Evaluation Of Heat Tolerance Of Perennial Ryegrass And Breeding Utilization Of HD-ZIP Transcription Factor

Perennial ryegrass(Lolium perenne L.)is widely distributed in temperate and semi-temperate regions in China and around the world.It is loved as a high quality cool season turf grass and forage grass with the advantages of fast growth,mowing and pruning resistance and high yield.The suitable temperature for perennial ryegrass is 16～24℃,and it has poor heat resistance,so it cannot cope with the long and hot summer in southern China.The inability of perennial ryegrass to over-summer leads to the need for replanting the following year,which in fact loses its perennial characteristics.In this study,we analyzed the genetic diversity of 201 perennial ryegrass germplasm resources for four morphological structural traits(plant height,stiffness,leaf width,and leaf length)and heat tolerance in all,and the results showed that these 201 perennial ryegrass germplasm differed widely from each other and had extensive genetic diversity.In this study,two LpHOX22 and LpHOX24 genes induced by high temperature stress were screened for homologous gene cloning and functional analysis based on the transcriptome data of perennial ryegrass stress in the pre-laboratory period.The specific results are as follows.1.Evaluation of heat tolerance of perennial ryegrass:Seven phenotypic trait data of201 perennial ryegrass germplasm resources were analyzed by frequency distribution histogram,and all seven phenotypic trait data approximately obeyed normal distribution;the coefficients of variation in descending order were:heat tolerance(72.88%)>leaf area(49.58%)>leaf width(31.55%)>natural plant height(24.42%)>Leaf length(23.13%)>stretched plant height(22.65%)>plant stiffness(21.95%);Pearson correlation analysis showed that the germplasm with low values of stretched plant height,small leaf area, narrow leaf width and short leaf length had higher heat tolerance;the higher the stretched plant height,the higher the natural plant height,the higher the stiffness and the wider and longer leaves.The cluster analysis by the Euclidean distance furthest distance method classified the 201 perennial ryegrasses as a whole into three groups(Ⅰ-Ⅲ):Cluster Ⅰ is tall and characterized by high mean values of plant height and stiffness.Cluster Ⅱ is the dwarf type,characterized by low mean values of plant height and uprightness and narrow leaf width.Cluster Ⅲ was intermediate,with data in the middle category.2.Cloning and functional analysis of LpHOX22 and LpHOX24 genes:In this study,based on the transcriptome data of perennial ryegrass,two unigene of homeobox induced by high temperature stress were screened.ZIP Ⅰ gene family of LpHOX22 and LpHOX24genes,which are involved in regulating plant responses to environmental stress.Amino acid sequence analysis showed that both LpHOX22 and LpHOX24 have a highly conserved homeodomain(HD)domain and a HALZ domain(a leucine"zipper" immediately attached to the HD),and the proteins of the HOX22 and HOX24 genes are highly Conserved.The LpHOX22 and LpHOX24 genes were cloned and functionally validated in perennial ryegrass,and the results showed that LpHOX22 and LpHOX24transgenic Arabidopsis had reduced heat tolerance relative to the wild type,and plant survival was significantly lower than that of the wild type after a 3-d recovery at 42℃ for52 h,while electrical conductivity was significantly higher than that of the wild type.The transgenic Arabidopsis seeds also showed more sensitivity to osmotic stress than the wild type at the germination stage.This suggests that LpHOX22 and LpHOX24 genes may act as negative regulators of high temperature stress.