Analysis Of Erianthus Fulvus WRKY Gene Family And Gene Overexpression Vector Construction

The exploitation of sugarcane germplasm resources is the key to achieve breeding,among which wild germplasm is the main source of sugarcane resistance lineage.E.fulvus is an important and closely related wild species of sugarcane with strong drought and cold tolerance,while WRKY transcription factors have important roles in response to abiotic and biotic stresses.To address the key problems of shortage of sugarcane breeding resistance lineage donor resources,unclear genetic background,and lack of precise identification techniques,and to investigate the functions of WRKY transcription factors in drought in sucrose,this study conducted a genome-wide identification of the WRKY gene family in sucrose based on published genomic data of sucrose using bioinformatics;Analyze the physicochemical properties,protein secondary structures,evolutionary relationships,conserved domains,gene structures,cis acting elements,and GO annotations among members of the WRKY family of E.fulvus;The results of the analysis and the evolutionary tree constructed were used to screen out genes that might be relevant to drought resistance in sugarcane,to construct preliminary overexpression vectors for the candidate genes,and to transform the constructed overexpression vectors into rice,with the following main results:(1)Identification and genetic analysis of WRKY gene family members in the genome of E.fulvus Using the Hidden Markov Model of WRKY,89 WRKY gene family members were identified in the genome of E.fulvus and named in order from Ef WRKY01 to Ef WRKY89.Chromosomal localization results showed that the 89 E.fulvus WRKY genes were unevenly distributed on 10 chromosomes,with the largest number of WRKY genes distributed on Chr4,reaching 20,and the lowest distribution being Chr5,6,and 9,all of which were 5;The number of protein amino acids corresponding to the members of the E.fulvus WRKY gene family ranges from 213(Ef WRKY13)to 2738(Ef WRKY85);The distribution analysis of conserved motifs found that motif 1 motifs are highly conserved,except for Ef WRKY51,Ef WRKY87,and Ef WRKY89,which are not present in these three genes,all other 86 Ef WRKYs are present;gene structure analysis revealed that Ef WRKY12,Ef WRKY14,Ef WRKY33 and Ef WRKY54 had no introns,while all other Ef WRKYs family members had introns,ranging from 1-9 in number,and some Ef WRKY gene family members do not contain an untranslated region(UTR)at the 5? end and 3? end;(2)GO enrichment analysis of potential target genes of E.fulvus WRKY gene family members Based on Uniprot’s Swiss-Prot file,and using TBtools software to compare protein sequence files corresponding to 89 E.fulvus WRKY gene family members,GO enrichment analysis of potential target genes was performed.GO functional enrichment revealed that these genes are mainly involved in sequence-specific DNA binding functions and transcriptional regulation in cellular processes,thus influence processes such as regulation of biosynthesis and gene expression;they are also mainly focused on biological processes such as bioregulation,cellular processes and metabolic processes;catalytic activity,binding functions in molecular functions;(3)Overexpression vector construction and genetic transformation research To better investigate the role of E.fulvus WRKY genes in the drought tolerance process in plants,five drought tolerance candidate genes were screened using the results of the analysis and the constructed evolutionary tree,and p BWA(V)HS-Ef WRKY46,p BWA(V)HS-Ef WRKY55,p BWA(V)HS-Ef WRKY60,p BWA(V)HS-Ef WRKY61 and p BWA(V)HS-Ef WRKY69 overexpression vectors,and the recombinant plasmids of these five candidate genes were cultured by co-culture with rice(Zhonghua11)seed-induced healing tissues until seedlings were established.Positive tests on rice seedlings revealed that the transformation rate of p BWA(V)HS-Ef WRKY46,p BWA(V)HS-Ef WRKY55,p BWA(V)HS-Ef WRKY60,and p BWA(V)HS-Ef WRKY69 was 100%,while the transformation rate of p BWA(V)HS-Ef WRKY61 was 91%,thus indicating that the candidate genes screened from E.fulvus were successfully transformed into rice.In this study,we used bioinformatics to perform a series of analyses on the WRKY gene family in the E.fulvus genome,including member identification and physicochemical properties,to understand the information on the origin evolution or kinship of the WRKY gene family in E.fulvus,and to explore the functions of WRKY gene family members in E.fulvus;on this basis,five drought-related candidate genes were screened,and overexpression vectors were constructed and successfully transferred into E.fulvus.and constructed overexpression vectors,which were successfully transferred into rice(Zhonghua 11).This has laid the foundation for the exploitation of drought-resistant germplasm in E.fulvus,and also provided a scientific basis for the precise identification of E.fulvus and the utilization of sugarcane breeding.