Functional Identification Of BpFLA Family Genes And Expression Analysis Of Their Promoters In Betula Platyphylla

Fasciclin-like arabinogalactan proteins(FLAs)are a subclass of arabinogalactan proteins(AGPs)involved in plant growth,development and response to abiotic stress.FLAs are widely distributed in plant tissues and cells.At present,there is little research on FLA gene expression and function regulation.We cloned the FLA family gene and promoter in the birch,and study the expression characteristics and transformation of the FLA family gene in Arabidopsis in order to identify some members of the initial function and lay the foundation for functional research in Birch.Based on the data of the established birch xylem transcriptionsgroup and birch genomic data analysis,we identified 24 Birch FLAs,named BpFLAs.The multiple sequence alignment results show that the BpFLA family of proteins contain the FLA gene-specific FAS domain except BpFLA16,and BpFLA11,BpFLA12,BpFLA13,BpFLA21,BpFLA22 and BpFLA23 contain two fasciclin(FAS)domain.It is worth mentioning that there are three conserved regions(H1,H2,and[Y/F]H)in FAS domains.System evolution analysis results show that BpFLAl、BpFLA2、BpFLA3/17、BpFLA4、BpFLA5、BpFLA6、BpFLA7、BpFLA9/18、BpFLA10、BpFLA19、BpFLA20、AtFLA6、AtFLA7、AtFLA9、AtFLA11、AtFLA12 and AtFLA13 gather for a group.In this group,BpFLA20 has a close evolutionary relationship with AtFLA11 and AtFLA12,we can speculate it may be associated with the development of the secondary cell wall;BpFLA11、BpFLA21、BpFLA22、AtFLA15、AtFLA16、AtFLA17 and AtFLA18 gather for a group;BpFLA8、BpFLA12、BpFLA13、BpFLA14、BpFLA15、BpFLA16、AtFLA4、AtFLA 1、AtFLA2、AtFLA3、AtFLA5、AtFLA8、AtFLA10 and AtFLA14 gather for a group.In this group,BpFLA8 has a close evolutionary relationship with AtFLA4,it may be associated with tolerance,salt ions in the cell expansion,and may produce a synergistic effect with the ABA;BpFLA 12 evolution is closer to AtFLA1,and it may be related to root development and cell differentiation;BpFLA16 evolution is closer toAtFLA3,it may be related to the development of microspores;BpFLA23、BpFLA24、AtFLA19、AtFLA20 and AtFLA21 gather for a group;Transmembrane analysis showed that BpFLA 1,BpFLA6,BpFLA7,BpFLA10,BpFLA12,BpFLA13,BpFLA16 and BpFLA23 have transmembrane regions,other FLA gene does not exist transmembrane region,and is present outside the membrane.We also analyzed the expression pattern of BpFLAs in root,stem and leaf tissues.Results demonstrated that the expression level of FLAs gene in roots was higher.The expression levels of BpFLA1、BpFLA4、BpFLA6、BpFLA7、BpFLA8、BpFLA9/18、BpFLA10、BpFLA19、BpFLA20、BpFLA21 and BpFLA24 in roots were higher than those in stem and leaves,the leaf expression is the lowest;The expression levels of’ BpFLA2_BpFLA3/17.BpFLA5、BpFLA11、BpFLA13、BpFLA14/15、BpFLA16、BpFLA22 and BpFLA23 in roots were higher than those in stem and leaf,the stem expression is the lowest;The expression level of BpFLA12 in leaves was higher than that of stem and root,the stem expression is the lowest.The analysis of quantitative real-time reverse transcription polymerase chain reaction in dicated that BpFLA1O、BpFLAI11、BpFLA12、BpFLA14/15、BpFLA19、BpFLA20 and BpFLA21 was up-regulated induced by 200mM NaCl,we speculated that these genes may be involved in birch response to salt stress.And BpFLA4、BpFLA9/18、BpFLA10、BpFLA11、BpFLA12 and BpFLA13 was up-regulated induced by 300mM mannitol,we speculated that these genes may be involved in birch response to drought stress.We obtained 10 FLA family genes by RT-PCR.Constructed the overexpression vector pROK Ⅱ-FLA3/17、pROK Ⅱ-FLA4、pROK Ⅱ-FLA5,and transformed into Arabidopsis by transgenic experiments.Ultimately,we succeeded in obtaining pure and BpFLA3//17、BpFLA4、BpFLA5 transgenic Arabidopsis.The phenotypic observation of transgenic Arabidopsis showed that:BpFLA3/17、Bp FLA4、BpFLA5 overexpressing lines rosette leaves were larger than wild type plants,pumping stems early,The height of inflorescence stems is higher than that of wild type.This suggests that these three genes are involved in the growth regulation of Arabidopsis thaliana and promote the growth of leaves and stems.Based on the data of the established birch xylem transcriptionsgroup and birch genomic data analysis,we identified 24 promoters corresponding to the FLA gene of birch.The promoter of five FLA genes of birch was cloned by RT-PCR.The five promoters were targeted to replace the 35S promoter in the pBI 121-GUS expression vector to construct the FLA promoter to drive the GUS gene to the plant expression vector,and through transgenic stability into Arabidopsis thaliana.The BpFLA1、BpFLA2、BpFLA3 and BpFLA9 gene promoter was obtained by GUS expression of transgenic Arabidopsis thaliana.The transgenic Arabidopsis thaliana was stained by GUS histochemical staining,the results of temporal and spatial expression show that:The activity of the BpFLA1 promoter was weak and the transgenic plants were not stained with GUS.The BpFLA2 promoter was expressed only in cotyledons,and with the growth of plants,the senescence of cotyledons and the smaller staining area were observed.The BpFLA3 promoter and the BpFLA9 promoter were expressed at all stages of the plant.In the veins of mature leaves,the pods had the highest activity at both ends,and the expression activity of the growing site was higher than that of the mature site,This suggests that the BpFLA promoter is involved in the regulation of expression of Arabidopsis thaliana at different developmental stages and in different tissues.At the same time,the cis-acting elements of these four promoter sequences were predicted by using the Plant CAER website.The results showed that in addition to the basic transcriptional elements TATABOX and CAATBOX contained in the promoter sequences of the BpFLA1,BpFLA2,BpFLA3 and BpFLA9 genes,there are also homeopathic component HSE involved in the thermal stress response,the cis-acting regulatory element Sknl-Motif,a cis-acting element that regulates circadian rhythm regulation,and a large number of light-responsive elements(BOX I,BOX 4,G-BOX,etc.).BpFLA2,BpFLA3 and BpFLA9 genes are also present on the promoter sequence for the anaerobic induction of the cis-acting element ARE and the fungal inducer reaction element BOX-W1.BpFLA1,BpFLA2 and BpFLA3 genes also contain MYB binding sites involved in drought-inducible element MBS.The promoter sequence of the BpFLA2 and BpFLA9 genes also contains the cis-acting element ABRE involved in the abscisic acid reaction.The promoter sequence of the BpFLA1 and BpFLA3 genes also contains the homeopathic component TCA-element involved in the salicylic acid reaction.The promoter sequence of the BpFLA1 gene also contains MYBHvl binding site element CCAAT-box,an element HD-Zipl involved in mesophyll differentiation,involving element HD-Zip2,a leaf morphological control.The promoter sequence of the BpFLA2 gene also contains the TGACG-motif,which regulates the MeJA reaction and cis-acting,and the MYB binding site involves the light response element MRE,the auxin response element TGA-element and the wound response element WUN-motif.The promoter sequence of the BpFLA3 gene also contains the cis-acting element GCN4-motif,which is involved in the expression of endosperm,and the component TATCBOX of gibberellin response.The promoter sequence of the BpFLA9 gene also contains the AT-rich element of the AT-enriched DNA-binding protein(ATBP-1),the GARE-motif of the gibberellin response,and the cis-element LTR involved in low temperature reactivity.These indicate that BpFLA1,BpFLA2,BpFLA3 and BpFLA9 genes may be involved in these controls.