| Pitaya is belongs to Cactaceae Hylocereusundadus.It was introduced to Taiwan in the 1990s then introduced to Guangdong,Guangxi,Hainan and other areas.Pitaya has become a new economic tropical fruit in Hainan.Stresses including drought,high temperature and so on seriously affect plant growth and yield.Although pitaya has good drought tolerance and survivability,drought stress results in decreased production of pitaya,lower quality,and not even bear fruit.In this study,pitaya of Dahong was used as the experimental material,we compared pitaya under different drought periods with pitaya under regular watering.On one hand we had measured free proline(Pro),soluble protein,soluble sugar,malondialdehyde(MDA),peroxidase(POD),catalase(CAT),superoxide dismutase(SOD)and analyzed the differences between samples.On the other hand a new generation of high-throughput sequencing technology(RNA-Seq)was used to compare the root and stem of pitaya in the treatment group and control group.We screened specific expressing genes,verified the expression of the screening genes,analyzed function of the screening genes which related to the molecular mechanism of drought stress,explored regulatory molecular mechanism of pitaya under drought stress.The results are as follows:1.It had significant differences in physical and biochemistry idexes of Dahong pitaya between treatment group and control group.After forty days of drought stress,the content of free praline(Pro)and soluble sugar were significantly increased in the treatment group;the content of soluble protein and malondialdehyde(MDA)went down at first and then rose;the activity of peroxidase(POD)significantly increased and then declined which was similar with the activity of the catalase(CAT).Yet,the activity of superoxide dismutase(SOD)continued to decline.The results showed that pitaya had a good drought resistance and pitaya can adapted drought stress environment by regulating physiological and biochemical changes in vivo.2.Samples were stem under drought stress(DS),root under drought stress(DR),control group of stem(CS),control group of root(CR).Each group was set with three biological replicates,named DS1,DS2,DS3,DR1,DR2,DR3,CS1,CS2,CS3,CR1,CR2,CR3.The number of high quality reads we received were 65826242(91.15%),65700694(90.98%),65465498(90.65%),65572056(90.98%),65830600(91.16%),65178148(90.35%),66068390(91.49%),67040900(92.83%),66499560(92.08%),65328888(90.46%),66303274(91.81%),66597036(92.22%)respectively.The number of high quality reads we can assembly were 22770,22560,13975,34016,35953,44050,15538,37881,21739,42799,43262,53218 items,and we totally got 52717 Unigene which mean length was 1102bp,N50 was 1792bp,length was between 596-1451bp.The number of Unigene which mapped to the twelve samples that we respectively received were 16742,16759,10042,25916,27312,33105,11289,28457,16168,32056,32950,40007 items.In Nr,Nt,Swiss-Prot,KEGG,KOG,GO,Inter-Pro databases,the number of Unigene which mapped to the seven databases were 37658(71.43%),24248(46.00%),25596(48.55%),28168(53.43%),30047(57.00%),21628(41.03%),32354(61.37%)items respectively and there were 19763 Unigene mapped to all seven databases among them.3.Analyzed all Unigene from assembly:In Gene Ontology,molecular function mainly enrichment to cellular component,biological process,molecular function,respectively had 40294,42436,24236 Unigene;we got 25 categories by KOG database comparison and the maximum number of Unigene we got in general function prediction only was 7131,the minimum number of Unigene in cell motility was 43;we divided KGEE into 19 groups and 11 groups were related to metabolism;there were 1600 Unigene that mapped to TF and most of them were enriched to AP2-EREBP,WRKY,MYB,M YB-related and so on,these Unigene were all taking part in abiotic stress response.4.We screened differentially expressed genes(DEGs)by Pvalue<0.05 and |log2FC| ≥1 and totally we got 215 genes.There were 194 DEG from CR-VS-DR that including 70 up-regulated expression genes and 124 down-regulated expression genes which responsed to drought stress.There were 21 DEGs from CS-VS-DS that including 12 up-regulated expression genes and 9 down-regulated expression genes which responsed to drought stress.DGEs were mostly mapped to metabolism,Genetic information processing,Environmental information processing,Cellular processes,Organismal systems by analyzing Pathway between treatment group and control group.Metabolism included biosynthesis of other secondary metabolites,carbohydrate metabolism,Amino acid metabolism,Lipid metabolism,Glycan bosynthesis,metabolism and so on,these pathways were all related to abiotic stress response.It showed that the ways pitaya responsed to drought stress was complicated and the main way to adapt to dry environment was to maintain normal metabolic levels.5.Based on the analysis of Pathway,we screened 19 Unigene in CR-VS-DR database,5 Unigene in CS-VS-DS database and verified their expression levels by qRT-PCR.Finally we got the same results by sequencing and prediction results.It showed that sequencing results can be trusted and we were thus able to gain a deep insight into the function of these genes.We screened Unigene69_All and CL218.Contigl_All,verified their differential expression in different tissues,analyzed Unigene69_All by bioinformatics analysis and predicted its function. |
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