| Objective: In this study,Gardenia jasminoides Ellis was subjected to different levels of drought and waterlogging stress,and various physiological indicators and endogenous hormone contents were measured to study the physiological characteristics of gardenia response to water stress.At the same time,in order to explore the molecular mechanism of gardenia responding to water stress,and provide the basis or ideas for the research of gardenia or other Medicinal plants against water stress,high-throughput sequencing was used to obtain the transcriptome data of gardenia under water stress,and the differentially expressed genes were analyzed by a variety of software and technologies.Methods: Different concentrations of PEG-6000 were used to simulate gardenia to suffer from light drought(LD),moderate drought(MD)and severe drought(SD),and“double basin method” was used to simulate gardenia to suffer from light waterlogging(LW),moderate waterlogging(MW)and severe waterlogging(SW).Mature leaves of gardenia at the same leaf position were picked every 5 days for follow-up study.1.Relative water content of gardenia leaves was measured by drying and weighing method,chlorophyll content was measured by acetone extraction method,SOD activity was measured by NBT riboflavin microplate method,POD activity was measured by guaiacol method,and MDA content was determined by TBA colorimetry.The content of PRO was determined by the ninhydrin microplate method,and the content of soluble sugar was determined by the anthrone colorimetric method.2.The content of four endogenous hormones(ABA,IAA,GA and ZR)in gardenia was determined by enzyme-linked immunosorbent assay.The 80% methanol solution was used to roughly extract the endogenous hormones from gardenia,the crude extract was passed through a C-18 solid-phase extraction cartridge,followed by nitrogen drying,and the volume of the sample diluent was adjusted to obtain a sampleto be tested.3.The total RNA of gardenia leaves was extracted by Trizol method,and then the library construction,sequencing,quality control,and De Novo assembly were completed in order to obtain All-Unigene,which was compared with six major databases using Blast software.4.DEGseq software was used to analyze the differential expression between groups,GO enrichment and KEGG enrichment of differentially expressed genes were performed,and metabolic pathways were screened and studied significantly.At the same time,the reliability of the transcriptome data was verified by q RT-PCR.Result: 1.Compared with the CK group,the relative water content and chlorophyll content of gardenia leaves under drought stress generally decreased,the SOD enzyme and POD enzyme activities increased slightly and then decreased significantly,and the MDA content began to increase significantly in the later stage of stress,the PRO content and soluble sugar content increased significantly at the beginning of the stress,and then gradually decreased.The relative water content,chlorophyll content,the SOD enzyme activity,and the PRO content of gardenia under waterlogging stress were consistent with those under drought stress.The POD enzyme activity had a significant upward trend followed by a downward trend,and the MDA content had no significant change before 20 days,the soluble sugar content in the LW group increased first and then decreased,and the soluble sugar content in the MW and SW groups decreased first and then increased.2.Compared with the CK group,the ABA content of gardenia leaves significantly increased and maintained for a long time under drought stress,the IAA and GA contents first increased and then decreased,and the ZR contents showed high-low-high fluctuations,IAA/ABA,GA/ABA values of gardenia in the late stage of stress gradually decreased.In LW group,the ABA content in gardenia leaves first decreased and then increased,while in MW and SW groups,the IAA content in gardenia leaves first increased and then decreased,the GA content in gardenia leaves gradually decreased and the ZR content fluctuated in waterlogging stress.At the same time,the IAA/ABA,GA/ABA and ZR/ABA values in gardenia leaves generally increased and then decreased.3.It was found that each group of samples had more than 37,620,388 Raw reads.After quality control,more than 36,274,838 Clean reads were obtained,the Q20 valuewas more than 97%.124192 Unigenes were assembled by De Novo,the average length of Unigene was 537.88 bp,and N50 was 753 bp.There are 68648 Unigene notes in NR database,the most of which are coffee canephora,accounting for 32.33%,and both coffee and gardenia belong to Rubiaceae,which has reference value.In GO database,genes are mainly annotated into cell components,and in KEGG database,genes are mainly annotated into genetic information translation process,carbon metabolism,energy metabolism and lipid metabolism.4.The results showed that 12842,5576 and 20137 genes were differentially expressed in gardenia on the 5th,10 th and 15 th day of drought stress,and 19631,23693 and 15045 genes were differentially expressed on the 5th,10 th and 15 th day of waterlogging stress.Through in-depth analysis of GO and KEGG enrichment,we found that the differential genes were mainly enriched in signal transduction,transcription factors,genetic materials and processes,as well as various metabolic pathways.The genes closely related to drought stress include STKc MAP3K-like,ABA receptor PYR / PYL / RCAR protein,PP2 C,b ZIP transcription factor 53,MYB12 and other genes.The metabolic pathways closely related to drought stress are phenylpropanoid biosynthesis,plant-pathogen interaction,circadian rhythm-plant.The genes closely related to waterlogging stress include proteolipid membrane potential modulator,PP2 C,EIN3 proteins,Salicylic acid-induced protein homologs,SNAC1,and WRKY65 PE.The pathways closely related to waterlogging stress are phenylpropanoid biosynthesis,glutathione metabolism,glycolysis/gluconeogenesis.Conclusion: 1.When gardenia suffered from water stress,the relative water content and chlorophyll content of leaves decreased rapidly to reduce their own activities,and the activities of the SOD enzyme and POD enzyme were adjusted to reduce the oxidative damage caused by stress,the fact that MDA content did not increase in the early stage of stress indicated that SOD enzyme and POD enzyme played a role in coordination with other antioxidant substances,and the content of PRO and soluble sugar increased to maintain the homeostasis of gardenia internal environment.2.In the early stage of drought stress,ABA,IAA,and GA in gardenia played an important role in signal transduction,growth and development regulation,these three endogenous hormones had a downward trend in the later stage,indicating that gardenia response to drought stress may be limited by the time of stress.In the later period,the increase of ZR content can delay the senescence of leaves and maintain thelife activity of gardenia.The changes of endogenous hormones in gardenia under moderate and severe waterlogging stress were basically the same as that under drought stress,but the response of endogenous hormones in gardenia under light waterlogging stress was relatively slow,and the response of hormones in gardenia would be similar at the later stage of stress.3.The annotated results of gardenia All-Unigene in the GO and KEGG databases under water stress indicate that gardenia responds to water stress by actively adjusting cell structure,regulating the translation process of genetic information and various metabolic pathways.The genes and pathways that are mainly enriched in gardenia under drought stress include signal transduction of stress,transcription regulation,cell wall strengthening,adhesion and defense,stomatal closure,reduction of oxygen free radical generation,allergic reactions and defense responses.The genes and pathways that are mainly enriched in gardenia under waterlogging stress involve signal transduction of gardenia stress,transcriptional regulation,the use of non-enzyme substances to remove oxygen free radicals to reduce oxidation,adjust their own energy supply,and regulate cell osmotic pressure. |
