| Drought stress is one of the most important environmental stress factors in plant growth.When plants are subjected to drought stress,their growth and reproduction will be adversely affected.In recent years,hemerocallis plays an increasingly important role in landscape greening in China.Therefore,exploring the candidate genes of hemerocallis in response to drought stress and analyzing its genetic regulation basis can provide new gene resources and genetic basis for molecular design breeding of hemerocallis for drought tolerance.In this study,drought-tolerant hemerocallis’ Mengzihua ’and drought-sensitive hemerocallis’ Shy Tiger’ were used as experimental materials,which were treated with drought stress for 0 d,7 d and 14 d,and the treated leaves were sequenced by high-throughput sequencing technology.Transcriptome data quality control(Fast QC),sequence alignment between sequencing data and genome(HISAT2),quantitative gene expression analysis(String Tie),differential expression analysis(edge R)and gene function enrichment analysis(top GO)were carried out according to the results of sequencing data,which provided new gene resources and mechanism for molecular breeding of hemerocallis for drought resistance The theory basis.The main research results are as follows:1.On the whole,the gene expression levels of both drought-tolerant and sensitive hemerocallis showed different expression profiles after normal growth and drought stress treatment,indicating that drought stress can strongly affect the gene expression of hemerocallis.2.In order to verify the reliability of sequencing results,we randomly screened 8differentially expressed genes for fluorescence quantitative PCR detection,and the results showed that the expression of these 8 genes was consistent with the transcriptome results,indicating that the transcriptome sequencing data were highly reliable.3.Differential expression analysis of transcriptomic data showed that after 7 days of drought treatment,the differentially expressed genes of ’Mengzihua’ and ’Shy Tiger’ were 913 and 1209 respectively,among which 29 and 47 transcription factors were differentially expressed,respectively.The number of up-regulated genes of ’Shy Tiger’ was 385,and the number of down-regulated genes of ’shy Tiger’ was 824.The number of up-regulated genes was 345 and down-regulated genes was 568.After 14 days,1777 genes and 1440 genes were differentially expressed in ’Mengzihua’ and ’shy tiger’,including 50 and 44 transcription factors.The number of up-regulated genes was 755 in 1777 differentially expressed genes,and the number of down-regulated genes was 1022,and the number of up-regulated genes was 521 and 919 in 1440 differentially expressed genes.4.Further analysis revealed that a total of 1777 differentially expressed genes were identified from the transcriptomic data of ’Mengzihua’ for 14 days under drought stress,from which 10 candidate genes for inhibiting expression,12 candidate genes for inducing expression and 22 candidate genes for diversifying expression were screened out.Multiple sequences of these candidate genes were compared with arabidopsis thaliana genome,and it was found that these genes improved the drought resistance of hemerocallis by participating in water stress response,osmotic stress response,ABA response,protein dephosphorylation and proline biosynthesis.5.Our previous study showed that the stomatal conductance of ’shy tiger’ was significantly higher than that of ’wild flower’,and the stomatal density of ’shy tiger’ was slightly lower than that of ’Mengzihua’.Therefore,we analyzed the differentially expressed genes related to stomata,and found four differentially expressed genes Hf MYB2,Hf MKK9,GPA and Hf MYB4 in the transcriptome data of ’shy tiger’ under drought treatment.Nine drought-related differentially expressed genes Hf MYB102,Hf MYB2,Hf MKK9,Hf CCA1 and CYCA2;1 were found in the two cultivars treated with drought for 7 d and 14 d.GPA,Hf MYB4,Hf ERF4 and Hf WRKY30.Hf MYB102,CCA1 and CYCA2;1 were differentially expressed which related to plant hormones.6.In the screening of differential genes affected by drought stress,it was found that the expression of GRAS family genes in hemerocallis was affected by drought stress,so the relevant situation of GRAS transcription factor family in hemerocallis was further explored.The results showed that 12 GRAS transcription factor family genes were differentially expressed in hemerocallis under drought stress.The GRAS family proteins of hemerocallis hemerocallis vary greatly in physical and chemical properties.Twelve proteins are alkaline and hydrophobic.The GRAS family proteins of hemerocallis hemerocallis contain ten conserved domains.Evolutionary analysis showed that GRAS family genes were divided into8 subgroups,and hemerocallis GRAS genes accounted for 4,among which the ⅷ subgroup contained the most GRAS members,which was 6.The GRAS family protein sequence of hemerocallis GRAS is highly conserved.The analysis of secondary structure of hemerocallis GRAS protein showed that for hemerocallis GRAS protein,α helix accounted for the largest proportion of secondary structure(39.54～52.28%),and β fold(3.43～6.97%)accounted for the least proportion.Twelve genes were divided into four main categories.The tertiary structure of hemerocallis GRAS protein was analyzed,and it was found that the constituent forms of conservative domain of hemerocallis GRAS protein in the same subfamily were very similar,but the number of Hemerocallis GRAS gene and Arabidopsis GRAS gene were different in different subfamilies.The GRAS genes of 12 hemerocallis hemerocallis showed differential expression after drought stress,Hf GRAS1,Hf GRAS3,Hf AS6,Hf GRAS8,Hf GRAS11 and Hf GRAS12 were up-regulated genes.Hf GRAS2,Hf GRAS4,Hf GRAS5,Hf GRAS7 and Hf GRAS9 were down-regulated genes.It is speculated that Hf GRAS3,Hf GRAS4 and Hf GRAS5 may play an important role in the drought resistance of hemerocallis. |
