Identification Of Hypericum Perforatum WRKY Gene Family And Functional Study Of HpWRKY85 Gene

Hypericum perforatum L.is a perennial herb of the family Hypericaceae,and its dry aerial part is used as medicine.With the in-depth study of pharmacological effects,its commercial value is increasingly prominent,and the market demand has continued to increase.The collection of wild resources and the existing artificial planting scale are difficult to meet the market demand,so it is urgent to expand the planting area of Hypericum perforatum.The adaptability of Hypericum perforatum to the environment is strong,and it can be popularized in most parts of the country.However,the humid and semi humid areas are the main production areas of grain crops and economic crops in China,the land area available for planting medicinal plants such as Hypericum perforatum is limited.Therefore,promoting the cultivation of Hypericum perforatum in arid and semi-arid areas has become one of the important means to alleviate its shortage of resources.To understand the molecular mechanism of drought resistance of Hypericum perforatum is the premise and basis for its large-scale planting in arid and semi-arid areas.As an important class of transcription factors,WRKY gene plays an important role in regulating plants against stress.Therefore,based on the whole genome database of Hypericum perforatum,this study carried out a comprehensive bioinformatics analysis and expression analysis of WRKY family genes,and screened out a gene HpWRKY85 that responded to drought stress.Further exploring the phenotypes,physiological characteristics and molecular mechanisms of over-expressing HpWRKY85 transgenic Arabidopsis thaliana plants in response to drought treatment,in order to provide information for understanding the role of HpWRKY85 gene in the molecular mechanism of drought resistance of Hypericum perforatum.The main research contents and results are as follows:1.By using the genomic database of Hypericum perforatum sequenced and completed in our laboratory,86 members of the WRKY gene family of Hypericum perforatum were screened through comparison,named HpWRKY1～HpWRKY86,and a series of bioinformatics analysis including physicochemical properties,phylogeny and domain analysis,gene structure and conserved motif analysis,promoter cis-acting element analysis and gene ontology annotation were carried out,thereby classifying 86 HpWRKY proteins into three major groups of Group 1 to 3,and the second group can be further divided into five subgroups of Group 2a to 2e;The distribution and characteristics of WRKY domain,zinc finger structure,gene structure and conserved motif in the same group of HpWRKYs have a very high similarity.Many cis-acting elements related to hormone,biotic and abiotic stress responses are found in the promoter region of the HpWRKY genes;The functional classification of proteins shows that HpWRKYs are widely involved in the biological processes in plants.Secondly,the gene expression patterns of different tissues and different stress treatments were analyzed.The results show that HpWRKY gene expression is specific and diverse,and the gene Hp WRKY85 is screened out.The expression levels of HpWRKY85 are low in tissues but increase to 85 times,58 times and 188 times of the control under drought,SA and MeJA stress,respectively.It is suggested that the gene may be the key gene in response to SA,MeJA and drought stress in Hypericum perforatum.2.The cloned primers were designed to amplify the gene HpWRKY85 cDNA sequence,which is 567 bp in length and encodes 188 amino acids.At the same time,the signal peptide,transmembrane domain,hydrophilicity/hydrophobicity,secondary and tertiary structure and characteristics of HpWRKY85 protein were analyzed by software.As a typical zinc finger protein,HpWRKY85 protein has strong hydrophilicity,but does not have signal peptide and transmembrane domain.Furthermore,the Gateway technology was used to construct the HpWRKY85-pEarleyGate103 subcellular localization vector.The transient expression results of onion epidermal cells show that the HpWRKY85 protein is located in the nucleus,which is in line with its characteristics of nuclear transcription regulation.3.The Gateway technology was used to construct the HpWRKY85-pEarleyGate202 overexpression vector,which was further transformed into Arabidopsis thaliana to obtain transgenic lines overexpressing HpWRKY85.It is observed that the primary root length of the HpWRKY85 over-expressed lines is 1.45 times as long as that of the wild type on average under the mannitol simulated drought condition.Drought treatment of Arabidopsis thaliana plants in the soil,it is detected that the MDA content in the wild-type Arabidopsis thaliana treated group is increased by 105.31%compared with the control group,while the over-expressed lines are only increased by 28.96%on average;In addition,the content of ROS in wild-type Arabidopsis thaliana leaves is higher than that in over-expressed lines.These results show that Arabidopsis thaliana over-expressing HpWRKY85 show better drought resistance compared with the wild type.Further through qRT-PCR technology,it is found that the transcriptional abundance of stress-related genes AtDREB2A,AtNCED3,AtRD29A,and AtRD29B in Hp WRKY85 over-expressed lines is significantly higher than that of wild type under drought condition.It is speculated that the transcription factor HpWRKY85 enhances drought resistance by up-regulating the expression of these genes.In conclusion,this study lays the foundation for the study of WRKY gene function in Hypericum perforatum through the systematic analysis of WRKY family members.It is clear that the overexpression of the gene HpWRKY85 can reduce the accumulation of reactive oxygen species and malondialdehyde in the plant under drought stress,so as to reduce the degree of damage to the plant caused by adversity,and play a positive role in the process of plant drought resistance.The above research provides a scientific basis for further study on the drought resistance mechanism of Hypericum perforatum.