Studies On Dof Transcription Factors From Moso Bamboo (Phyllostrachys Edulis) Responding Abiotic Stress And Influencing Flowering Initiation

Moso bamboo(Phyllostrachys edulis)is the most widely distributed and extensive use among the bamboo species,it is one of the dual-use bamboo species that combines economic,ecological and social benefits.However,moso bamboo is a plant that flowers only once in the lifetime,and it has a long flowering cycle,and the flowering period is uncertain,and collectively die after flowering,causing serious economic losses and ecological damage.The molecular mechanism of the regulation of flowering time is still unclear until now,the synergistic effect of drought and photoperiod on the flowering mechanism of moso bamboo has not been reported.Therefore,it is not only of great scientific value but also of great biological significance to study the regulation mechanism of moso bamboo flowering initiation.Based on the genomic database of moso bamboo,this study uses bioinformatics as a starting point to identify and express pattern analysis of the Dof transcription factor family from the whole genome.The key Dof genes related to abiotic stress and flowering initiation of moso bamboo were screened for functional verification,the downstream flowering regulatory factors and interaction proteins were preliminary detected and isolated,preliminary exploration of the molecular mechanism of drought and photoperiod regulation moso bamboo flowering.The main results are as follows:1.Identification and expression pattern analysis of Phe Dof transcription factor family in moso bambooThrough the 2018 version of moso bamboo genome data,the Dof transcription factor family was comprehensively identified and analyzed.A total of 65 Phe Dof transcription factors were identified,and 39 new Dof transcription factors were identified compared with the 2013 version of the genome.The 65 Dof genes were divided into four subfamilies,all of which contained a conserved zf-dof domain;and the gene structure and protein motif,and separation time were further comprehensive analyzed.The results of tissue-specific assay showed that most of Phe Dofs had the highest expression in the flowering leaves,flower buds and the earlystages of moso bamboo flower development,and were tissue-specific.Under the conditions of photoperiod treatments,the Phe Dofs showed a significant diurnal oscillation expression pattern,most of the Phe Dofs reached a peak in the initial stage of light,and the transcription levels were reduced to the lowest at dusk.Most of Phe Dofs genes were up-regulated after drought and low temperature treatments,and down-regulated after 200 m M Na Cl treatment.It is indicated that Phe Dofs were involved in the growth and abiotic stress response of moso bamboo and has many biological functions.2.Isolation and function preliminary research of the Phe Dof transcription factors from moso bambooSix Phe Dofs genes(Phe Dof1,Phe Dof2,Phe Dof4-1,Phe Dof12-1,Phe Dof12-2,Phe Dof26)were isolated from moso bamboo by molecular biology method,having a conserved single zinc finger domain.Structural analysis revealed that only Phe Dof1 contained an exon,the remaining 5 genes contained two exons and one intron.Subcellular localization showed that six Phe Dofs genes were all located in the nucleus and had transcriptional activity.Phe Dofs took part in ABA and GA3 stress responses.3.Abiotic stress function research of Phe Dof transcription factors in transgenic ArabidopsisGUS staining showed that Phe Dof4-1 was expressed in the roots and hypocotyls of transgenic Arabidopsis,and Phe Dof12-1 was detected in the roots,hypocotyls,leaf vascular bundles,stamens and petals of transgenic plants.Under drought stress,over-expressing Phe Dof12-1 transgenic Arabidopsis seeds had earlier germination time,faster growth rate and had longer roots than wild type,which increased the drought tolerance of transgenic Arabidopsis.Under the condition of 150 m M Na Cl,salt stress tolerance of transgenic Arabidopsis was enhanced after overexpression of Phe Dof26 and RNAi interference,however,antisense expression of Phe Dof26 reduced salt stress tolerance in transgenic Arabidopsis.Under drought and salt stresses,overexpression of Phe Dof4-1 resulted in low germination rate,slow growth and seedling yellowing of transgenic Arabidopsis,stress-related genes CBF2,CBF3,COR15 A,KIN1,RD22,DREB2 A,ERD10,and ZAT12 genes were down-regulated,reducing resistance of transgenic Arabidopsis.In contrast,antisense expression of Phe Dof4-1leaded to up-regulation of stress-related genes and increased tolerance to drought stress in transgenic Arabidopsis.By Yeast two-hybrid library,the resistance-related protein Phee IF5 A was initially screened to interact with Phe Dof4-1.4.Study on the flowering initiation function of Phe Dof genes in transgenic ArabidopsisThe results of q RT-PCR showed that Phe Dof12-1 was highly expressed in the flower bud formation stage during the flower development of moso bamboo,non-flowering leaves and bracts.Overexpression of Phe Dof12-1 promoted early flowering of transgenic Arabidopsis,and flowering formation factors FT,SOC1,AGL24 were up-regulated,and the flowering inhibitors FLC and FVP were down-regulated,indicating that Phe Dof12-1 was a flowering promoting factor.In contrast,overexpression of Phe Dof4-1 and Phe Dof26 delayed the flowering time of transgenic Arabidopsis,they played a negative regulatory role;however,Phe Dof26 RNAi interference plants had significantly earlier flowering time,and induced flowering-related gene expression.Phe Dof26 not only affected the flowering time of transgenic Arabidopsis,but also played a role in leaf senescence.Compared with wild-type Arabidopsis,RNAi interference plants resulted in leaves yellowing,and most of rosette leaves were dry,induced leaf senescence.While overexpression of Phe Dof26,transgenic plants with green leaves and delayed senescenc.Yeast one-hybrid assay,the regulatory relationship between Phe Dofs transcription factors was preliminarily identified,Phe Dof1 combined the Phe Dof12-2promoter;and Phe Dof1,Phe Dof12-2,and Phe Dof26 could combine with the promoter of Phe Dof4-1.Phe Dof1 and Phe Dof26 could interact with each other.Phe Dofs regulated the transcription of downstream genes Phe COL4,Phe COL3 and Phe FD.It is speculated that Phe Dofs may regulate the flowering of moso bamboo by regulating the expression of Phe COLs and Phe FD genes.5.Preliminary identification of Phe Dofs regulating flowering pathway of moso bambooAt CDFs binded to the promoters of CO and FT to inhibit genes expression in Arabidopsis and were negative regulators of CO and FT.Y1 H results showed that multiple Phe Dofs can be combined with Phe COLs and Phe FD promoters.It is speculated that the regulation of CO andFT by Dof is conservative in moso bamboo,therefore,the Phe COL gene family was identified and excavated.14 Phe COLs genes were screened,and most of the Phe COLs were expressed in the leaves of the bamboo,Phe COLs showed diurnal oscillation expression pattern and participated in the photoperiod response of moso bamboo.The homologous genes Phe COL14 and Phe FT of Arabidopsis CO and FT were isolated from moso bamboo,Phe COL14 was localized in the cell membrane and had no transcriptional activity.Phe FT was both localized in the nucleus and cell membrane,and had transcriptional activity.The results of Yeast one-hybrid showed that Phe COL14 and Phe FT could bind to the promoters of Phe COL3 and Phe FD.Yeast two-hybrid experiments showed that Phe Dof12-1 and Phe Dof4-1 interacted with Phe COL14 and Phe FT,respectively.It was speculated that Phe Dofs may regulate the flowering of moso bamboo by regulating the expression of Phe COLs and Phe FD genes.This study initially revealed a possible network pathway of Dof,CO and FT interaction in regulating moso bamboo flowering.In summary,moso bamboo was used as the research object in the study,and the functions of moso bamboo Dof transcription factor to regulate abiotic stress response and flowering initiation were studied by bioinformatics and molecular biology methods.The results showed that six key Phe Dof genes played an important role in abiotic stress and photoperiod flowering regulation.Phedof12-1 improved drought stress tolerance and induced early flowering of transgenic Arabidopsis,which played a positive regulatory role;Phe Dof4-1 and Phe Dof26 prolonged the flowering time of Arabidopsis,they were negative regulation factors.Phe Dof4-1could interact with the resistance-related protein Phee IF5 A and the flowering inducing factor Phe FT,Dof transcription factors regulated each other and the transcription of CO and FD.The results of this study provided scientific basis for the research on resistance breeding and molecular regulation mechanism of moso bamboo flowering.