Study On Drought Resistance Of Medicago Ruthenica Based On Transcriptome And Small RNA Analysis

Drought is among the main environmental factors that adversely affect the growth,development,and yield of forage grass.It is meaningful for the breeding of forage droughtresistant varieties to study the morphological,physiological,and molecular mechanisms of forage grass response to drought stress and to mine drought-responsive genes.Medicago ruthenica is a perennial legume forage characterized by cold tolerance,drought resistance,and strong adaptability,and is accordingly considered an ideal material for studying stress resistance mechanisms and a source for mining leguminous plant stress resistance genes.Moreover,it also has the potential for further development into an excellent forage resource.In this study,we used the M.ruthenica variety Turmot as a test material,and the germplasms 02982 and 03056 as control materials.On the basis of an analysis and comparison of the morphological and physiological responses of these three materials to drought stress,we used transcriptome,small RNA,and degradome sequencing techniques to mine key genes and mi RNAs involved in the response to drought stress in the leaves of Turmot seedlings and identified the associated biological pathways.Using the information thus obtained,we conducted a preliminary analysis of the mi RNA mtr-mi R156b-5p and its interactions with target genes by constructing a transient expression system of M.ruthenica;and SPL gene family members,the target genes of mtr-mi R156b-5p,were identified and analyzed by bioinformatics methods.Subsequently,we heterologously overexpressed M.ruthenica mtr-mi R156b-5p in Nicotiana benthamiana plants to examine the effects on plant morphology.The main results obtained are as follows.(1)In response to continuous exposure to drought stress,we observed significant increases in the root-to-shoot length ratio and root-to-shoot weight ratio of the three M.ruthenica varieties(Turmot,02982,and 03056),whereas in contrast,we detected significant reductions in plant height stress indices,dry matter stress indices,and leaf relative water content.Furthermore,with a prolongation of drought stress exposure,we observed a continuous accumulation of the contents of proline and malondialdehyde in the leaves of the three varieties.On the basis of comprehensive analyses of the aforementioned indices,we established that among the three assessed materials,Turmot was characterized by strongest resistance to drought.(2)Transcriptome sequencing analysis and study was performed on Turmot seedlings under drought stress(DS)and rewatering treatments(RW).The sequencing data obtained from de novo analyses of transcriptome assemblies yielded a total of 147,957 transcripts,which were assembled into 52457 unigenes.Reference-based analyses of the transcriptome assemblies revealed that 33,457 of the transcripts thus obtained were aligned with those in the M.ruthenica reference genome,of which 27,402 and 10,297 genes were annotated based on GO and KEGG analyses,respectively.The findings of gene differential expression and functional analyses indicated that the drought response-associated genes of M.ruthenica were mainly enriched in three biological pathways,namely,“response to water deprivation”(GO:0009414),“response to chitin”(GO:0010200),and “response to wounding”(GO:0009611),and the two KEGG pathways “plant hormone signal transduction”(ko04075)and “MAPK signaling pathway-plant”(ko04016).(3)By performing small RNA sequencing analysis and study for Turmot seedlings exposed to the same drought and rewatering regimes,we identified a total of 669 conserved mi RNAs(belonging to 92 mi RNA families)and detected 1089 novel mi RNAs from nine c DNA libraries(CK＿1/2/3,DS＿1/2/3,and RW＿1/2/3).Among the conserved mi RNA families,mi R166 and mi R171 were found to have the most members.Furthermore,we identified 141,36,and 111 mi RNAs that were differentially expressed(DEMs)in comparisons between DS and CK,DS and RW,and RW and CK,respectively,and among these,the number of down-regulated DEMs in each comparison group was significantly greater than that of the up-regulated DEMs.(4)Degradome sequencing analysis and study performed for Turmot receiving drought and rewatering treatments yielded a total of 1099 mi RNAs(523 conserved and 576 novel),and 5943 target transcripts,forming 9906 mi RNA–target pairs.Among these mi RNAs,ppeMIR169i-p3＿2ss17GT19TG was characterized as having the maximum number of target genes in M.ruthenica.On the basis of an integration of the data obtained using transcriptome,small RNA,and degradome sequencing analyses,we were able to generate mi RNA–target gene–function Sankey and Network plots depicting the drought responses in M.ruthenica.(5)We constructed the recombinant vector p NC-CAM1304-35S-mtr-mi R156b-5p,and transiently expressed in the leaves of Turmot seedlings.q RT-PCR analysis of the expression patterns of mtr-mi R156b-5p and its targets in transiently expressed M.ruthenica leaves revealed that evm.TU.original＿scaffold＿933＿pilon.102,evm.TU.frag Scaff＿scaffold＿126＿pilon.825,and evm.TU.original＿scaffold＿994＿pilon.229 are negatively regulated by mtrmi R156b-5p,and may exert feedback regulation on this mi RNA.(6)Bioinformatic analyses enabled us to identify a total of 21 SPL gene family members in the M.ruthenica genome,which were named Mru SPL1–Mru SPL21.These Mru SPL genes are distributed among seven chromosomes(chr1,chr2,chr3,chr4,chr7,chr8,and unanchor421＿1＿112456),with the number of genes on each chromosome differing.Phylogenetic analysis revealed that members of the Mru SPL family could be divided into nine distinct groups(1 to 9),the members of each of which are closely related.Analyses of the gene structure and conserved motifs of these Mru SPLs revealed high similarities among group members with respect to exon number,length,and distribution,as well as a high conservation of motif type and number.These findings would accordingly tend to indicate that members of the same Mru SPL group are highly conservated in terms of gene structure,and that the encoded proteins may thus perform similar functions.(7)We also examined the heterologous expression of M.ruthenica mtr-mi R156b-5p by overexpressing the recombinant vector p ART-CAM-mtr-mi R156b-5p in transformed leaf explants of sterile N.benthamiana seedlings,for which we obtained 21 transgenic tobacco overexpression lines.Among these,we found that the level of mtr-mi R156b-5p expression in line OE-11 was 36 times higher than that in control plants.Phenotypic comparisons revealed that OE-11 plants have slightly higher plant height,slightly thicker stems,a larger number of branches,a significantly larger number of leaves,lighter colored leaves,and smaller mature leaves compared with the controls.Moreover,the squaring stage of OE-11 was later than control N.benthamiana plants.The findings of this study complement and enhance our current understanding of the molecular mechanisms underlying the responses of M.ruthenica to drought stress.These findings will provide a foundation for the further functional characterization of the key genes and mi RNAs involved in the response to drought and other abiotic stresses in M.ruthenica.Having characterized the regulatory mechanisms underlying the drought stress response,these genes and mi RNAs could have potential utility,not only as early assessment indicators for breeding but also by contributing to the development of novel approaches and methods for the future breeding of legume forage with excellent stress resistance.