genome of potato.The length of amino acid sequence was 182～695,the isoelectric point was 5.41～9.11,and the hydrophilic value was-0.934～-0.372,The sequences of the nine BES1 genes were amplified and sequenced using the cDNA of potato variety"Desirée" as a template.The results showed that in addition to StBES1-8,StBES1-2,StBES1-3,StBES1-4 and StBES1-5 proteins had one mutation in amino acid,and StBES1-1,StBES1-6,StBES1-7 and StBES1-9 had synonymous mutations.2.The characteristics of StBES1 protein sequence were analyzed.and the results showed that except StBES1-7 which had no basic motif.the other 8 StBES1 sequences all had bHLH domain that could bind to the e-box region of the target gene promoter.All StBES1 proteins had different BIN2 phosphorylation sites(6-15).The analysis of StBES1 gene structures revealed that StBES1-1 and StBES1-5 had 10 and 12 exons respectively,while the remaining seven StBES1 sequences all had two exons.Analysis of the promoters of StBES1 genes revealed that they contained cis-acting elements capable of responding to hormones and stresses.3.Expression patterns of StBES1 genes were analyzed by qRT-PCR.Tissue expression analysis showed that StBES1-2 and StBES1-8 had high expression level in roots,stems,leaves,stolons and tubers,among which StBES1-2 had the highest expression level in tubers,indicating that StBES1-2 may be involved in potato tuber formation.StBES1-1 and StBES1-5 were generally low in each organ.StBES1-4 and StBES1-9 were highly expressed in stem and may play a role in stem elongation.The expression of StBES1-3 was the highest in roots,which was nearly 20 times higher than that in tubers.The expression of StBES1-6 was the highest in leaves.Hormone treatment essays showed that the expression of StBES1 was significantly down-regulated by BR.Except StBES1-4,the expression of other StBES1 genes was up-regulated or down-regulated under the induction of IAA.SA,GA and ABA treatment up-regulated or down-regulated the expression of all StBES1 genes.Stress treatment essays showed StBES1-2,StBES1-6 and StBES1-8 were significantly up-regulated under the treatments of cold,heat,salt and drought,and the most significant up-regulation was observed under the stress of heat.StBES1 StBES1-5 and StBES1-9 were significantly down-regulated under heat stress.These results suggest that StBES1 may play an important role in the regulation of potato hormone and the resistance to stress.4.Subcellular localization of 9 StBES1 transcription factor proteins was conducted,and the results showed that all these proteins were located in the nucleus and cytoplasm.Yeast monoheterozygotic test showed that all 9 StBES1 had transcriptional activity,among which StBES1-1 and StBES1-5 had a lower growth rate on the SD/trp-his plate,and a light blue color on the SD plate containing x-a-gal,indicating that the transcriptional activity of StBES1-1 and StBES1-5 was lower than that of other transcription factors.5.Three PCR-positive overexpressed StBES1-2 potato lines,A3,A4 and A8,were obtained through genetic transformation.QRT-PCR analysis showed that the expression of StBES1-2 in the three transgenic potato lines was significantly higher than that in the control.