| Germin-like proteins(GLPs)are a class of stress response proteins in plants,which participate in a variety of biotic and abiotic stress response processes in plants.GLPs are highly similar to germin gene in wheat(Triticum aestivum).It is a soluble glycoprotein located in the extracellular matrix.S.portulacastrum is a kind of halophyte,which grows in the tropical coastal areas.It is a dicotyledonous and perennial sandy herb facultative halophyte of Aizoaceae.It is also called salt collector because it can collect a large amount of salt in cells and tissues and survive under extremely high salinity conditions.In order to study the salt tolerance mechanism of S.portulacastrum and explore its salt tolerance related proteins and genes,this study used multi-omics data and bioinformatics analysis methods to identify and analyze the GLPs gene family of S.portulacastrum obtained,and carried out subsequent transgenic functional verification studies on the selected SpGLP19 family members.The main research contents and results are as follows:1.In this study,an important salt-responsive candidate protein,namely germinin-like protein(GLP),was screened out among 96 salt-responsive proteins identified by two-dimensional electrophoresis(2DE)proteomic data of S.portulacastrum leaves in response to NaCl stress at different concentrations(0,300 and 600 mM)obtained in the laboratory.The protein sites identified in 2DE gels were 45,86,89 and 92.The results showed that:These protein loci is highly sensitive to NaCl stress,and in 600 mM after treatment showed markedly enhance the expression level of tip may exist in the plants of the genus S.portulacastrum similar GLP,play an important role in salt tolerance response,so choose the GLP gene further analyzed the gene families,verify that the protein in the S.portulacastrum salt-tolerant regulation of biological functions.2.A total of 50 SpGLPs genes were systematically identified and distributed on 17 chromosomes with different densities.Subcellular localization prediction showed that these GLP family members were distributed outside the cell.Based on phylogenetic tree analysis,GLP proteins in S.portulacastrum,rice,and Arabidopsis were classified into three major groups(A,B,and C).The results showed that these GLP families are highly diverse in terms of function and trait evolution.Based on the analysis of exon-intron organization,it was found that SpGLP members had similar exon-intron structure,but there was a large deviation between intron length and exon size,which provided more information for studying the gene function and evolutionary relationship between SpGLP members.Multiple sequence alignment and conserved motif analysis showed that all SpGLP protein families had conserved domains from germinating protein-like protein families.According to the analysis of these conserved motifs by MEME online tool,cupin motifs were found in all SpGLP member sequences,and the distribution of conserved motifs in members of the same branch of the evolutionary tree was more similar,indicating that proteins of S.portulacastrum GLP had similar functions.The identification of GLP family provides important information for the screening of key candidate genes of S.portulacastrum salt tolerance.In addition,SpGLP gene may play different roles in the growth and development of S.portulacastrum.Therefore,tissue-specific GLP gene expression patterns of S.portulacastrum with different salinity and different tissue and transcriptome data of S.portulacastrum treated with different salinity were constructed by analyzing the transcriptome data.The results showed that the expression levels of GLP gene members were different in different tissues and stages.SpGLP26 was the most abundant in mature leaves,SpGLP22,SpGLP37,SpGLP38 and SpGLP39 were mainly expressed in young leaves.The expression levels of SpGLP49,SpGLP40 and SpGLP42 were the highest in young stems.Notably,the expression of SpGLP 19 was lower in the leaves of the control group,while it was induced by different concentrations of NaCl and the expression level increased with the increase of NaCl concentration,which may indicate that SpGLP 19 plays a role in the response to salt.The key candidate salt tolerance genes were screened and verified by physiological,biochemical and molecular biological methods.3.The physiological indexes of S.portulacastrum treated with different salt gradients(0,300 and 600 mM)under stress were measured,and the selected candidate genes were verified by qRT-PCR experiment.The results showed that with the increase of salinity stress,the plant height in the control group was not significantly different from that under 300 mM NaCl stress,but significantly decreased under 600mM NaCl stress.However,chlorophyll and water content increased first and then decreased,malondialdehyde content and proline content increased significantly,and soluble sugar content did not change significantly,indicating that S.portulacastrum could still grow normally within a certain salinity range,and only when the stress of NaCl exceeded 300 mM began to affect the growth of S.portulacastrum.This is already much higher than the simulated growth value of seawater concentration(200 mM NaCl).In addition,the results of qRT-PCR of several selected candidate genes showed that:The fluorescence quantitative results of SpGLP19,SpGLP22,SpGLP26,SpGLP38,SpGLP50 and SpGLP36 were basically consistent with the transcriptome sequencing results,which proved the high reliability of the sequencing.Significantly increased 4-fold and 8-fold,respectively,which further indicated that SpGLP19 may play a key role in the response to salt.Therefore,SpGLP19 was used as a candidate gene to construct an overexpression vector for the subsequent functional verification of Arabidopsis transgenic.4.Build the S.portulacastrum salt stress response of the candidate genes SpGLP19 plant expression vector,to model plant Arabidopsis thaliana as a material for genetic transformation experiment to obtain transgenic Arabidopsis seeds,through the medium resistance screening and purpose for a positive strain band augmentation of T3 generation of seeds,respectively for medium and salt stress of soil culture experiment,to observe the root length and growth,the results showed that the growth status and root length of SpGLP19 positive plants were significantly better than those of wild-type plants under different concentrations of salt stress.The above results will play a theoretical foundation for expanding the excavation of salt tolerance genes in halophytes S.portulacastrum in the future,so as to obtain more salt tolerance genes for transgenic research,and bring practical significance for soil salinization management in China. |
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