| Soil salinization has seriously inhibited the growth of agricultural and forestry crops,which is a common problem faced by all countries in the world.The use of genetic engineering and other technologies to genetically improve forest trees or to efficiently breed good varieties is the main goal of forest genetics and breeding works,which can effectively promote the use of saline-alkali lands.Because of complex regulatory network,halophytes are widely used as important materials to explore the mechanism of salt-tolerance in plants and improve the salt-tolerance ability of agricultural and forestry crops.As a kind of typical halophyte,Spartina alterniflora Loisel are widely distributed in the coastal basins of many provinces in China as they can endure high salt stress.At the same time,as an common forest and grass plant,the invasion of Spartina alterniflora seriously affected the growth of coastal shelter forests,which has caused huge damage to the forest economy and ecological environment.Exploring of the genetic theoretical basis of salt tolerance is fundamental to understand the crazy invasion and an important prerequisite for the ecological control of Spartina alterniflora.However,due to the lack of reference genome,studies on the molecular mechanism of salt-tolerant regulation at the level of post-transcription are relatively fewer,and the application of excellent genes of Spartina alterniflora on forest genetic improvement has not been effectively carried out.In order to fully and systematically analyze the molecular mechanism of salt tolerance in Spartina alterniflora,in this study,we treated the seedlings of Spartina alterniflora with different concentration(0,350,500 and 800 m M)of Na Cl,and the regulatory mechanism of salt tolerance in Spartina alterniflora was comprehensively analyzed at transcriptional and post-transcriptional level through the sequencing of full-length transcriptome,RNA-seq and PAS-seq at the 3' end of m RNA.At the same time,the salt-tolerrant related genes of Spartina alterniflora were applied to the genetic improvement in Populus L and the main results obtained are as follows:(1)A total of 24,670 Unigenes and 90,587 non-redundant transcripts were found through sequencing on the full-length transcriptome of Spartina alterniflora.The average length of transcripts is 2,405 bp,of which 84,715 transcripts have the ability to encode proteins.By prediction,3,494 nc RNAs and 4,997 transcription factors were obtained from all transcripts and the gene sequences of Spartina alterniflora was more similar to the monocotyledons such as Oryza sativa,Zea mays and Sorghum bicolor through homology analysis.Finally,by functional enrichment,the genes in Spartina alterniflora were found with functions such as signal transduction,ion transport and catalytic activity,and participating in various biological processes related to salt tolerance regulation such as transcriptional regulation,growth and development and stress response.(2)By analyzing the RNA-seq data of Spartina alterniflora under different treatment of salt concentrations,3,870 differential expressed Unigenes and 6,676 differential expressed transcripts were found,and the number increased gradually with the rise of salt concentration.Further analysis showed that a large number of stress-related kinases(ITPK2,CPK1,MAPK33,etc.),transcription factors(HSF,WRKY,b ZIP,etc.)and nc RNA(linc RNA and mi RNA mainly included)were included in the differential expressed genes.By functional enrichment analysis,the differential expressed up-regulated genes were found mainly involved in the processes of ion transport,stress response and oxidation-reduction reaction,while the down-regulated genes were related to the photosynthesis,chlorophyll synthesis and translation.By KEGG enrichment,the differential expressed genes were also found involved in important pathways for salt tolerance regulation like secondary metabolite biosynthesis,photosynthesis and plant hormone signaling,etc.OST1,CHIA,CIPK10,SUS4 and other possible core regulatory genes in response to high salt stress were subsequently obtained by use of WGCNA(weighted gene co-expression network analysis).Finally,comparing with the RNA-seq data of rice under high salt treatment,it is found that the reason why Spartina is more salt-tolerant may be related to the difference of the expression patterns.(3)A total of 5,328 genes with AS(alternative splicing)occurred corresponding to 14,058 non-redundant AS events were found through the analysis on RNA-seq data under salt stress in Spartina alterniflora,among which 7,903 events belong to the intron retention(IR)type,accounting for the largest proportion(56%).Through analysis of the AS events under different salt treatment,the number of differential expressed AS(DE-AS)events were found increasing gradually with the rise of salt concentration.Some DE-AS genes include Cluster6109,Cluster6352,Cluster8608 and Cluster11561 were verified by RT-PCR and q RT-PCR.Functional enrichment analysis showed that DE-AS genes were mainly involved in the cell process,metabolism process and synthesis of cell related components,and also had catalytic activity,transport activity,and transcription factor activity.Finally,The expression of AS factors was analysed combined with the RNA-seq data,and important factors like U11/U12-65 k,SR33,SR34,PRP39 A,PRP18A and SRZ21,were significantly un-regulated under high salt stress,which further exhibited that AS might have certain regulatory role in the process of responsing to salt stress in Spartina alterniflora.(4)Through the construction and sequencing analysis of PAS-seq libraries,a large number of poly(A)sites at the 3' end of Spartina alterniflora genes under salt treatment were obtained.A total of 47,176 PACs were obtained by regarding all the poly(A)sites adjacent at 24 nt as a PAC(poly A cluster),and most of them were distributed at the 3'UTR region of the genes,of which 9,248 genes contains two or more PACs.Through analysis of the 3'UTR-PAC under different salt stess,it was showed that the number of differential expressed PAC(DE-PAC)increased gradually with the rise of salt concentration and 7,339DE-PACs corresponding to 3,383 genes were found under 800 m M Na Cl treatment,and these DE-PAC genes were mainly involved in biological processes such as carboxylic acid metabolism,chlorophyll biosynthesis,redox reactions,and copper ion transmembrane transport.For ease of analysis,IGV visualization was performed on all DE-PAC genes,and3'RACE verification was also carried out on some genes such as Cluster16664.Finally,the expression of some poly(A)factors were regulated by high salt stress based on the RNA-seq and q RT-PCR data,indicating that APA also had potential regulatory role in the process of responsing to salt stress in Spartina alterniflora.(5)Taking the genes containing multiple PACs at the 3'UTR region as the research object,the 3'UTR length of all DE-PAC transcripts under different salt stress were calculated,and the results showed that salt stress generally promoted the lengthening of 3'UTR on Spartina alterniflora genes.207 genes with 3'UTR lengthened under 800 m M high salt stress were mainly involved in stress response and various ion transport processes after functional analysis,and some important genes like Cluster16664 were validated by RT-PCR,q RT-PCR and Northern blot.With the help of dual-luciferase report system,ion transport related genes Cluster9684(KT2),Cluster10136(CIPK23),Cluster16664(HKT1)and Cluster30331(ZTP29)were demonstrated being able to inhibit the translation efficiency of proteins after 3'UTR lengthening,and it is proved that this inhibition might be related to the mi RNA binding sites and AU-rich elements of the 3'UTR region through further experiments.Finally,RNA degradation experiments showed that high salt stress could increased the stability of ion transport related genes in Spartina alterniflora,which initially revealed the possible regulatory role of3'UTR length in response to salt stress in Spartina alterniflora.(6)Multiple stress-related genes were selected combined with the full-length transcriptome and RNA-seq data of Spartina alterniflora,and the transgenic Arabidopsis materials were obtained by the application of flocculation mediated by Agrobacterium and five stress-related genes were preliminarily selected through different salt treatment experiments.The salt-tolerant genes were further transformed into poplar,and Sa ABI5,Sa Hsp70 and Sa ERF1-1 transgenic poplars with certain salt tolerance ability were finally obtained through salt treatment and detection including observation of phenotypes and determination of related content.Generally speaking,the full-length transcriptome,differential expressed genes,alternative splicing,polyadenylation and 3'UTR length of Spartina alterniflora under salt stress were systematically analyzed by multiple sequencing technologies in this study,not only full-length transcriptome of Spartina alterniflora with high quality was obtained and moreover,it was found that salt stress could regulate the expression of stress-related genes at the transcriptional and post-transcriptional level.To a certain extent,it explains the molecular mechanism of the response to salt stress in Spartina alterniflora,which provides a certain genetic theoretical basis for enriching the genetic resources of Spartina alterniflora and controlling its ecological invasion.At the same time,the stress-related genes of Spartina alterniflora were cloned into Populus and transgenic materials with certain salt tolerance were initially obtained,which made certain contribution to the improvement of forest genetic breeding. |
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