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Sequencing Of Alfalfa Transcriptome And Preliminary Exploration Of MsMYB4 Salt Stress Response Function

Posted on:2021-01-22Degree:MasterType:Thesis
Country:ChinaCandidate:X J LiuFull Text:PDF
GTID:2430330605963919Subject:Cell biology
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Soil salinization is a worldwide resource and ecological problem.Salinized land covers more than 100 countries and regions in the world,accounting for about 20% of the world's total cultivated land area.China is one of the countries with the most serious soil salinization problem.Salinized soil seriously affects normal plant growth and crop yield through ion toxicity,oxidative stress and osmotic stress.However,in the long process of evolution,plants have developed a whole set of tolerant mechanisms to salt stress.Alfalfa is an important forage resource in the world,but the alfalfa industry in China is also seriously affected by soil salinization,so it is particularly important to screen alfalfa salt-tolerant strains,dig out its stress tolerant genes,explore its stress tolerant mechanism and cultivate new high quality stress tolerant alfalfa strains by means of molecular biology.Many literatures have shown that MYB transcription factor plays an important role in plant tolerant to abiotic stress.In this paper,transcripome sequencing analysis of relative salt tolerant strain HD and salt sensitive strain SD in alfalfa under NaCl treatment was carried out to screen potential salt tolerant functional genes.RNA-Seq analysis under NaCl treatment was carried out on these two strains to screen out possible salt tolerance genes.Given MYB family plays an important role in salt stress response and its isolation identification in alfalfa is very limited,we selected the 17 MsMYBs of salt stress response genes and analysising their expression under the salt stress,to testing the RNA-Seq results and clone the full length cDNA sequence.For MsMYB4,which is the most significantly upregulation of salt stress and with a higher upregulation range in HD,is overexpressed in Arabidopsis thaliana,to preliminary identifies the salt tolerance function and preliminary analysis of salt tolerance mechanism for MsMYB4.The results are as follows:1.RNA-Seq and analysis of alfalfa treated with NaClRNA-Seq was performed on the whole root material treated with NaCl for 0,1 and 24 h in two strains of HD and SD,and a total of 279397 transcripts and 158126 unigenes were obtained through screening.83.57% of unigenes was the coding protein.A total of 39338 SSR fragments were detected in RNA-Seq data,with Mone,Di,Tri fragments accounting for more than 98% and Tetra,Penta,Hexa less than 2%.DEGs clustering analysis show that there are two main groups of classes,the first group was unigenes in HD-24 and SD-24,and the second group was unigenes in HD-0,HD-1,SD-0 and SD-1,which were divided into two subgroups,one was unigenes in HD-0 and SD-0,and the second subgroup was unigenes in HD-1 and SD-1.It was found that there were 935 DEGs in HD-1 vs HD-0 and 2809 DEGs in HD-24 vs HD-0.There were 1456 DEGs in SD-1 vs SD-0,and 3356 DEGs in SD-24 vs SD-0.There were 629 DEGs in HD-0 vs SD-0,681 DEGs in HD-1 vs SD-1,and 657 DEGs in HD-24 vs SD-24.During the whole NaCl treatment,178 genes in HD strain were always up-regulated and 341 genes were always down-regulated.In SD,244 genes were always up-regulated and 694 genes were always down-regulated.There were also 113 genes that showed differences in HD and SD in the control and treatment groups.DEGs' GO analysis proved that 35 GO terms were enriched in HD-1 vs HD-0,50 GO terms were enriched in HD-24 vs HD-0,45 GO terms were enriched in SD-1 vs SD-0,49 GO terms were enriched in SD-24 vs SD-0,7 GO terms were enriched in HD-0 vs SD-1,27 GO terms were enriched in HD-1 vs SD-1,and 19 GO terms were enriched in HD-24 vs SD-24.KEGG pathway analysis showed that in HD-1 vs HD-0,367 DEGs enriched in 85 pathways;HD-24 vs HD-0,1334 DEGs enriched in 108 pathways;SD-1 vs SD-0,534 DEGs enriched in 99 pathways;SD-24 vs SD-0,1447 genes enriched in 114 pathways;HD-0 vs SD-0,207 DEGs enriched in 64 pathways;HD-1 vs SD-1,222 DEGs were enriched in 73 pathways;HD-24 vs SD-24,244 DEGs were enriched in 76 pathways.2.Dig out the MYB transcription factors of response to salt stress in alfalfa17 MsMYBs transcription factors were selected from the RNA-Seq DEGs,and their RNA-Seq data were tested by Realtime-PCR.The full-length c DNA sequences were further cloned and named as MsMYB1-17.Sequence analysis revealed that all the 17 MYBs contained conserved R domain and related conserved residues and belonged to R2R3 subfamily,which were located in S2,S3,S4,S8,S14,S15,S17,S20,S22,and S24 subfamily in the phylogenetic trees.3.Preliminary identification of salt tolerance function for alfalfa MsMYB4 geneThe study found that MsMYB4-OE Arabidopsis thaliana plants under NaCl treatment than wild type show better seed germination and root growth phenomenon: In the absence of NaCl,the WT and MsMYB4-OE seed germination rate were all 100%,after 150 m M NaCl treatment,WT's seed germination rate was 60% and the two OE lines' seed germination rate up to 84.4% and 82.3%;In the absence of NaCl,the root growth status of WT and OE lines' was similar,while the root length of OE lines' is better than the WT significantly after treated with NaCl.When treated with exogenous ABA,MsMYB4-OEs also showed higher tolerance: the seed germination rate of WT decreased from 100%(0 ?M ABA)to 80%(1 ?M ABA),and then decreased to 60%(2 ?M ABA),while the germination rate of OE lines was significantly higher at the above three ABA concentrations,are100%,95%,and 80%.After 2 ?M ABA treatment,the root growth of WT was only 23% of that of control group,while the root growth of OE lines was still 45%.To sum up,Alfalfa varieties HD and SD were treated with NaCl,a large number of involved in transcription regulatory factors of primary and secondary metabolism,signal transduction factors and encoding protein gene,its expression was induced or inhibit in different degrees,and some of those genes,the expression's amplitude or pattern for up or down by salt stress in HD and SD was different.Through analysis and speculation,we selected and cloned 17 MsMYBs which may play a role in signal transduction pathway of alfalfa salt stress.Through the overexpression experiment MsMYB4 of Arabidopsis thaliana,it was found that the MsMYB4 gene significantly improved the plant's salt and ABA tolerance,which indicated that this gene was probably a key salt tolerance gene in alfalfa and played a role through the aba-dependent tolerance pathway.
Keywords/Search Tags:Alfalfa, salt stress, RNA-Seq, MYB, overexpression
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