| Hypericum perforatum L.,also known as St.John’s wort and melaleuca,is not only a perennial herb of Hypericum and Hypericum,but also an important medical plant in China.St.John’s wort has antiviral,antioxidant,anti-inflammatory,antibacterial and other pharmacological effects,and its extracts are widely used in the treatment of depression,hepatitis a,hepatitis b and other diseases.At present,the molecular mechanism of drought response in st.John’s wort is seldom studied.Studies have reported that bZIP transcription factors are involved in gene regulation in plant growth and development,photopogenesis and other biological processes,and play an important role in abiotic and biogenic stress tolerance.In this study,systematic bioinformatics analysis of the bZIP gene family of hyperforin perforatum was carried out based on the whole genome data of hyperforin perforatum and the two key genes named HpbZIP14 and HpbZIP69 response to drought stress were preliminarily determined.By further studying the function of these two genes,the molecular mechanism of their response to drought and the influence on the growth and development of hyperforin perforatum were explored.The main research contents and results are shown as follows:1.A total of 79 HpbZIP genes were identified based on the whole genome database of hyperforin perforatum.Through phylogenetic and evolutionary analysis,the HpbZIP gene family was divided into 10 subfamilies.Bioinformatics software was used to carry out physiological and biochemical analysis,gene structure analysis,protein conserved motif analysis and GO annotation on all members of the family.Real-time fluorescence quantitative PCR was used to analyze the expression patterns of all genes in different tissues.Representative 1-2 genes were selected from different subfamilies,and the gene expression pattern of these genes under stress was analyzed.In order to further study the function of genes in the transformant bZIP family,the cis-acting elements in promoter of the gene family were statistically analyzed,and the result showed that there were a large number of genes in the transformant bZIP family with various biological and abiotic stress response elements,among which the number of genes with drought response elements was 64.2.With the reference of relevant research results on gene function of arabidopsis thaliana bZIP family and the drought resistance mechanism of AtbZIP36,HpbZIP14 and HpbZIP69 in the bZIP gene family of hyperforin perforatum are the most closely clustered with AtbZIP36 in arabidopsis thaliana through evolutionary analysis.HpbZIP14 and HpbZIP69 also have bZIP conservative domain,and the similarity with AtbZIP36 reaches 96%.It is speculated that the two genes HpbZIP14 and HpbZIP69 also have similar functions with AtbZIP36,and their corresponding functions are further studied.3.The gene expression of HpbZIP14 and HpbZIP69 was analyzed by real-time fluorescence quantitative qRT-PCR:the expression of HpbZIP14 and HpbZIP69 under drought,high salt and ABA treatment were detected.The results showed that the expressions quantity of HpbZIP 14 and HpbZIP69 were significantly up-regulated under the induction of ABA.And the two genes showed the stress response to drought and high salt.Especially during different time periods,HpbZIP14 and HpbZIP69 have a high expression level.We screened out HpbZIP14、HpbZIP69,speculating that they response to stress abduction and play an important role in the process of growth and development.4.The pEarleyGate103 subcellular localization vector of HpbZIP 14 and HpbZIP69 was constructed,and the localization vector was transferred into the onion epidermal cells by gene gun transfer method.It was found that HpbZIP14 and HpbZIP69 were located in the nucleus,which consistents with the general characteristics that transcriptional regulation of transcription factors in the nucleus.5.The Gateway technology was used to construct HpbZIP 14 and HpbZIP69 overexpression vectors,which were genetically transformed by agrobacterium-mediated inflorescence impregnation and transformation of arabidopsis thaliana,and 18 HpbZIP 14overexpressed plants and 22 HpbZIP69overexpressed plants were obtained,which were verified from DNA and RNA levels.6.Real-time quantitative PCR and semi-quantitative detection were conducted on transgenic seedlings cultured in soil for 20d.The results showed that the gene expression of transgenic arabidopsis thaliana plants increased significantly compared with wild type.Three highly expressed lines(HpbZIP14:oe-2,oe-5 and oe-9)were selected from the overexpressed plants of HpbZIP 14 and HpbZIP69(HpbZIP69:OE-1,OE-3 and OE-6)to conduct follow-up functional research.7.Tissue test-tube plantlet of Arabidopsis thaliana mutants bzip14/bzip69,WT and T3 transgenic arabidopsis thaliana strains were cultured in mannitol medium with concentrations of Ommol·1-1,100 mmol·1-1 and 250mmol·1-1 for 7 days.The results showed that the mutant,transgenic and wild-type seeds germinated in the medium without mannitol.In the mannitol medium with a concentration of 100 mmol·l-1,the root length of mutant,wild type and transgenic plants all decreased to different degrees,and the growth of the taproot of mutant and wild type plants decreased greatly.In the mannitol medium with a concentration of 250mmol·l-1,the growth of main roots of mutants,wild-type and transgenic plants was significantly inhibited,the leaves of the plants became smaller,the color became darker,and the leaf edges became yellow.However,the roots of transgenic plants grew up in the wild-type,the number of lateral roots was more than that of the wild-type,and the growth status was better than that of the wild-type.8.Phenotypic observation and related physiological indexes were performed on arabidopsis thaliana plants treated with drought for 15d.(1)The phenotypes of HpbZIP14/HpbZIP69 overexpressed lines and control lines(CK:wild type and mutant)under drought stress were observed.The results showed that the leaves of HpbZIP 14 and HpbZIP69 in overexpressed strains were in good condition,and most of the leaves were still green,but the leaves were yellow at the edges,and the plants showed a preliminary flowering trend.The leaves of the control lines were obviously wilted,the leaves were small,most of the leaves had lost water,yellowed,even shed,and the plants had developed to late flowering period.Three days after rehydration,the phenotype of the drought-treated overexpressed lines recovered,while that of the wild-type and mutant did not.Compared with the HpbZIP14 and HpbZIP69 transgenic soil plants under drought stress,the stem of the plants under drought stress was fine and soft,the growth and development was rapid,the leaves were thin and the edges’color were changed to yellow color.The stem of the plants without drought stress stood erect,the vegetative growth can sustain for a long time,the leaves were large and thick,and there was no obvious yellowing of the leaves.(2)The content of melatonin in CK strains under drought stress was detected by LC/MC.The results showed that the content of melatonin in HpbZIP14 and HpbZIP69 overexpressed strains was higher than that in the control strains under drought stress and normal culture condition.The content of melatonin in HpbZIP14 and HpbZIP69 overexpressed strains without drought stress was slightly higher than that of overexpressed strains under drought stress.(3)The physiological indicators of overexpressed and control strains were detected.The results showed that the activity of superoxide dismutase(SOD)in overexpressed lines was significantly higher than that in control lines(P<0.05).The MDA content was significantly lower than that of control strain(P<0.05)and the content of hydrogen peroxide(H202)and reactive oxygen(ROS)contents were lower than that of control strain.In summary,this study conducted a basic bioinformatics analysis on the bZIP gene family of hyperforin perforatum,which laid a foundation for further research on the function of the bZIP family of hyperforin perforatum.The functions of HpbZIP14 and HpbZIP69 in drought resistance and growth and development regulation were preliminarily explored,which laid a foundation for further research on the drought resistance of st.John’s wort and the molecular mechanism of its growth and development regulation. |
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