The Identification Of Drought-resistance Gene In Perennial Ryegrass(Lolium Perenne L.)

Drought stress is responsible for half of all meteorological catastrophes,which account for 70%of all worldwide disasters,according to data,and it poses a severe danger to both food and environmental security.A good strategy to deal with the problem is to breed drought-resistant cultivars and improve plant drought tolerance.It’s critical to use biotechnology to mine drought-resistant genes and enhance plant tolerance to adversity stress in a short amount of time.The perennial ryegrass（Lolium perenne L.）,one of the most popular cool-season perennial herbs in temperate regions of the world,is severely affected by drought stress on its turf quality and other traits.Its genetic diversity is considerable since it is a self-incompatible diploid.Based on this,the relative drought resistance and non-drought resistance variations of 18 perennial ryegrass types were determined in this study by a detailed comparison of their performances under simulated extreme and moderate drought circumstances.Under dehydration circumstances,transcriptome analysis evaluated differentially expressed genes in these two cultivars.Through GO annotation and the KEGG pathway analysis,we mine candidate genes of perennial ryegrass regulating water deprivation on the cellular level（transcription factors,protein kinases,etc.）and the physiological level（ROS scavenging by antioxidant enzymes,osmotic regulation of soluble sugar and proline）.The main findings of this work are as follows:（1）In the cultivar screening experiment,we measured the survival rate and relative water content of 18 perennial ryegrass cultivars under extreme drought treatment after rehydration,and the relative growth rate as well as the relative biomass when the soil moisture content was maintained at 40%and 50%.We also looked at the rate of water loss in isolated leaves during normal development.Thus,by rating the test results,the relative drought resistance of all cultivars was evolved.Sopin is the most drought-resistant type（scoring 32）,whereas Charging is the least drought-resistant type（scoring 11）.（2）Transcriptome analysis showed that there were 7851 differentially expressed genes（DEGs）between these two varieties under dehydration treatment,4618 of which were up-regulated in Sopin.The DEGs were mainly enriched in signal transduction,translation,amino acid metabolism and carbohydrate metabolism pathways.GO and KEGG identified 143 water loss response genes from the aforementioned genes,including DREB,NAC,MYB,WRKY,and other transcription factors involved in signal transduction,osmotic regulation,and reactive oxygen species scavenging.When compared to Charging of non-drought-resistant cultivar,there are 97 up-regulated in Sopin.These findings suggest that perennial ryegrass regulates the expression of those genes in response to water loss stress.（3）By measuring the stress physiological parameters such as ROS（O₂^-,H₂O₂）,MDA,antioxidant enzyme activities（SOD,CAT,POD）,soluble sugar and free proline,the drought-resistant cultivar had lower levels of ROS and MDA,higher antioxidant enzyme activity,higher soluble sugar content,and more rapid proline accumulation rate than the drought-sensitive cultivar,implying that ROS scavenging and osmotic regulation in perennial ryegrass played key roles in plant response to water loss.（4）By combining transcriptome data and physiological results,35 genes were identified from water loss response genes,including transcription factors and genes involved in signal transduction,antioxidation,starch degradation and proline biosynthesis.The expression levels of most genes were greater in the drought-resistant cultivar than in Charging under water loss stress,according to the results of q RT-PCR investigation.Since some photosynthetic proteins can indirectly scavenge ROS,the expression of 5LHC-related genes and 1 CA gene were analyzed.It was found that there was no significant difference in the expression of LHC-related genes in these two varieties under control or water loss conditions,indicating that the antioxidant enzymatic system and osmoprotectant played a leading role in scavenging ROS when perennial ryegrass seedlings lost 40%-50%water.Taken together,we found that transcriptional regulation,reactive oxygen species scavenging,and osmoregulation of soluble sugars and proline play important roles in the response of perennial ryegrass seedlings to water loss stress.This study provides candidate genes for future research on molecular breeding for drought resistance in perennial ryegrass,as well as a foundation for studying drought resistant processes in perennial ryegrass.