Cloning And Function Studies Of IbWRKY44 And IbWRKY75 Genes In Ipomoea Batatas

WRKY transcription factor plays an important role in plant growth and development,response to stress,and secondary metabolism.Analyzing the functions of Ib WRKYs in Ipomoea batatas by means of bioinformatics and molecular biological,which is of great significance to cultivate high-quality Ipomoea batatas with good stress resistance.In our study,the genes Ib WRKY44 and Ib WRKY75 were cloned from Ipomoea batatas cv.‘XZS-3’ and ‘XS-18’,respectively,and their biological functions were preliminarily investigated.Firstly,the amino acid sequences of Ib WRKY44 and Ib WRKY75 were analyzed for bioinformatics and phylogenetic analysis.And the promoter sequences of Ib WRKY44 and Ib WRKY75 genes were analyzed for cis-acting elements.Secondly,the expression patterns of Ib WRKY44 in root tubers of purple and non-purple Ipomoea batatas varieties and the expression characteristics of Ib WRKY75 in different tissues,leaves at different development stages,and tissues treated with PEG and Na Cl were analyzed by q RTPCR.Then,the expression vectors of 35S::Ib WRKY44::GFP and 35S::Ib WRKY75::GFP were constructed,and they were instantaneously expressed in tobacco leaves for gene subcellular localization.At the same time,35S::Ib WRKY44::GFP was respectively transformed into Arabidopsis thaliana and the embryogenic suspension cells of ‘XS-18’.Furthermore,the CRISPR/Cas9 knockout expression vector of Ib WRKY44 was constructed and transformed into the embryogenic suspension cells of ‘XZS-3’ The main results were listed as follows:1、Related results of Ib WRKY44 geneThe Ib WRKY44 gene was successfully cloned from Ipomoea batatas cv.‘XZS-3’.The ORF of Ib WRKY44 gene was 1401 bp and encoded 446 amino acids.The Ib WRKY44 protein,belonged to the class Ib WRKY transcription factor family,were alkaline unstable hydrophobic protein,without signal peptide and transmembrane domain,and had the typical 4 β-pleated sheet structure.Phylogenetic analysis showed that Ib WRKY44 had the highest similarity with At WRKY44 in Arabidopsis WRKY family,were the closest relatives with WRKY44 s in Ipomoea nil and Ipomoea trifida,which belonged to Convolvulaceae,and was closely related to WRKY44 in Solanaceae potato and tomato.In the promoter of Ib WRKY44,there were various specific cis-acting elements such as plant hormone response elements,photoinduction elements and anaerobic induction elements,as well as binding sites of transcription factors such as AP1,MYB and MYC.It was speculated that the transcription of Ib WRKY44 can be activated or inhibited by these factors,thus extensively regulating the growth and development of plants.Subcellular localization results suggested that the protein located in the nucleus.The q RT-PCR analysis showed that the expression level of Ib WRKY44 gene in root tubers of purple Ipomoea batatas was significantly higher than the non-purple Ipomoea batatas,suggesting that it might be involved in the synthesis of anthocyanin in Ipomoea batatas.About the part of genetic transformation,a total of 11 overexpressed transgenic Arabidopsis thaliana strains of Ib WRKY44 were obtained,and the seed coat of its T0 generation seed became lighter and the seed size increased,suggesting that Ib WRKY44 might be involved in the development of Arabidopsis thaliana seeds and the synthesis of proanthocyanins in their seed coat.2、Related results of Ib WRKY75 geneThe Ib WRKY75 gene was successfully cloned from Ipomoea batatas cv.‘XS-18’.The ORF of Ib WRKY75 was 597 bp and encoded for 198 amino acids.The Ib WRKY75 was an alkaline unstable hydrophobic protein,without signal peptide and transmembrane domain.Phylogenetic analysis showed that Ib WRKY75 belonged to the class IIc protein of the WRKY transcription factor family and had high homology with In WRKY75 in Ipomoea nil.Subcellular localization results suggested that the protein located in the nucleus.In the promoter of Ib WRKY75,there were a variety of specific cis element,such as plant hormone response element and photoinduced components,as well as MYC and MYB transcription factor binding sites.The plant hormone response element included ABRE,CGTCA-motif and the photoinduced components included AE-box and box-4.It was speculated that the transcription of Ib WRKY75 could be activated or inhibited by these factors and play a wide role in the biological regulatory network mediated by these factors.The q RT-PCR results showed that Ib WRKY75 had highest expression in stem,followed by leaf,root and petiole.The expression of Ib WRKY75 was significantly increased after induction by PEG and Na Cl treatments,the most significant up-regulation was in roots,indicating that Ib WRKY75 might mediate drought resistance and salt tolerance pathways in sweet potato.In addition,the transcriptional level of Ib WRKY75 was significantly up-regulated with the natural aging of leaves,indicating that Ib WRKY75 might be positively regulating the aging of leaves in sweet potato.In conclusion,Ib WRKY44 and Ib WRKY75 played regulatory roles in anthocyanin synthesis,growth and development,morphogenesis,and stress response in sweet potato,respectively.The results will provide theoretical basis for parsing anthocyanin synthesis mechanism in the underground of plants and provide molecular targets for the art quality of sweet potato genetic improvement.And which has important theoretical and practical significance to the development of sweet potato industry.