| Songnen Plain is located in the northeastern region of China,with saline-alkali land about 2.73 million hectares,mainly containing sodium carbonate and sodium bicarbonate,commonly known as soda saline soil,which extreme soil environment PH value can be as high as10.5 or more.Most plants can not grow or only sporadic distribution,while the Puccinellia tenuiflora is one of the few plants that can naturally grow in it.For years,scientists have never stopped studying the mechanism of salt tolerance in plants,but the Puccinellia tenuiflora as a typical saline-alkaline tolerance plants,the special saline-alkali tolerance mechanism is not clear,it is worth our further study and discussion.Seed germination period is one of the most critical periods in the process of plant growth and development.Therefore,it is of great significance to study the mechanism of saline-alkaline tolerance in seed germination period.In the case of unknown genomic information of species,based on the reverse genetics,the transcripts of the study species can be directly sequenced by high-throughput sequencing technology RNA-Seq,and genes with significant differences before and after salt treatment were screened.In this study,the germination period of Puccinellia tenuiflora seeds was used as the material,on the condition of water and simulated natural salinity-alkalinity stress respectively as control group and treatment group for the transcriptome sequencing and analysis.Through the screening of the high quality of the sequencing data,the transcription group de novo constructed a high quality Puccinellia tenuiflora transcriptome,obtained a total of 4,1043 unigene.The establishment of the transcriptome laid the foundation for further screening of saline-alkaline tolerance genes of Puccinellia tenuiflora.And the use of NR,Swiss-Prot,Pfam,COG,GO,KEGG and other public databases on the construction of unigene for gene function,biological pathways and other annotations.In addition,in view of the Puccinellia tenuiflora is one of the Poaceae plant,this study also individualized the establishment of the Poaceae database Poafor further annotation.In order to clarify the changes of gene expression profile during the seed germination period,the results showed that there were significant differences in the expression of 3,310 genes,among which 2,196 genes were up-regulated and 1,114 genes were down-regulated.Go enrichment of function analysis was performed on all the differentially expressed genes.The up-regulated genes were enriched into 14 GO terms,distributed in 8 GO groups,267 nodal genes.The downregulated genes yielded 24 enriched GO terms,distributed in 12 GO groups,with a total of 19 nodal genes.GO functional enrichment of differentially expressed genes and the analysis of nodal genes found that the pathways of "substance transport","redox" and "substance metabolism" were closely related to Puccinellia tenuiflora's saline-alkaline tolerance.In order to further explore the salt-alkali stress functional genes,the high expression genes in the above-mentioned differential genes were analyzed.The results showed that there are 120 and 39 high expressed genes in up and down regulated genes respectively,functional classification includes "transporter","signal transduction","stress response protein","stress response enzyme" and other biological processes or functions.A total of 26 candidate genes were screened by analyzing the genes with high expression and high fold change.Based on the expression characteristics and enrichment analysis of up-regulated genes showed that similar to other plants the transport of energy material is significantly enhanced under salt-alkali stress.The energy metabolism process of photosynthesis and respiration is also active,and the reactive oxygen species produced in this process are eliminated by its huge antioxidant system.However,there are some special genes in this study,such as the high expression and high differences of nitrogen metabolism-related genes have shown a great relationship with salt-alkali stress.Analysis of down-regulated genes revealed that in the seed germination period of Puccinellia tenuiflora was down-regulated partial genes expression to adapt to salt-alkali stress,such as reducing the amount of amino acid hydrolysis by reducing the decomposition of storage protein by reducing the storage protein gene,so as to protect it from NH3 poisoning and improve its saltalkali tolerance.Next,the specific genes of Puccinellia tenuiflora is digged.Puccinellia tenuiflora can be distinguished from the vast majority of Poaceae plants and has salt-alkali tolerance,in addition to its different regulatory mechanisms,it is likely that in extreme salt and alkali environment Puccinellia tenuiflora evolved out of special genes that other Poaceae plants do not have.So we need to dig out these specific genes.We look for Puccinellia tenuiflora unique genes from all the differentially expressed genes,a total of 26 candidate specific genes were found,17 of the upregulated genes and 12 of the down-regulated genes.These unigenes have a complete open reading frame,and in NR,Swiss-Prot,COG,Poa comparison have not comment results,indicating that other species does not have these genes.The validation of the specific genes' function of Puccinellia tenuiflora is of great significance for revealling its salt-alkali resistance.In order to certify the reliability of data analysis,we select 14 differentially expressed genes for expression pattern validation.The results shows that the analysis is reliable.In summary,this study was conducted to study the germination of Puccinellia tenuiflora,the aim of this study was to study the causes of salt-alkali tolerance of Puccinellia tenuiflora on the genetic level,so as to screen out a series of candidate genes related to salt-alkali tolerance,which will help to guide the research of the follow-up molecular biology experiments,thus revealing the mechanism of Puccinellia tenuiflora. |
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