Analysis On The Salt Tolerance Of Nitraria Sibirica Pall.and Functional Verification Of NsRabE1c Gene

Nitraria sibirica Pall,a deciduous shrub of the genus Nitraria L.of Zygophyllaceae,is a typical halophyte and a typical vegetation in desert ecosystem.It has the characteristics of strong drought resistance,cold resistance,barren resistance,wind and sand resistance and strong saline-alkali resistance.At the same time,its fruits and leaves have high nutritional,medicinal and feeding value,so it is a"golden"tree species with both ecological and economic benefits.However,the research on the mechanism of salt tolerance at the molecular level is still in its infancy,which limits the development and utilization of its salt tolerance advantage.Therefore,In this study,one-year-old N.sibirica treated with 300 mmol·L^-1Na Cl was used as experimental material,and the leaves were sequenced by transcriptome.Firstly,the full-length transcript was obtained based on Pacbio sequencing,and the CDS sequence was predicted by Trans Decoder software,and the transcript was annotated through six major databases;secondly,the transcript was analyzed by De Seq2 to screen the differential genes;finally,the key genes of salt tolerance were screened by WGCNA and the key gene NsRabE1c was transferred into Arabidopsis thaliana for functional verification.The main conclusions are as follows:（1）After filtering out incomplete CGs,1,030,629 full-length non-chimeric reads（FLNC）were obtained.After removing redundancy and clustering,89,017 full-length transcripts（average length 2721.43 bp,N50 3009 bp,Q30 values 98%）were obtained.A total of 86,482 CDS sequences were predicted,among which 4000 genes without CDS may be small RNA and t RNA.41,724 transcripts can be annotated in 6 databases（GO、KEGG、NR、SWissport、Tr EMBL and egg NOG）.84,632 transcripts were annotated in at least one database.A large number of genes are annotated to“signal transduction”,“intracellular trafficking,secretion,and vesicular transport”,“response to stimulus”,“metabolic process”,“organelle”“transcription factor activity”and“carbohydrate metabolism”et al.（2）By differential expression analysis,6561 differential genes with more than 2 times difference（|log2FC|≥1,FDR<0.05）between salt treatment and control and 756 differential genes with more than 16 times difference（|log2FC|≥4）were obtained.GO enrichment analysis showed that many single genes were enriched in the pathways of“response to stimulus”,“hydrogen peroxide response”,“ion binding”,“cell communication”,“growth”,“carbohydrate metabolic process”,“cell wall organization or biogenesis”,“photosynthesis”and“polysaccharide metabolism process”.KEGG enrichment analysis showed that a large number of single genes were enriched in the pathways of“phosphatidylinositol signaling system”,“starch and sucrose metabolism”,“carbon metabolism”,“peroxisome”,“MAPK signaling pathway-plant”and“plant hormone signal transduction”.（3）WGCNA analysis showed that N.sibirica enhances salt tolerance by regulating ion balance,increasing permeability,scavenging reactive oxygen species,regulating cell wall structure and signal transduction.A total of 66 genes in the top 10%of connectivity were screened,and 34 have been verified in other crops.（4）The total length of NsRabE1c sequence was 1108 bp,and the CDS region was 651 bp,encoding 216 amino acids.Based on the analysis of its evolutionary relationship,it was found that the species with high homology with NsRabE1c was coffee arabica.Subcellular localization analysis showed that NsRabE1c was located in nucleus and cytoplasm in N.sibirica.（5）NsRabE1c overexpressed A.thaliana（OE）germinated earlier,grew faster and had the longest root length than mutant A.thaliana（atrabe1c）and wild type A.thaliana（Col-0）without salt application,and the content of K⁺in OE was much higher than that of atrabe1c and Col-0.Through the determination of the expression of NsRabE1c in N.sibirica,it was found that the expression of NsRabE1c decreased in 1-4 h after salt application（growth stagnation）,and increased significantly in 6-24 h（growth recovery stage）.The above results suggest that Rab E1c can increase the content of K⁺and promote the growth and development of N.sibirica to regulate salt stress.（6）Under salt stress,the growth of NsRabE1c overexpressed A.thaliana（OE）was worse than that of mutant A.thaliana（atrabe1c）and wild type A.thaliana（Col-0）,and the germination rate,root length and survival rate of OE were lower.After the application of salt,the content of Na⁺in the leaves of OE increased rapidly,while the content of K⁺decreased gradually,and the ratio of K⁺/Na⁺decreased,which seriously destroyed the balance of K⁺/Na⁺.The content of Na⁺in the leaves of atrabe1c changed little,but the content of K⁺increased gradually and the ratio of K⁺/Na⁺increased.The above results showed that NsRabE1c decreased the salt tolerance of plants by reducing the ratio of K⁺/Na⁺.