Study On The Development Of Salt Gland And Trichome In Limonium Sinuatum

At present,soil salinization is increasing yearly that causes to the gradual reduction of agricultural land worldwide,it is urgent to improve the utilization of saline-alkali land and cultivate salt-tolerant crops.Studies have shown that the salt glands and trichomes of all recretohalophytes do not coexist,and they have similar differentiation patterns and their development-related genes are relativele similar.We speculated that salt glands and trichomes may have a common"ancestral gene".Some of the plants evolved salt glands to adapt to high-salt environments,while others evolved trichomes to adapt to drought environments.Therefore,it is importantto look for transitional species with both salt glands and trichomes.This transitional species is of great significance for screening key genes that control the development of salt glands and trichomes,revealing the molecular mechanism of salt glands and trichomes development,and cultivating stress-resistant crops.The salt tolerance of L.sinuatum was compared with L.aureum,L.gmelinii and L.otolepis,which have only salt glands and no trichomes.The salt tolerance threshold values of L.aureum,L.gmelinii,L.otolepis and L.sinuatum were 350.9,417.7,295.5 and 401m M Na Cl,respectively.These results indicated that the salt glands of L.sinuatum had secretory function and endowed the plants with salt tolerance.The data were reliable and representative of L.sinuatum as the material for screening the key genes of salt gland development.In order to determine the development sequence and development time of trichomes,salt glands,stomata and pavement cells.The development of the first true leaf of L.sinuatum was tracked.The development of leaf epidermis of L.sinuatum is divided into six periods:undifferentiated stage(Stage A),trichome differentiation stage(Stage B),salt gland differentiation stage(Stage C),stoma differentiation stage(Stage D),pavement cell differentiation stage(Stage E)and mature stage(Stage F).After clarifying the development stages and corresponding development times,a large number of true leaves of L.sinuatum at each stage were collected,and transcriptome data of each stage were obtained using a high-throughput sequencing platform to screen the key genes that control the development of salt glands and trichome.The number of Stage A-Stage E materials is9000,5500,4000,2800 and 1500.After the transcription group data were obtained,the differential expression genes of Stage A,Stage B,and Stage C of L.sinuatum were compared with the gene sequences of salt gland development candidate genes of L.bicolor(Limonium bicolor(Bag.)Kuntze)and trichome development genes,a total of 1387 genes which may control the development of salt gland and 40 genes which may control the development of trichome were screened.Leaf transcriptome sequencing cannot distinguish the gene expression of different types of cells.However,there are great differences in the regulation of gene expression in different epidermal structures of L.sinuatum.Single-cell sequencing can obtain gene expression pattern from single-cell level,and gain a deeper understanding of the key genes and functions of salt gland development.The biggest challenge of plant single-cell sequencing is to separate single plant cells from tissues.Therefore,it is very important to study how to prepare protoplasts of L.sinuatum and select the optimal enzymatic hydrolysis conditions.The conditions and methods of the protoplast preparation in the Stage B of L.sinuatum were established.The results showed that the yield of true leaf protoplasts obtained at Stage B of L.sinuatum by this method was19.66×10~5/m L,and the viability was 88.23%,which laid the foundation for identifying unique genes controlling salt gland differention or trichome development by single-cell sequencing.