| Herbaceous peony(Paeonia lactiflora Pall.)is a famous traditional flower in China.It is of high ornamental and garden application value because of its colorful flowers,rich flower patterns and elegant qualities.In recent years,it has been widely used in potted gardening,fresh cut flower cultivation and garden landscape.The northern region of China is the main producing area of peony,with the lack of water resource,often drought and less rain.Drought is the main adversity stress factor affecting the cultivation of P.lactiflora in northern China.To systematically study the physiological and molecular responses of P.lactiflora to drought stress,screening the genes closely related to drought resistance of P.lactiflora,and exploring the molecular mechanism are of great significance for cultivating drought-tolerant varieties of P.lactiflora and promoting the cultivation of P.lactiflora in arid and semi-arid areas of northern China.Therefore,we studied the physiological response with five-year-old P.lactiflora ’Da Fugui’ potted seedling and analyzed the molecular response of P.lactiflora to drought stress,based on analysis of the transcriptome sequencing and screened the key genes,with candidate genes PlPM19L and PIMYB108 cloned and their preliminary verification to the drought function carried.The main results are as follows:(1)After treated by drought stress,the leaf of P.lactiflora became withered and yellow.Compared with the control,drought stress reduced the relative water content of leaves,soil relative water content and chlorophyll content,and increased soluble sugar content,soluble protein content,abscisic acid(ABA)content,protective enzyme activity(superoxide dismutase(SOD),peroxidase(POD),catalase(CAT),ascorbate peroxidase(APX)),ascorbic acid-glutathione(AsA-GSH)related enzyme activities in the circulatory system(reduced gluten)Glutathione(GSH),Ascorbic Acid(AsA),Glutathione Reductase(GR),Dehydroascorbate Reductase(DHAR),Monodehydroascorbate Reductase(MDHAR)),Carotenoids content and Flavonoids content.Drought stress inhibited P.lactiflora net photosynthetic rate(Pn),stomatal conductance(Gs),transpiration rate(Tr),potential quantum yield(Fv/Fo)and the actual photosynthetic efficiency of photosystem Ⅱ(Y(Ⅱ)).Besides,with continuous drought treatment,the chloroplast and mesophyll tissue were destroyed in P.lactiflora leaf,which resulted in stomatal closure,and thus inhibited the photosynthesis.(2)A total of 86,257 unigenes of high quality were obtained by transcriptome sequencing(RNA-seq)from P.lactiflora leaves and controls on the 21st day after drought stress treatment,and 39,309 unigenes were annotated into four databases.A total of 22,582 differentially expressed genes(DEGs)were identified.The expression patterns of 17 randomly selected DEGs were detected by real-time quantitative fluorescence PCR(qRT-PCR),which confirmed the accuracy and reliability of RNA-seq data.Pathway analysis of all DEGs showed that a total of 3,179 DEGs were annotated into 128 pathways,of which only 33 pathways met the p-value<0.05,and could be divided into five major metabolic categories:the reactive oxygen species(ROS)system,chlorophyll degradation and photosynthetic competency,secondary metabolism biosynthesis,fatty acid metabolism and sugar metabolism.Of these,the expression levels of DEGs associated with ROS metabolism,photosynthesis,carotenoid biosynthesis,fatty acid metabolism,and glycometabolism pathway were down-regulated,and the expression levels of DEGs associated with flavonoid biosynthesis and ABA synthesis and signal transduction pathways were up-regulated.(3)The RACE cloning technology was adopted to clone candidate genes,which were in response to drought and screened from P.lactiflora,and the full length of PIPM19L cDNA of AWPM-19 family plasma membrane protein gene was 910 bp,encoding 178 amino acids,having high homology with other plants PlPM19L sequence.The PlPM19L fusion Green fluorescent protein(GFP)expression vector was constructed and targeted on the cell membrane.The vector was transferred into tobacco by Agrobacterium-mediated leaf-plate method,and PlPM19L transgenic plants were obtained.The positive transgenic plants were detected by PCR and qRT-PCR.The wild-type and transgenic tobacco were subjected to natural drought stress.After 10 days of treatment,the leaves of wild-type tobacco showed serious wilting and drooping phenotype,while the transgenic tobacco kept normal growth state.Under drought stress,the accumulation of H2O2 in transgenic tobacco was lower than that in wild-type,but the relative water content in leaves,ABA content and the activities of SOD,POD,CAT and APX in transgenic tobacco were higher than those in wild-type.In addition,compared with wild-type tobacco,transgenic tobacco always maintained higher Pn,Gs,intercellular CO2 concentration(Ci),Tr,maximum fluorescence(Fm),Y(Ⅱ),the maximum light efficiency(Fv/Fm),Fv/Fo under drought stress.(4)The RACE cloning technology was adopted to clone candidate genes,and the MYB transcription factor family PlMYB108 gene sequence was obtained whose full-length sequence was 1314 bp,encoding 291 amino acids,having high homology with other plants MYB108 sequence.The PlMYB108 fusion GFP expression vector was constructed and infected with tobacco,which was mainly located on the nucleus.The vector was transferred into tobacco by Agrobacterium-mediated leaf-plate method,and the transgenic plants with PlMYB108 gene were obtained.The positive transgenic plants were detected by PCR and qRT-PCR.The wild-type and transgenic tobacco were subjected to natural drought stress.After 10 days of treatment,the transgenic tobacco grew normally,while the wild-type tobacco leaves wilted and drooped seriously.In addition,under drought stress,the accumulation of H2O2 in transgenic tobacco was relatively low,while the relative water content of leaves,flavonoid content,SOD,POD,CAT,APX,Pn,Gs,Ci,Tr,Fm,Y(Ⅱ),Fv/Fm and Fv/Fo in transgenic tobacco were higher than those in wild type tobacco. |
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