Transcriptional Regulation Mechanism And Function Of DiASR1 Gene In Davidia Involucrata

Dove tree（Davidia involucrata Baill.）is a single genus in the family Nyssaceae.It is both the endemic species to China and the world-famous ornamental plants.Both male and bisexual flowers can be found on the dove tree at full-blossom stage,and its inflorescence is a subglobular capitate with paired papery,rectangular,obovate bracts at the base.It is famous for the bracts,which are shaped like the wings of a dove.However,the regulatory mechanism of specific character formation in the bracts of dove tree is still unclear.To study the regulatory mechanism of bracts development in dove tree,a transcriptome sequencing of the bracts and leaves was performed,and an ASR homologou gene（Di ASR1）encoding Abscisi acid Stress Ripening protein（ASR）with specific expression in the bracts was identified.The 35S promoter was used to drive the overexpression of Di ASR1 gene in Arabidopsis,and the leaves of transgenic Arabidopsis showed obvious albino similar to that of bracts.Besides,the transgenic Arabidopsis could not obtain offspring due to the albino of leaves,suggesting that the constitutive expression of this gene has a lethal effect on the transgenic plants.Therefore,in this study,the natural promoter P_{Di ASR1} was used to drive the genetic transformation of Di ASR1 in Arabidopsis.Base of previous studies,the regulation mode and function of this gene were further explored to reveal its role in the development of bracts in dove tree.The main findings are as follows:1.The results of subcellular localization showed that Di ASR1 was localized in the nucleus and cell membrane.2.Yeast hybridization confirmed that Di ASR1 had transcriptional activator activity,and Di ASR1 might be act as a transcription factor with its transcriptional activator located at the N terminal.3.GUS gene was transformed into Arabidopsis driven by the promoter of Di ASR1,which was named as P_{Di ASR1}.GUS histochemical staining showed that the chromogenic sites were concentrated in young leaves,bud and stem,indicating that P_{Di ASR1} was mainly expressed in tissues with strong division.Besides,the analysis of cis-acting elements showed that P_{Di ASR1}has cis-acting elements involved in environmental responses such as light,temperature,GA₃,and drought.And the P_{Di ASR1} transgenic Arabidopsis was treated with different treatment such as light,high illumination intensity,darkness,high temperature,low temperature,high salt,drought and hormones,and then stained and observed.The results showed that the expression of the P_{Di ASR1} was induced by high illumination intensity,high temperature and ABA,inhibited under dark,low temperature,high salt,drought and GA₃ treatment.4.Phenotypic observation of transgenic Arabidopsis showed that the Di ASR1 gene caused early reproductive growth,abnormal inflorescence structure,shortened filaments,elongated styles,and low seed setting rate.In addition,the leaves of transgenic plants showed obvious bleaching.The bleaching area started from the tip of the leaf and gradually covered the entire leaf.The process was similar to bracts that of dove tree.In the initial albino area,there was no differentiation of barrier tissue,and its cell structure was more compactly arranged than the normal area,but most of the cells were wilted and shrunken.In the mid-albino phase,the number of chloroplasts in the leaf cells decreased,the chloroplast membrane ruptured,the thylakoid ablated,the matrix lost,and the osmophilic droplets became larger.In the late albino period,there were two different cell structures formed by leaf epidermal cells.One was the puzzle-shape structure similar to that of bracts epidermal cells in dove tree,and the other was a polygonal structure common to epidermal cells in ordinary leaves.In summary,the expression of Di ASR1 gene is strictly regulated by environmental factors,and the expression pattern of the P_{Di ASR1} was consistent with the change of environmental factors in the early development of bract,suggesting that the Di ASR1 gene was specifically accompanied by the function of the bract development.The heterologous expression of this gene in Arabidopsis can shift vegetative growth into productive growth earlier and lead to leaf bleaching,similar to the bleaching of bracts in the dove tree.It proved that the Di ASR1 gene plays an important role in the occurrence and growth of the sliver,especially in the process of albino.