Mining And Functional Validation Of Flowering-regulated WRKY Transcription Factors In The Iris Laevigata

Flowering regulation is one of the important directions in landscape plant breeding research.Regulating early or late flowering of landscape plants can extend the ornamental time of plants and improve the economic and ornamental value of landscape flowers.Nowadays,more and more studies have found that WRKY transcription factors in different tissue parts of plants can regulate growth and developmental processes,including flowering regulation,in the adaptation of plants to environmental changes.In this study,we firstly analyzed the WRKY transcription factor family in Iris laevigata perianths and identified two genes related to flowering regulation,Il WRKY11 and Il WRKY22,and performed gene cloning,bioinformatics analysis,and verification of flowering regulation by heterologous transformation in tobacco and arabidopsis.The results of this study laid the foundation for the analysis of the mechanism of flowering regulation in I.laevigata and the exploitation of WRKY transcription factors,and also provided new genetic resources for flowering regulation breeding practice.The research results are as follows.(1)By analyzing the transcriptome data of I.laevigata perianths,68 members of the Il WRKY transcription factor family were screened.Analysis of the conserved structural domains and zinc finger structures revealed that they were distributed in three major groups of the WRKY family,with 22 sequences in group I;35 sequences in group II,of which 5sequences belonged to group IIa,7 sequences belonged to group IIb,only 1 sequence in group IIc,13 5 sequences belong to group IIa,7 sequences belong to group IIb,only 1 sequence in group IIc,13 sequences belong to group IId and 9 sequences belong to group IIe;there are 11 sequences in group III.They may be involved in the response to biotic and abiotic stresses and regulation of growth and development and other functional regulation.(2)Those involved in flowering regulation were mainly distributed in groups IIc,IId and IIe.Through the functional analysis of homologous WRKY proteins with high similarity,isoform 287969 and isoform 51755,named Il WRKY11 and Il WRKY22,which might be screened for possible involvement in flowering regulation,were selected as the study objects.(3)The c DNA obtained from the reverse transcription of total RNA of I.laevigata was used as the template to clone the gene sequences of Il WRKY11 and Il WRKY22,which were888 bp and 840 bp in length,encoding 295 and 279 amino acids,respectively.Bioinformatics analysis showed that the molecular weight of Il WRKY11 protein was 32.291 k Da,the theoretical isoelectric point was 9.76,the molecular formula was C1398H2237N419O421S20;the molecular weight of Il WRKY22 protein was 30.604 k Da,the theoretical isoelectric point was5.02,the molecular formula was C1314H2065N383O435S13。 Both are unstable hydrophilic proteins with no transmembrane structure and signal peptide,and both have two glycosylation sites and are mainly phosphorylated by serine.It is predicted that the Il WRKY11 protein may play a role in biotic stress,abiotic stress,growth and development and flowering regulation in plants;the Il WRKY22 protein may be involved in the regulation of biotic stress,leaf senescence and flowering time.(4)The p CAMBIA1300-Il WRKY11-GFP and p CAMBIA1300-Il WRKY22-GFP plant overexpression vectors were constructed and transferred into Agrobacterium tumefaciens GV3101 by freeze-transfer method,and the localization of Il WRKY11 and Il WRKY22 proteins in the nucleus was confirmed by injection into native tobacco leaves,which was consistent with the predicted results The target genes were successfully inserted into the genomes of arabidopsis and tobacco by PCR,and the highly expressed lines of Arabidopsis T3 and tobacco T1 generations were obtained by q RT-PCR screening.(5)Identification of Il WRKY11 and Il WRKY22 genes involved in flowering regulation:Il WRKY11 gene was able to positively regulate flowering in Arabidopsis,and the time to shoot and flowering were 2 to 3 days earlier than the control;Il WRKY22 gene delayed flowering in Arabidopsis,and the time to shoot and flowering were 3.5 days later than the control;control and trans-Il WRKY11 and Il WRKY22 gene also delayed flowering in tobacco.The two genes play opposite roles in the regulation of flower formation.(6)The q RT-PCR assay of the expression of genes related to flowering stage in transgenic Arabidopsis and trans nulled control revealed that Il WRKY11 and Il WRKY22 genes are both involved in flowering stage regulation through the combination of high expression of GA20 OX gene and signals from other pathways,which may affect the expression of age pathway SPL3 to determine the length of juvenile period in Arabidopsis,or the vernalization pathway which is influenced by the differential expression of VRN1,leading to different action results.