Transcriptome Analysis Of Leymus Mollis Response To Salt Stress

As one of the most important global food crops,Leymus mollis L.is affected by increasingly serious soil salinization and climate change?such as intensified drought and high temperature?.Breeding resistant wheat varieties is an effective way to increase the yield of wheat.Some wild relatives of wheat have developed unique mechanisms to adapt adverse environment during the long-term evolution,such as salt tolerance,drought resistance,pest and disease resistance,which are excellent genetic resources for genetic improvement of wheat.L.mollis,which belongs to the Triticeae of Gramineae Leymus,is a allotetraploid plant that is distributed in the coastal beach and barren land.L.mollis is a near-source species of wheat and has good salt tolerance,which is regarded as an important genetic resource for the genetic improvement of wheat.Although L.mollis is considered to be of great application value in studying the genetics and breeding of wheat,the genetic information and the mechanism of stress resistance of it is virtually unknown at present.The genetic information for the physiological and biochemical responses of L.mollis to salt stress is unclear.The new generation of high-throughput sequencing technology is able to quickly and completely obtain the sequence information and content of almost all transcripts in a particular sample.Transcriptome information can be used to study functional components of genome,cutting mode,transcriptional structure,post transcriptional modification,classification of transcriptional products and differential expression of stress conditions.Therefore,transcriptome sequencing has become the main way to study gene expression.The transcriptomes of plant,animal and micorbial have been reported previously.In this study,Illumina HiSeq^TM 2000 high-throughput sequencing technology was applied to perform transcriptome sequencing of L.mollis leaves under normal and salt stress conditions to obtain complete transcriptome of L.mollis.Bioinformatics methods were used to comprehensively and systematically analyze the expression profiles of L.mollis response to salt stress,and to reveal the salt-tolerant mechanism of L.mollis by comparing the differentially expressed gene profiles between the treated and control samples.The results are as below:?1?There were 46154210 and 46406704 Clean reads in the control group and the treatment group,and Clean reads were assembled by Trinity software.218966 and228637 Contig were obtained in the control group and the treatment group,respectively.The N50 length was 466 bp and 438 bP,respectively.Contig was further assembled,and 110323 and 112846 U were obtained.The length of Unigene and N50is 1 379bp and 1 319bp,respectively.?2?Functional annotations were performed on Unigene,where a total of 151,538,124,074,78,554,47,358,81,400,74,200 genes were annotated to the Nt,NR,Swiss-Prot,COG,GO,and KEGG databases,respectively.By comparing Unigene with COG database,15786 annotation results were obtained and belonged to 25 major categories,such as"general function prediction".The riparian leaf transcriptional group was annotated to the GO database,and 20350 Unigene were classified into"biological process","cell component"and"molecular function"three.In a large class,18550 Unigene were annotated for all the Unigene of the riparian leaf transcriptional group to the KEGG library,involving 128 metabolic pathways,including the metabolic pathways involved in the plant's salt resistance.The above annotation results have important reference value for revealing the salt tolerance mechanism of Leymus.?3?The differential expression analysis was performed between the treatment and the control group,and the differential expression of the selected Unigene was analyzed by GO and KEGG enrichment.The results indicated that Unigene play roles in the response to salt stress by regulating the accumulation of osmotic pressure substances,participating in the regulation of oxidase system,participating in secondary metabolite pathways,and encoding salt-tolerant genes such as Na⁺/H⁺antiporter.?4?A total of 32 Na⁺transporters related genes and 94 oxidative stress responsive genes were obtained from the riparian transcriptome.The Na⁺transporters which maintain Na⁺/K⁺balance and osmotic pressure are located on the tonoplast and plasma in cells.The function of oxidative stress responsive genes are to clear up the reactive oxygen species?ROS?in time and to reduce their damage to cell membrance system.In addition,the gene of Na⁺/H⁺antiporter?NHX?in vacuolar membrance is one of the most important salt-tolerant genes in plant cells.In this study,the cDNA sequence of Na⁺/H⁺antiporter 2 gene?LmNHX2?was obtained from transcriptome sequencing.The gene-encoded protein and the expression of LmNHX2 under salt stress were carried out with bioinformatics analysis.The results showed that LmNHX2 cDNA included an ORF of 1617 bp encoding 538 amino acids,most of which are polypeptide chain hydrophobic amnio acids and has the structural characteristics of a typical transmembrane protein.The structures of LmNHX2 protein,TaNHX2,TeNHX2 and HvNHX1 have high similarity in amino acid,belonging to the same branch in the evolutionary relationship.The results of real-time PCR showed that the expression of LmNHX2 was induced by salt stress.These results will be helpful to further study the function of LmNHX2.