Identification Of Alfalfa-specific Gene MsASG166 And Analysis Of Its Function In Response To Drought Stress

Alfalfa（Medicago sativa）is a dominant and characteristic species in China’s grass and pasture germplasm innovation industry,but the drought is a key bottleneck affecting its yield and geographical distribution.At present,research on drought-resistant functional genes and molecular regulatory mechanisms in alfalfa are still seriously lagging behind.Linage-specific genes are genes that are specific to a species and have important functions in regulating plant responses to adversity stress,but whether they regulate alfalfa responses to drought stress remains unclear.In this study,we identified alfalfa-specific genes based on the whole alfalfa genome for the first time and analyzed their sequence characteristics,chromosome distribution,and expression patterns in response to abiotic stresses.The alfalfa-specific gene MsASG166（Ms G0780040165.01.T01）was identified to have a positive regulatory function in response to drought stress by q RT-PCR,semi-quantitative PCR,yeast heterologous expression and Arabidopsis overexpression experiments.This study is the first time to identify alfalfa-specific genes and analyze the physiological and molecular functions of MsASG166 in response to drought stress,which can provide excellent genetic resources for creating new germplasm of alfalfa with high drought tolerance using genetic engineering technology.The main results of this study are as follows:1.A total of 199 alfalfa-specific genes（MsASG）,3054 legume-specific genes（LSG）,and 45912 evolutionarily conserved genes（ECG）were identified in the genome of "Zhongmu No.1" by BLAST alignment.Compared with ECGs,MsASGs and LSGs had shorter gene length,protein length,exon length,fewer introns,and slightly higher GC content.Among them,81% of MsASGs and 68% of LSGs had no introns,and MsASGs and LSGs were evenly distributed on the eight chromosomes of " Zhongmu No.1" with no chromosome distribution preference.2.The expression patterns of seven MsASGs significantly responding to abiotic stresses were further analyzed based on RNA-seq results.The results of q RT-PCR showed that MsASG111,MsASG165,MsASG097,MsASG166,MsASG125,MsASG151,and MsASG016 significantly responded to drought,salt,and ABA stresses,and The expression levels could reach several thousand-fold of the control.The semiquantitative PCR results showed that MsASG111,MsASG165,MsASG097,MsASG166 and MsASG125 showed significantly higher transcript levels in response to drought stress.The CDS sequences of MsASG111,MsASG165,MsASG097,MsASG166 and MsASG125 have been cloned,and they were heterologously expressed in yeast,and it was found that yeast overexpressing MsASGs had a better growth trend under drought stress conditions compared to the null yeast.3.MsASG166 showed superior drought resistance in all the above experiments.To further analyze the molecular function of MsASG166,a PHG-MsASG166 overexpression vector was constructed and MsASG166 was observed to be localized in the intercellular space and cytoplasm after transfer into onion epidermal cells.Root length phenotypic evaluation on drought stress plates showed that the root length of MsASG166 overexpressing Arabidopsis was significantly longer than that of the wild type,and the seedlings have stronger drought resistance than the wild type.