Cloning And Functional Analysis Of LB128867 And LB125251 Genes In Early Development Stage Of Limonium Bicolor

Salt stress is one of the main factors that limit plant growth and development.With the changes of climate and the increase of irrigated agriculture,the environmental problems,such as soil salinization,are becoming more and more severe.These abiotic stresses will inhibit the growth of plants and reduce the yield of crops.Halophytes are one of kind of plants which have evolved a series of adaptive mechanisms to grow and survive in saline environments,including the adaptations in physiological,biochemical and molecular levels.Therefore,it is an important way to utilize saline soils effectively by improving the salt tolerance of crops,and it can be achieved by studying the salt tolerance mechanism of halophytes.The halophyte Limonium bicolor is a typical recretohalophyte with salt glands.The physiological mechanism of salt tolerance of L.bicolor has been extensively studied,but it is still unknown that the molecular mechanism of salt tolerance and the key genes that control salt gland development.Therefore,two unknown high-specific expressed genes at the developmental stage of salt gland were selected and the gene function was analyzed.In the present study,the specific high-expressed unknown genes LB128867 and LB125251 were selected based on the transcriptome sequencing results of the early developmental stage of L.bicolor leaf,which are unique to the early developmental stage of L.bicolor leaf,and the gene function in salt gland development was analyzed:(1)the full length of the two genes were cloned and their bioinformatics were analyzed;(2)subcellular localization of the two genes were detected in tobacco;(3)the overexpression lines of L.bicolor and A.thaliana were obtained;(4)the knocked out lines of L.bicolor were obtained using CRISPR-Cas9 technique,the function of these two genes in salt tolerance were preliminary studied.The main results are as follows:(1)According to the results of the transcriptome of L.bicolor,two unknown genes with high expression in the stage A and stage B were screened,which were named as LB128867and LB125251,respectively.The full length of the gene was cloned using 3'and 5'RACE techniques.The full length of LB128867 gene is 1634 bp,the coding sequence is 1239 bp,encoding 412 amino acids;the full length of LB125251 gene is 2009 bp,the coding sequence is 1581 bp,encoding 527 amino acids.Their functions were unknown by blasting on NCBI.LB128867 protein is a hydrophobin and LB125251 is a hydrophilic protein;the protein sequence coding by LB128867 contains three low complexity regions,LB125251 contains three low complexes and a coiled-coil domain analyzed using SMART;the LB128867 gene is closely related to an unknown gene of Beta vulgaris L.,Chenopodium quinoa Willd,and Spinacia oleracea L.,and the LB125251 gene are closely related to Spinacia oleracea L.(2)The subcellular localization of LB128867 is on the plasma membrane and LB125251is in the nucleus based on the detection by tobacco.(3)The coding regions of LB128867 and LB125251 were ligated to the expression vector p CAMBIA1300,respectively,and were transformed into L.bicolor and wild-type A.thaliana to obtain the overexpression line.The expression levels of each gene in the overexpressed Arabidopsis lines were identified by Real-time PCR,and the homozygous lines LB125251-OE4?LB125251-OE9?LB125251-OE10 and LB128867-OE4?LB128867-OE6?LB128867-OE7 were obtained.There was no significant difference in the germination of overexpression lines and wild Arabidopsis seeds under normal condition.(4)Seeds were harvested from WT,LB125251-OE4?LB125251-OE9?LB125251-OE10?LB128867-OE4?LB128867-OE6 and LB128867-OE7,and were cultivated in 1/2 MS culture medium containing 0,50,80 and 120 m M Na Cl,respectively.The results showed that under different salt concentration treatments,the germination rate of seeds from WT,LB125251-OE4?LB125251-OE9?LB125251-OE10?LB128867-OE4?LB128867-OE6 and LB128867-OE7 were decreased with the increase of salt concentration,as well as the germination index and the cotyledon growth rate.Compared with WT,the reduction in the overexpression lines of LB128867 and LB125251 were significantly higher.The result indicated that in A.thaliana,plants overexpressed with LB128867 and LB125251 were salt sensitive.Under the treatment of 80 m M Na Cl,the Na~+content in A.thaliana plants overexpressed with LB128867 and LB125251 were significantly higher than that in WT,and the Na~+/K~+was also significantly higher than that of WT.The salt tolerance in overexpression lines of A.thaliana was significantly reduced with the expression of genes,which indicates that the genes were negative regulators of plant salt tolerance.(5)The CRISPR-Cas9 knockout vector was constructed and transformed into the leaves of L.bicolor,and the knockout lines of LB128867 and LB125251 were obtained.