Using Rna-seq Platform Revealing Molecular Mechanism Of Freezing Tolerance In Alfalfa

Alfalfa is a perennial high quality forage grass,which is rich in protein and other nutrients.It has the reputation of "the king of pasture".In China,the growth of alfalfa is mainly concentrated in the northern regions.In these areas,the climate is cold,and it is prone to low-temperature and freezing damage,because of insufficient freezing tolerance,hard winters(with extremely low temperatures)are a major limitation to alfalfa production.Therefore,in alfalfa,how to discover the resources of freezing tolerance gene and cultivate new varieties of freezing tolerance has become an urgent question to be solved in the high yield and high quality of alfalfa industry in our country.Zhaodong alfalfa is widely planted in Northeast China,which is suitable for the local freezing climate.It is an excellent freezing tolerance variety in Medicago sativa.Therefore,in this study,Zhaodong alfalfa was used as the research material,performed the freezing transcriptome sequencing,small RNA sequencing and degradome sequencing were carried out to explore the miRNA genes,functional genes and their regulation related to freezing stress in alfalfa.At the same time,we integrate the information of gene regulatory network of plants,construct the gene regulatory network of alfalfa,and analyze the molecular mechanism of freezing tolerance in alfalfa.The main results are as follows:1.Transcriptome sequencing analysis of alfalfa reveals CBF genes potentially playing important roles in response to freezing stress.Transcriptome sequencing analysis of alfalfa response to freezing stress was performed,and 75551 transcripts have been identified based on high-throughput sequences,and identified 5767 transcripts differentially expressing response to freezing stresses.These genes annotation results showed that they were mainly classified as response to stress,transcription regulation,hormone signaling pathway,antioxidant,and so on.Further analysis found that AP2/ERF,WRKY,MYB and other transcription factor family genes play an important role in the regulation of alfalfa cold stress.Among them,the expressed of 10 AP2/ERF transcription factors were characterized by qRT-PCR,confirming that the transcriptome sequencing analysis results are reliable.2.Genome-wide investigation of microRNAs and their targets in response to freezing stress in alfalfa,based on high-throughput sequencing.Using small RNA sequencing,the sequencing results revealed that 197 mi RNAs,and included 24 novel miRNAs,and that the expression of 35 miRNAs was affected by freezing stress.Among them,12 miRNAs were identified by qRT-PCR.Meanwhile,105 target genes cleaved by these miRNAs were characterized by degradome sequencing and established a regulatory pattern between miRNA and mRNA.3.Mechanism of freezing tolerence regulation in alfalfaBy systematic biology methods,we found the network data of model plant gene regulation,construct the gene regulation network of alfalfa,integrate the regulation mode between miRNA and mRNA,establish the gene regulation network of alfalfa freezing stress,analyze the regulation mechanism of alfalfa freezing tolerance and provide genetic resources and scientific basis for alfalfa varieties.