| Pueraria montana var.thomsonii(Bentham)M.R.Almeida is a perennial vine of the genus Puerariae in the family Leguminosae,which has drought and cold tolerance characteristics,but there are few studies on drought resistance in Pueraria montana var.thomsonii,and the mechanism of drought resistance is not clear.Therefore,in this experiment,1-year-old Pueraria montana var.thomsonii seedlings were used as experimental materials to observe and determine the changes in leaf morphological structure(leaf stomata,anatomical structure),physiological and biochemical parameters using pot planting to simulate drought stress and rewatering experiments,and transcriptome sequencing analysis of Pueraria montana var.thomsonii during drought stress and rewatering.Main study results are as follows:(1)With the aggravation of drought stress,the relative water content of Pueraria montana var.thomsonii leaves gradually decreased,the biomass increased in the early stage of drought,and then gradually decreased;the leaves appeared curling,dry,and even shedding,and the stem and vine withered.After rehydration,the leaves turned green and grew vigorously,and the plant morphology was not significantly different from that of the control group.(2)Under normal environment,most of the stomata of Pueraria montana var.thomsonii leaves are in an open state.With the aggravation of drought stress,the stomatal density and opening of epidermis in the leaves of Pueraria montana var.thomsonii increased firstly and then decreased,and the stomatal length and width gradually decreased.After restoration of watering,the stomatal density,opening rate,length and width gradually returned to the normal state,indicating that Pueraria montana var.thomsonii can resist the water shortage environment caused by drought stress by regulating the number,size and opening and closing degree of stomata during drought stress,and can react rapidly after rehydration and return to normal.(3)Leaf anatomy is important for plants to respond to environmental changes.With the aggravation of drought stress,the mesophyll cells of Pueraria montana var.thomsonii leaves gradually became smaller and tightly arranged,and the palisade tissues were disorganized and loosely arranged;the upper and lower epidermal thickness,palisade tissue thickness,and sponge tissue thickness gradually decreased with the aggravation of stress,decreased to the lowest at 18 days of drought,and rapidly increased after rewatering,returning to normal;the palisade tissue and sponge tissue thickness ratio did not change significantly compared with the control group under drought stress and rewatering conditions.(4)During drought stress and rehydration,the antioxidant enzyme activities of the leaves and roots of Pueraria montana var.thomsonii changed significantly,and the MDA content changed less.The POD activity,SOD activity,and CAT activity in roots were higher than those in leaves,indicating that powdered Pueraria montana var.thomsonii root was more sensitive to water deficit.With the aggravation of drought stress,POD activity and SOD activity in roots continued to increase,CAT activity gradually decreased,and MDA content showed a trend of first increase and then decrease;POD activity in leaves gradually increased,SOD and CAT activities gradually decreased,and MDA content showed a trend of first decrease and then increase and then decrease.After rewatering,the activities of POD and SOD in roots continued to increase,the activity of CAT first decreased and then increased,but did not return to the level of the control group,the content of MDA first increased and then decreased,and gradually returned to the normal state;the activity of POD in leaves continued to increase,and the activity of SOD,the activity of CAT and the content of MDA gradually returned to the level of the control group.(5)For transcriptome sequencing analysis of powdered Pueraria montana var.thomsonii root,a total of 195.79 Gb clean data were obtained,and the clean data of each sample reached more than 6.01 Gb;after quality control processing of the original data,a total of 1310143,772 raw reads were obtained,with a sequencing error rate below 0.0271%.A total of 62,981 unigene with an average length of 1246.25 bp and 122,718 transcripts with an average length of 1478.06 bp were obtained after assembly optimization of the resulting raw reads.The obtained unigene was compared with six major databases(NR,Swiss-prot,pfam,COG,GO,and KEGG databases),and a total of 36,255 unigene were annotated,accounting for 57.56% of the total unigene number,which were mainly annotated to pathways such as binding,catalytic activity,and cellular processes in the GO database and to pathways such as carbohydrate metabolism,translation,fold classification,and degradation in the KEGG database.(6)Through the screening and analysis of differentially expressed genes(DEGs),to explore the metabolic pathways and genes associated with drought stress.Differentially expressed gene analysis between the normally developed control group and the drought stress treatment group indicated that drought stress had a greater effect on plant growth and development,and the plants were able to respond rapidly after rewatering and returned to the normal control.The number of differential genes between the control group and continuous drought for 12 days,18 days,and 6days and 12 days after rewatering was 3363(1090 up-regulated and 2273 down-regulated),5986(2120 up-regulated and 3866 down-regulated),4690(2486 up-regulated and 2204 downregulated),and 5281(1925 up-regulated and 3356 down-regulated)DEGs,respectively.The number of differentially expressed genes was the highest at 18 days of drought,indicating that stress had the most significant effect on plants at this time,and nine KEGG metabolic pathways were significantly enriched,mainly including phenylpropanoid biosynthesis,starch and sucrose metabolism,flavonoid biosynthesis,phytohormone signal transduction,MAPK signaling pathway-plant,α-linolenic acid metabolism,cyanoamino acid metabolism and linoleic acid metabolism pathways,and DEGs genes were up-regulated.After 12 days of rehydration,a total of 19 KEGG pathway metabolic pathways were significantly enriched,mainly including starch and sucrose metabolism,phytohormone signal transduction,and phenylpropanoid biosynthesis.These results indicate that different response mechanisms are adopted to cope with stress in Pueraria montana var.thomsonii after drought stress and rewatering,forming a complex network regulation,which can be rapidly recovered after rewatering.Two CAT related genes,two SOD genes,two GPX genes,and six POD genes were found to be significantly differentially expressed during drought and rewatering.A total of 426 transcription factors were identified to be associated with drought stress and belong to 28 transcription families,mainly associated with transcription families such as AP2/ERF,MYB,and b HLH.To sum up,the dynamic changes of pueraria lobata in the process of drought stress and rehydration were explained by the changes of leaf morphology,physiological indexes and the results of transcriptome sequencing,the results showed that pueraria lobata responded to drought stress through complex regulation network,which laid a foundation for better understanding of the molecular mechanism of drought resistance and provided a theoretical basis for breeding good drought-resistant varieties. |
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