| Medicago falcata L.,an excellent leguminous perennial forage with higher low temperature resisitance than Medicago sativa L.,is an important genetic resources for M.sativa L.cold resistance breeding.In this study,the changes of cell membrane permeability,osmotic adjustment and antioxidants in M.falcata L.cv.Hulunbeier seedlings under low temperature stress were observed to investigated the physiological responses in M.falcata L.under low temperature stress.Then,the excellent genes with low temperature resistance were explored through the combined analysis of RNA-seq and Pacbio full-length transcriptome sequencing.The molecular regulation mechanism of M.falcata L.under low temperature stress was illustrated by weighted gene co-expression network analysis（WGCNA）.The main results were as follow:（1）The cell membrane permeability in M.falcata L.root was increased with the decrease of treated temperature,as a result of the increase of electrolyte leakage rate and malondialdehyde（MDA）content.The highest values were 89.45%at-15℃treated for 12 h and 93.26μM·g-1DW at-15℃treated for 8 h,respectively.The damage was more serious,but M.falcata L.can resist the damage caused by low temperature stress by increasing the content of cell osmotic substances and antioxidant activity.（2）The contents of proline,soluble protein and soluble sugar in M.falcata L.root were increased significantly with the decrease of temperature.The contents of proline and soluble protein were the highest at-15°C treated for 12 h and 8 h,reached 9.35μmol·g-1DW and 34.52mg·g-1DW,which were 0.54 and 0.219 times higher than that at control check（CK）.The content of soluble sugar reached maximum of 90.61 m M·g-1DW at-10°C treated for 2 h,which was 0.843times higher than CK.（3）The activities of superoxide dismutase,peroxidase and catalase and the content of reduced glutathione of M.falcata L.root increased under low temperature stress.The highest value of SOD activity was 5893.21U·g-1·h-1DW at 0°C treated for 8 h,the highest value of POD activity was9621.07 U·g-1·min-1DW at-10°C treated for 2 h,the highest value of CAT activity was 1335.53U·g-1·min-1DW at-5°C treated for 2 h,and the highest value of GSH content was 3.76 mg·g-1DW at-5°C treated for 2 h,which was respectively 0.498,0.799,1.184 and 1.228 times higher than that in CK.It is an effectively way to resist the damage caused by the increase of superoxide anion content at low temperature.（4）A total of 19.27Gb clean data was obtained by Pacbio full-length transcriptome sequencing in M.falcata L.roots under control check and 4,0,-5,-10 and-15℃treated for 2 h.A total of 115,153 full-length transcript sequences without redundancy were obtained,and 8,849alternative splicing（AS）,73,149 Simple Sequence Repeats（SSR）,76,015 complete Open Reading Frame（ORF）and 4,189 Long non-coding RNA（Lnc RNA）were predicted.Finally,111,587unigenes were annotated,while 3,566 unigenes were not annotated.92%of the annotated unigenes in non-redundant proteins（Nr）database were highly similar to the known sequences,among which,97,831 unigenes were from Medicago truncatula,and accounted for 87.87%.（5）A total of 11,369 differentially expressed genes（DEGs）were observed based on the expression levels of transcripts that compared between RNA-seq data and full-length transcripts.These DEGs were clustered into six sub-cluseres.Then,we characterized 1,538 transcription factor（TF）genes into 45 TF families,and the most abundant TF family was the WRKY family（186 genes）,followed by ERF（165 genes）,MYB（143 genes）,b HLH（131 genes）and NAC（111genes）families.（6）A total of 134 differentially co-expressed genes were obtained by comparing the DEGs at all treated temperature points,including 101 up-regulated genes and 33 down-regulated genes.The Gene Ontology（GO）functional analysis found that 7 differentially co-expressed genes were annotated,and these genes mainly distributed in“cell parts”,participated in“Metabolic process”,and functioned in“binding”.Twenty-seven differentially co-expressed genes were significantly enriched in 16 metabolic pathways including“Circadian rhythm-plant”,etc.A total of 18differentially co-expressed genes were annotated with Clusters of Orthologous Groups（COG）,mainly involved 15 function classification such as“General function prediction only”.The gene regulation network of 134 differentially co-expressed genes were constructed using Cytoscape software,and three genes including F01.PB40804,F01.PB75011 and F01.PB110405 were found to be high connectivity genes which were predicted to be the hub genes of M.falcata L.in response to low temperature stress.（7）Twelve gene modules were identified by associating DEGs with measured physiological indexes using WGCNA.“Electrolyte leakage”was significantly and positively correlated with MEgrey60,MEplum1 and MEbrown,indicating that it could be used as the main physiological index of plant resistance to low temperature stress.MEbrown was significantly positively correlated with POD,superoxide anion,electrolyte leakage and soluble sugar,indicating that DEGs in MEbrown were closely related to low temperature resistance in M.falcata L..The DEGs in the MEBrown module were mainly localized in four GO-terms,such as“starch catabolic process”and 88 KEGG pathways,such as“Starch and sucrose metabolism”.689 genes were functionally annotated with COG,including 290 functionally unknown genes.F01.PB95141、F01.PB97693 and F01.PB106092 were selected from the MEbrown module by gene regulation network analysis.These three genes participated in the biological process of“maltose biosynthetic process”,“starch catabolic process”and“response to cold”.（8）Twenty differentially co-expressed genes related to low temperature resistance were selected and verified by real-time fluorescence quantitative PCR（q RT-PCR）.The results showed that the expression trend of selected genes were consistent with the results of transcriptional sequencing,which verified the accuracy and reliability of transcriptional sequencing results. |
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