Study On The Response Of Paeonia Ostii To Drought Stress And The Function Of CCoAOMT Gene

Fengdan(Paeonia ostii)is a perennial woody plant belonging to the genus Paeoniae in the Paeoniaceae family.It is a variant of Yangshan peony with high ornamental value,medicinal value and oil value.As a resource of oil tree peony approved by the Ministry of Health,Paeonia ostii has been promoted and planted in more than 20 provinces and regions in China due to its advantages of easy survival,high seed yield and high unsaturated fatty acid content,especially in arid or semi-arid areas such as mountainous areas and sandy wastelands.In arid or semi-arid environment,drought stress is the main abiotic stress factor which affects the growth of Paeonia ostii.How to alleviate the damage of Paeonia ostii caused by drought stress has become an urgent problem to be solved.In order to improve the growth of Paeonia ostii in arid or semi-arid areas,it is of great theoretical and practical significance to systematically study the effects of drought stress on Paeonia ostii,take appropriate measures to alleviate the damage of drought stress to Paeonia ostii and find the key genes of Paeonia ostii in response to drought stress.Therefore,Paeonia ostii was used as the material to analyze its physiological response to drought stress in this study,and to explore the mitigation effect of exogenous calcium chloride(CaCl2)on the damage of Paeonia ostii under drought stress.Moreover,based on the transcriptome sequencing,the key genes of Paeonia ostii in response to drought stress were identified,and the candidate gene PoCCoAOMT sequence was cloned and its function of regulating drought tolerance was verified.The main results were as follows:(1)Drought stress caused Paeonia ostii leaves to wither and dry,reduced the leaf relative water content,increased the relative electrical conductivity(REC),malondialdehyde(MDA)content,hydrogen peroxide(H2O2)and superoxide anion(O2·-)accumulation,and increased the antioxidant enzyme activities of superoxide dismutase(SOD),peroxidase(POD)and ascorbic acid peroxidase(APX).Moreover,drought stress decreased net photosynthetic rate(Pn),stomatal conductance(Gs),intercellular CO2 concentration(Ci),transpiration rate(Tr),the maximum light efficiency(Fv/Fm),actual photosynthetic efficiency(Y(?)),and increased non photochemical fluorescence quenching coefficient of chlorophyll(qN)and quantum yield of regulatory energy dissipation(NPQ).In addition,continuous drought stress could destroy the chloroplast structure and close the stomata of Paeonia ostii leaves,and then inhibit its photosynthesis.(2)Exogenous CaCl2 treatment could alleviate the wilting and drying symptoms of Paeonia ostii leaves under drought stress,reduce the rate of decline of leaf relative water content,and keep the plant with lower Pro content,REC,H2O2 and O2·-accumulation and higher antioxidant enzyme activities including SOD,POD and APX.In addition,the exogenous CaCl2 treatment could increase the photosynthesis and chlorophyll fluorescence parameters of Pn?Gs?Ci?Tr?Fv/Fm?Y(?)and qN,and decrease minimum fluorescence(Fo).Transcriptome sequencing was performed on Fengdan leaves and controls on the 12th day after natural drought treatment(3)Transcriptome sequencing was performed using Paeonia ostii leaves and control on the 12th day after natural drought treatment.Then 78,391 high-quality unigenes were obtained,and 33,877 unigenes were annotated.Based on these results,a total of 22,870 differentially expressed genes(DEGs)were identified,and 18 DEGs were selected randomly to verify the accuracy and reliability of RNA-seq data by real-time quantitative PCR(qRT-PCR).According to the pathway function analysis of DEGs,6,997 DEGs were located in 126 pathways,but only 23 pathways met Q?0.05,and 7 pathways were up-regulated and 16 pathways were down-regulated.These pathways could be classified into 6 categories:ROS system,biosynthesis of secondary metabolism,photosynthetic metabolism,proline metabolism,fatty acid metabolism and plant hormone metabolism.Among them,a large number of DEGs related to ROS clearance,photosynthesis and zeatin synthesis pathways were down-regulated,while other DEGs related to proline synthesis and linolenic acid synthesis pathway were up-regulated(4)RACE technology was used to clone Paeonia ostii candidate genes obtained in previous screening in response to drought stress,and PoCCoAOMT was successfully cloned The full-length sequence of PoCCoAOMT 987 bp,and the open reading frame was 744 bp which encoded 247 amino acids.It had high homology with other CCoAOMT sequences in plants.The amino acid sequence encoded by PoCCoAOMT was analyzed by biological information.The relative molecular mass of this protein was 27915.84 Da that was a stable protein and hydrophilic protein.Also,there was no signal peptide,and the protein was located outside the membrane.The expression levels of PoCCoAOMT in the leaves of Paeonia ostii treated with drought stress and control were detected by qRT-PCR.The results showed that the expression of PoCCoAOMT gene was induced by drought stress.(5)The overexpression vector pCAMBIA1301-PoC CoAOMT containing PoCCoAOMT sequence was constructed,and the target gene was mainly located in the cytoplasm and expressed slightly in the chloroplast by subcellular localization observation with the help of tobacco.The vector was transferred into tobacco by agrobacterium-mediated method and positive transgenic tobacco was detected by PCR and qRT-PCR.Natural drought treatment was carried out on transgenic tobacco and wild-type tobacco.After 23 days drought stress treatment,the leaves of wild-type tobacco were severely wilted and drooped,while the transgenic tobacco maintained normal growth.(6)Under drought stress,transgenic tobacco maintained lower REC,H2O2 and O2·-accumulation,higher leaf relative water content,SOD and POD activities,photosynthetic and chlorophyll fluorescence parameters such as Pn,Gs,Ci,Tr,Fv/FM and Y(?).And the expression levels of senescence-associated genes including harpin inducing protein 18 gene(HIN1),cysteine protease 1 gene(CP1),cysteine protease 2 gene(CP2)were also lower in transgenic tobacco.In addition,the expression level and lignin content of PoCCoAOMT gene in roots,stems and leaves of transgenic tobacco were higher than those of wild type under drought stress,and the highest level was in roots and the lowest level was in leaves,and drought stress also induced the high expression of genes related to lignin synthesis including phenylalanine ammonia gene(PAL),cinnamyl alcohol dehydrogenase gene(CAD),4-coumarate:CoA ligase gene(4CL)and caffeic acid-O-methyltransferase gene(COMT)in transgenic tobacco leaves.The above results indicated that PoCCoAOMT could improve the resistance of plants to drought stress by promoting lignin synthesis.