The Analysis Of The Differentially Expressed Genes In Alfalfa Under Cold Stress At Transcriptome Level

The severe cold in the winter is one of the most important limiting factors for the alfalfa cultivation in Northeast and Northwest China.In addition to the necessary field management measures,selection of the varieties with strong cold resistance is an alternative way to reduce this adverse effects on alfalfa production.At present,the aflafal breeders still use traditional breeding methods to improve the cold resistance of alfalfa varieties,while the understanding of the mechanisms of alfalfa response to cold resistance at molecular level are still limited.In this study,with the Illumina Hi-Seq 4000 high-throughput sequencing platform,the molecular mechanisms of the alfalfa response to cold stress were analyzed at the transcriptome level.At the same time,all the transcription factor families that response to cold stress in alfalfa were identified and the evolution,gene structure and expression profile of alfalfa bZIP family were further analyzed.The main results are as follows:1.The seedlings of alfalfa varieties ZG9830(cold resistant)and CUF101(cold sensitive)were subjected to 4?of low temperature stress for 0h(CK)and 48h(treatment),respectively,and used for RNA sequencing.A total of 106.38 Gb clean data was obtained,and 78,925 unigenes were de novo assembled,of which 43,930 Unigenes were annotated in the seven public databases(Nr,Swiss-Prot,KEGG,COG,KOG,GO and Pfam).A total of 3,448 and 2,912 differently expressed genes(DEGs)were found after low temperature treatment,and 3,235 and 2,713 Unigenes annotated in at least one database of the seven databases mentioned above.GO functional annotation analysis showed that these DEGs were mainly involved in cell composition,molecular function and biological process,and could be divided into 9 sub-categories.The annotation results of COG indicated that all these DEGs were mainly involved in genome functional prediction,signal transduction,transcription,replication,recombination and repair,amino acid transport and metabolism,carbohydrate transport and metabolism,translation,ribosome structure and formation.The most important metabolic pathways involved in KEGG pathway annotation were ribosome,amino acid biosynthesis,plant hormone signal transduction,carbon metabolism,eukaryotic ribosome biogenesis,starch and sucrose metabolism and so on.There were 2,122 common DEGs were found in the two cultivars,and the expression patterns of the up-regulated or down-regulated genes were shared the similar trend.There were 1,326 DEGs unique to the cold resistant cultivar ZG9830,of which 1,220 were annotated in the Nr and the other 6 databases.2.A total of 59 transcription factor families and 1,730 transcription factor genes were identified in the Unigenes obtained by sequencing.Among them,PHD,ABI3VP1,AP2 / EREBP and other 10 transcription factor family contained the largest number of genes.Besides,there were 251 genes encoding 34 transcription factors were identified in the cold resistant cultivars ZG9830,and of which 98 genes were unique to ZG9830.The expression profiles of these genes from different transcription factor families vary in different organs,indicated these genes may regulate the response of ZG9830 to the cold stress through different regulation pathways.3.A total of 138 genes,which could be divided into 10 subfamilies,were identified in alfalfa through the whole transcriptome level.In addition to the core domain,many other conserved domains,such as the R / KxxS / T domain,the S / TxxD / E domain and the proline enrichment domain were also identified.The codon of the MsbZIP genes is more inclined to use the A/T base,which might have a high correlation with their functions.The expression profiles of the MsbZIP genes from 10 subfamilies in different organs vary in different tissues,indicated that these MsbZIP genes may have different biological functions in the developmental process and response to cold stress in alfalfa.In conclusion,the results in the present research would provide a theoretical basis for the further understanding of the molecular mechanism response to cold stress and the discovery of the cold-resistant genes in alfalfa.