Functional Study Of LbSAD2 In Salt Gland Development Of Limonium Bicolor

China has a large area of saline land,two thirds of which can hardly be used,and the area of saline land is still expanding.Too high salinity makes it impossible for crops to grow normally,and also induces a series of environmental problems.The utilization or improvement of saline land can not only increase land resources,but also improve environmental conservation and water and improve environmental quality.Halophytes have been widely studied for their ability to grow normally on saline soils,and even for their ability to benefit from moderate amounts of salt.According to different mechanisms of salt resistance,halophytes can be divided into three types,among which recreto-halophytes adapt to the salt environment using special structures to expel excess salt out of the body.Our experimental material is a halophyte with special salt-secreting structure of salt glands.Limonium bicolor’s salt glands consist of a complex of sixteen cells and its secretory function and apparent characteristics have been studied in details,but its specific developmental mechanism remains to be further studied.In the current study,based on previous basic studies on LbSAD2 gene,the function of LbSAD2 gene in salt gland development of L.bicolor was further studied,and the downstream interacting genes and upstream regulatory genes of LbSAD2 gene were screened,for the purpose of exploring the function of LbSAD2 gene in salt gland and its effect on the development of salt gland.The main experimental results are as follows:1.LbSAD2 is located in the salt gland,and positively regulates the development and distribution of salt gland of Limonium bicolorLbSAD2 gene is highly expressed and sensitive to salt stress during the development of salt glands.In situ hybridization showed that LbSAD2 gene was localized in salt glands.LbSAD2promoter(2000 bp)was cloned,and a GUS expression vector driven by LbSAD2 gene specific promoter was constructed to transform Arabidopsis thaliana.Histochemical staining showed that LbSAD2 was expressed in the whole plant,especially in young leaves,lateral roots and epidermis.The results indicated that LbSAD2 gene is related to the growth and development of plant especially the early development of epidermal structure.The effects of LbSAD2 on the development of salt glands of L.bicolor were studied by VIGS gene silencing experiment and overexpression technique,supplemented by observing the spontaneous fluorescence of salt glands.The VIGS gene silencing technique was used to construct p TRV2-LbSAD2 silencing vector,and the expression level was identified and phenotype was observed after injection of L.bicolor.The experimental results were compared with the control group,and the number of salt glands,salt secretion ability and stress resistance were significantly reduced after LbSAD2 gene silencing.At the same time,the 1300 overexpression vector of LbSAD2 gene was constructed to transform the L.bicolor,and the expression level was identified after screening the overexpression strain on hygromycin.It was found that the number of salt glands increased after the overexpression of LbSAD2 gene.Compared with the control group,the number of salt glands at the paraxial surface increased significantly,which was significantly more than that at the distal end,while the number of salt glands at the proximal end of the control group was less than that at the distal end.It was comprehensively analyzed that LbSAD2 was related to the development of salt glands and salt secretion of salt glands.LbSAD2 played a positive role in the development of salt glands and affected the distribution of salt glands in leaves.2.Screening and verification of upstream regulatory genes of LbSAD2The p Ab Ai vector carried LbSAD2 gene specific promoter was constructed and the optimal concentration of Ab A was further confirmed.The candidate genes were selected through yeast nuclear library,and the full-length candidate genes were cloned and connected to the AD vector,and the yeast single hybrid was used for one-to-one verification.Finally,upstream regulatory genes Lb3G20661,Lb2G12077 and Lb1G07601 were obtained.Through bioinformatics analysis,it was found that Lb3G20661 gene encoded zinc finger protein,which was expressed in the nucleus and had high homology with tea tree,and played an important role in improving stress resistance,cell differentiation,embryonic development and initiation of epidermis development.Lb2G12077 contains conserved domains KNOX1,KNOX2 and HOX,among which KNOX,as a transcription factor with homologous heteromorphic domain,plays an important role in the regulation of plant growth and development.Lb1G07601 had no conserved domain and was the specific gene of L.bicolor.The screening of upstream regulatory genes of LbSAD2 provides important candidate genes for obtaining early regulatory genes of salt gland development.3.Screening and verification of LbSAD2 interacting protein gene Lb2G12567Since LbSAD2 had no self-activating activity,the full-length LbSAD2 was inserted into BD vector for nuclear yeast screening library,and the candidate interacting proteins were obtained.Based on the conserved domain of the interacting proteins and the high expression characteristics of salt glands during development,the target genes were further obtained,and the candidate genes were cloned.After the interaction was confirmed by yeast two-hybridization and bimolecular fluorescence complementization,LbSAD2 interacting protein Lb2G12567 was obtained,suggesting that LbSAD2 co-regulates salt gland development by interacting with Lb2G12567.The full length of Lb2G12567 gene is 549 bp,encoding 182 amino acids.Quantitative PCR showed that Lb2G12567 gene had high expression level during the development of salt glands,and was located in the nucleus of the cell.It is a non-transmembrane protein.The promoter sequence(2000 bp)of Lb2G12567 was obtained with cis-acting element(ABRE)involved in abscisic acid reaction and CGTCA-motif involved in Me JA reaction.Cis-acting element(TATC box)and salicylic acid reaction element(TCA-element)are involved in gibberellin reaction,and CGTCA-motif(MBS)is also present.A GUS expression vector induced by Lb2G12567 gene specific promoter was constructed and allogenetically transformed into Arabidopsis.Histochemical staining showed no expression in Arabidopsis.4.LbSAD2 interacting protein gene Lb2G12567 is involved in salt gland developmentWith VIGS gene silencing experiment and Crispr,the effect of Lb2G12567 on the development of salt gland of L.bicolor was investigated by observing the spontaneous fluorescence of salt gland.TRV-RNA2-Lb2G12567 vector was constructed,Lb2G12567 gene was knocked out by genetic transformation technology,and the number of salt glands was reduced by observing the regenerated buds.At the same time,the VIGS gene silencing technique was used to construct p TRV2-Lb2G12567 silencing vector,and the expression level was identified and phenotype was observed after injection of L.bicolor.Compared with the control group,the number of salt gland was significantly reduced after Lb2G12567 gene silencing.The leaf disc secretion test showed that the salt secretion ability of Lb2G12567 gene silencing lines was significantly decreased,and the stress resistance was significantly decreased observed by DAB and NBT staining.Based on the above results,the morphogenesis of the interaction gene Lb2G12567 and salt gland development was related to the number of salt glands,positively regulated salt gland development,and functionally cooperated with the gene regulatory pathway in the L.bicolor.In summary,thesis explores the mechanism of LbSAD2’s involvement in salt gland development,and improves the network of LbSAD2’s regulation of salt gland development by using the Y2 H and Y1 H library to obtain interacting proteins and upstream regulatory genes.Based on Lb3G20661,Lb2G12077 and Lb1G07601,and the interacting protein gene Lb2G12567,we further explained that LbSAD2 plays a key role in the development and early morphogenesis of salt glands.