| Pitaya(Hylocereus undatus)is a kind of Hylocereus undatus plants.It is nutritious,rich in a variety of beneficial substances.Due to its advantages of light and climate,Hainan is especially suitable for the cultivation of pitaya.However,May to November each year is the typhoon season in Hainan.Typhoons and other factors can lead to flooding,which will leads to the increase of soil water content.When the water content exceeds the field water holding capacity,it will cause flooding stress of pitaya.It will affect the normal growth and development of the pitaya,and it is not conducive to the production of pitaya.Therefore,"Taiwan Dahong" was used as a genetic material in this study.On the one hand,it was treated with flooding stress and samples were taken at different flooding times.Morphological indexes,seven physiological and biochemical indexes related to flooding stress were determined.Including SOD activity,soluble sugar content,soluble protein content,proline content,chlorophyll content,root activity etc.The difference and dynamic analysis among the samples with different duration of flooding were carried out to explore the macroscopic effect of flooding stress on pitaya.On the other hand,a new generation of high-throughput RNA-Seq sequencing technology was used for transcriptome sequencing to construct digital expression profiles of samples of different flooding durations.Through the comparative study of bioinformatics,the differentially expressed genes related to flooding stress were screened out,the important genes related to the response to flooding stress were cloned,and the regulation mechanism of the response to flooding stress wasexplored.The results of the study are as follows:1.The roots of the seedlings became shorter significantly between 0 and 10 days of stress,the lateral roots decreased significantly at 30 days of flooding,the lateral stems began to wrinkle and wilted between 30 and 40 days after stress;the base of the main stem began to appear disease spots between 30 and 40 days after stress,began to decay between 40 and 50 days after stress.The results showed that pitaya cutting seedlings had some tolerance to flooding stress,and the effect of flooding habitat on pitaya was the first to affect its root system.2.After the flooding stress,the SOD activity,POD activity,soluble sugar content,soluble protein content,proline content of pitaya increased significantly,the chlorophyll content remained basically unchanged,the root activity was decreased significantly.The results showed that pitaya can regulate some physiological substances in its body to resist the effects of adversity and enhancing the resistance of pitaya to flooding stress.3.According to RNA-Seq sequencing,the clean reads number of root samples CK1,CK2,CK3,which were not treated,and root samples FR1,FR2,FR3,which were not treated by flooding stress for 3 h,was 66469530(89.15%),66858484(89.59%),66364154(88.87%),66947570(89.70%),66976684(89.73%),63525616(89.59%).After clustering and removing redundancy,26466,25983,25780,26331,29186,23138 Unigenes were obtained respectively,accounting for 50.20%,49.29%,48.90%,49.95%,55.36%,43.89%,respectively.Its average length is 731.26 bp and N50 is 1792.4.The number of Unigene in comparison with the seven databases(Nr,Nt,GO,KOG,KEGG,SwissPprot,and InterPro)were 71.43%,46.00%,41.03%,57.00%,53.43%,48.55%,and 61.37%,respectively.Among them,the total number and proportion of Unigenes on all databases in the seven databases were 11357 and 21.54%,respectively.The total number and proportion of Unigenes annotated by any of the seven databases is 39239 and 74.43%.There were 19763 Unigene genes that were annotated in the Nr,KOG,KEGG,SwissProt,and InterPro databases.Compared with the 25 functional groups of the KOG database,the number of proteins involved in general function prediction only was the most,followed by the signal transduction mechanism;compared with the GO database,the functional distribution was mainly enriched in metabolic pathway and catalytic activity.5.The differences of gene expression between FR group and CK group were compared and analyzed.When the difference multiple of gene expression was 2,there were 1481 differentially expressed genes,of which 925 genes were up-regulated and 556 genes were down-regulated.When the difference multiple of gene expression was 3,there were 965 differentially expressed genes,of which 637 genes were up-regulated and 328 genes were down-regulated.When the difference multiple of gene expression was 4,there were 535 differentially expressed genes,among them 379 genes were up-regulated and 156 genes were down-regulated.Most of its functions are concentrated on sugar metabolism,nitrogen metabolism,alcohol metabolism and so on.In addition,some of the genes encoding transcriptional factors and some hormone related genes are also significantly different among different treatments.6.Through the GO enrichment analysis of differentially expressed genes between FR group and CK group,we can see that the differentially expressed genes are enriched in the single-organism metabolic process of themetabolic pathway,the pyruvate decarboxylase activity and thiamine pyrophosphate binding of catalytic activity and other aspects.By pathway enrichment analysis of differentially expressed genes,we can conclude that the differentially expressed genes are enriched in phenylalanine metabolism,stilbenoid,diarylheptanoid and gingerol biosynthesis,ubiquinone and other terpenoid-quinone biosyntheis.The number of differentially expressed genes related to energy metabolism,amino acid biosynthesis,ethylene metabolism,ROS metabolism and other metabolic pathways,secondary metabolite synthesis,and senescence related genes were more.This suggests that these differentially expressed genes are likely to be related to response mechanisms under plant flooding stress.7.It was predicted that there were 1600 Unigenes with the ability to encode transcription factors,among which MYB,MYB-related,WRKY,AP2-EREBP and bHLH family had more transcription factors,which were 190,149,100,125 and 117,respectively.The transcription factors of the NAC and C3H family are 70 and 88,respectively,and these transcription factors may be involved in the regulation network formed by the pitaya in response to flooding.When the fold change of differentially expressed genes was 4,the number of genes that can encode WRKY,MADS,bHLH,AP2-EREBP and other TF is large.These TF may be involved in the regulatory network formed by pitaya in response to flooding stress.8.According to the related reports,physiological data and KEGG database analysis results,26 Unigenes which may be related to flooding stress were selected for qRT-PCR verification.The results showed that the sequencing results were consistent with the predicted results.Furthermore,Glucose-6-phosphate isomeraseN Ribosome-binding ATPase YchF.Ethylene-responsive transcription factor 1B.Ethylene receptor 2.Ethylene insensitive 3.Cytochrome P450 87A3.LRR receptor-like serine/threonine-protein kinase FLS2.Squalene synthetase、WRKY transcription factor 3 and other genes that might be sensitive to flooding stress were selected for further digging and research.9.We selected gene Unigene15627_A11 and CL2218.Contigl_A11,these two genes were significantly different in plant flooding stress and closely related to flooding stress.We carried out bioinformatics analysis of the two genes to provide a theoretical basis for subsequent research. |
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