Sequencing Of Transcriptome And Small RNA And Expression Analysis Of Related Genes In Peanut Under Drought Stress

Peanut?Arachis hypogaea L.?is an important oil crop and plays an important role in the supply of edible oil in China.Drought is an important factor affecting the growth and yield of peanuts.Therefore,it is of great significance to clarify the mechanism of peanut response to drought stress for the cultivation of new peanut varieties with strong resistance.Currently,there are prevailing problems in peanut production in our country that seriously affect the peanut yield,quality,drought,plant diseases and insect pests.However,there are few literatures and reports on the changes of genes and miRNAs under drought-affected peanuts.By further studying the peanut transcriptome and small RNA,we can discover the related genes and miRNA of peanut drought resistance and study their differential expression under drought stress conditions.The results can provide theoretical basis for the research of peanut drought resistance and the cultivation of new drought resistance varieties.In this study,15% PEG6000 was used to simulate drought-stressed peanut roots?1³days?and untreated peanut roots?HS15₀,HS15₁,HS15₂ and HS15₃?for transcriptome and small RNA sequencing statistics,and analyzed the drought-related differentially expressed genes and miRNA;Determine the contents of chlorophyll,malondialdehyde,proline and soluble protein in peanut leaves and observe their dynamic changes under water stress;And the ahyNAC25 gene was cloned and genetically transformed into Arabidopsis thaliana to further study its function and corresponding molecular mechanism.The main results of the study are as follows:?1?After drought stress,the contents of chlorophyll a,chlorophyll b,and chlorophyll?a + b?of peanut seedlings decreased significantly with the increase of stress days.The contents of proline and soluble protein increased significantly on the first and second days of stress,and decreased slightly on the third day,but it still higher than those plants without drought stress.However,the content of malondialdehyde increased significantly with the increase of stress time.?2?Four libraries of HS15₀,HS15₁,HS15₂ and HS15₃ were constructed and transcriptome sequencing was carried out.43625768,44792456,43100476 and 36638768 clean reads were obtained respectively.Comparing the reference genome,the ratios were77.19%,74.76%,69.83% and 51.78%,respectively.?3?Some transcription factors and functional genes related to drought were selected in the transcriptome data: MYB transcription factor,WRKY transcription factor,NAC transcription factor,bZIP transcription factor,LEA gene,AQP gene,molecular chaperone,proline-related genes,SOD and betaine related genes.?4?Through the analysis of differentially expressed genes among samples,a total of13 differently expressed gene with significant and continuous up-regulated were screened out in three comparison groups,including seven HSP molecular chaperone family genes,two RING E3 ubiquitin ligases,a glutathione S transferase,a stigma-specific STIG1-like protein 1,an immunoglobulin binding protein,and a DnaJ homolog B subfamily member13;Meanwhile,13 significantly down-regulated differential genes were screened out.These may play a certain role in drought resistance of peanut.?5?Four libraries of HS15₀,HS15₁,HS15₂ and HS15₃ were constructed,and small RNA sequencing was carried out,obtaining 28005836,26862969,26668939 and25970037 clean reads respectively,.The proportion of reads in the compared small RNA database was 77.86%,81.14%,79.47% and 58.05% respectively.?6?From the small RNA sequencing data,8 miRNAs with continuous regulation expression were screened out.These miRNAs include ahy-miR156b-3p,ahy-miR159,ahymiR167-5p,ahy-miR156 a,ahy-miR156b-5p,novel_mir103,novel_mir83 and novel_mir70.They may participate in the drought resistance of peanut by increasing the expression of target genes through negative regulation mechanisms.?7?The bioinformatics of the ahyNAC25 gene?XM₀16087998.2?was analyzed.The gene has an open reading frame of 1032 bp and encodes 344 amino acids.Its encoded protein contains a NAM conserved domain,and the NAC25 gene homology with legume vine peanut and wild peanut is 99% and 97%,respectively.Genes belonging to the NAM superfamily.?8?The ahyNAC25 gene was constructed into a plant expression vector,which was transferred into Arabidopsis thaliana by dipping,and 16 transgenic plants and T1 generation Arabidopsis seeds were obtained.