| In order to study the effects of drought stress on the growth,physiological characteristics and content of medicinal components of Rehmannia glutinosa,drought germination experiments were carried out on the seeds of different varieties of R.glutinosa.According to the germination rate,drought resistance index and other indicators to select relatively drought-tolerant varieties,and the seedlings were further treated with drought,analyzed the physiological effects of R.glutinosa after drought at seedling stage.Meanwhile,the field pot experiment was designed to carry out drought stress on R.glutinosa by using water control method,determined the change of chlorophyll content,antioxidant enzyme activity and active ingredient content.Transcriptome sequencing was used to analyze the transcriptional characteristics of R.glutinosa related genes in response to drought stress,the selected drought related genes were cloned and analyzed for sequence characteristics and expression patterns.Constructed the overexpression vector to transform tobacco,and preliminarily analyzed the function of candidate genes in response to drought stress.The main research contents and results are as follows:1.Using PEG6000 simulate drought to treat the seeds of 9 R.glutinosa varieties,it was found that the germination rate of seeds decreased gradually with the increase of PEG6000 concentration after drought treatment.R.glutinosa varieties 1710,BJ1,QH-1 and 8N did not germinate at the concentration of 20% PEG6000,the germination rates of 301,HF and W4 treated with 5% and 10% PEG6000 were higher than those of CK.The germination index of HF,301 and 8N was higher than that of the control under mild drought stress.The drought resistance index of W4,888,8N,301 and 1710 increased first and then decreased with the increase of PEG6000 concentration,the hypocotyl length of W4 was increased in 5% PEG6000 compared with the control,and the radicles of 888,HF,1701,301 were longer than the control under 5% and 10%PEG6000 stress.Combining various indicators,it is concluded that 301 and HF are relatively drought-tolerant varieties,W4 and QH-1 are drought-sensitive varieties.The seedlings of relatively drought-tolerant R.glutinosa varieties HF,301 and droughtsensitive varieties W4,QH-1 were subjected to drought treatment,measured the activity of antioxidant enzymes.The results showed that the SOD and POD activities of the four cultivars were increased after drought treatment,indicating that R.glutinosa seedlings can resist drought by increasing their own antioxidant enzyme activities.2.The drought treatment of R.glutinosa varieties 85-5 and J9 at the adult stage was carried out by pot water control method,with the extension of treatment times,the soil water content decreased gradually,the drying rate of leaves and roots of the two varieties increased gradually.The contents of chlorophyll a,chlorophyll b,carotenoids and total chlorophyll in 85-5 leaves increased first,then decreased and finally increased again during drought treatment,the contents of chlorophyll a,chlorophyll b,carotenoids and chlorophyll in J9 leaves increased first and then decreased.The SOD activity of 85-5 and the SOD and POD activities of J9 increased first and then decreased,while the 85-5 POD activity increased first,then decreased,then increased and then decreased.The changes in the content of active components of R.glutinosa under drought stress were measured,the results showed that the content of catalpol in the roots of R.glutinosa increased after drought stress,moderate drought was beneficial to the increase of the content of total phenylethanoid glycosides and acteoside.3.Transcriptome sequencing of roots and leaves of drought-treated R.glutinosa cultivar 85-5,the results showed that the number of differentially expressed genes(DEGs)in leaves was more than that in roots after drought treatment,in droughtstressed leaves,the differentially expressed genes were mainly enriched in the photosynthetic-antenna protein,galactose metabolism and hormone signaling pathways,and the expression levels of many genes involved in the photosynthetic-antenna protein pathway were significantly decreased.The differentially expressed genes in the roots of R.glutinosa after drought stress were mainly enriched in the metabolic pathways of carotenoid synthesis,phenylpropane biosynthesis,sucrose and starch.Moreover,drought stress induced the up-regulated expression of transcription factor genes such as AP2 EREBP,b HLH,b ZIP,MADS and WRKY,which positively regulated the response of R.glutinosa to drought stress.The expression characteristics of the catalytic enzyme genes in the synthetic pathways of R.glutinosa acteoside and catalpol were analyzed,and candidate genes that may be involved in the synthesis of acteoside and catalpol in response to drought were screened out.It provides a reference for further research on the molecular mechanism of R.glutinosa responding to drought stress,and lays a foundation for the research on the molecular mechanism of drought-regulated quality formation of R.glutinosa.4.The Rg NCED and Rg CYP707A3 genes involved in the synthesis and decomposition of ABA in R.glutinosa were cloned by PCR amplification technology,their sequence lengths were 1 803 and 1 392 bp,respectively.The Rg NCED protein belongs to the RPE65 superfamily and has the conserved domain sequences of MIAHPKLDP and HDFAITE unique to the NCED family;The Rg CYP707A3 protein belongs to the P450 superfamily and has a relatively conserved heme iron ligand cysteine residue(PFGNGTHSCPG)structure of the CYP707 A family.QRT-PCR analysis showed that the expression of Rg NCED in the root,stem,leaf and flower of R.glutinosa was from high to low,followed by leaf,flower,stem and root.After drought treatment,the expression of Rg NCED in leaves first increased and then decreased,and gradually increased in root;The expression level of Rg CYP707A3 gene in the four parts of R.glutinosa roots,stems,leaves and flowers is from high to low,followed by leaves,roots,stems and flowers.After drought treatment,its expression levels in leaves and tuberous roots first decreased and then increased.The overexpression vectors of Rg NCED and Rg CYP707A3 were constructed and transferred into tobacco genome by Agrobacterium-mediated method.PEG6000 solution simulated drought treatment found that the activities of SOD and POD in transgenic tobacco leaves overexpressing Rg NCED were higher than those in wild-type tobacco,while the activity of SOD in transgenic tobacco leaves overexpressing Rg CYP707A3 was higher than that in wildtype tobacco,the activity of POD was lower than that in wild-type tobacco,it is speculated that the molecular regulatory mechanisms of Rg NCED and Rg CYP7073 in response to drought stress may be different. |
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