The Regulatory Mechanism Of ChIFN-α Gene For Adventitious Root Formation In Lotus Japonicus

The formation and development of roots is important for forage yield,which is regulated by several genes.ChIFN-ɑ,a regulator from animal,promotes plant root formation and development,but its mechanism of regulation in plant is unclear.Based on the electron microscopic observation,transcriptome sequencing and real-time PCR,based on the relationship between ChIFN-ɑand root development,the molecular regulation mechanism of the formation and development of adventitious roots in the Lotus japonicus was further explored.Analyze the action pathway of animal cytokines in plant,and provide a new choice for plant growth and development regulation.The main research contents and results are as follows:1.Identification and analysis of transgenic ChIFN-ɑgene Lotus japonicus.The target gene was detected by GUS histochemical activity assay and PCR amplification,and it was found that the exogenous ChIFN-ɑgene was stably present in the transgenic Lotus japonicus（TP）receptor plants.2.The effect of ChIFN-ɑon the development of adventitious roots of Lotus japonicus.The wild type（WT）plant was used as a control to observe the adventitious root development of the transgenic ChIFN-ɑgene.It was found that in the 12d observation period of adventitious root formation and its initial development,the adventitious roots of TP Lotus japonicus appeared 3 days earlier than WT.The adventitious roots of TP Lotus japonicus developed rapidly,and the number of root hairs was more than WT.ChIFN-ɑgene expression promotes the formation and development of adventitious roots in Lotus japonicus.3.ChIFN-ɑmediated molecular network of adventitious root development in Lotus japonicus.The RNA-seqanalysis of adventitious root development at 0d,3d,6d,9d and 12d showed that 16380 differentially expressed genes in the molecular regulation of root development of TP and WT.Biological processes such as transduction,DNA replication,and transcriptional translation are closely related.The gene expression patterns of 23 types of WT that are not significantly enriched are significantly enriched in TP.The most differentially expressed transcription factors are ERF,bHLH,MYB,WRKY,etc.Transcriptome data analysis showed that the developmental regulatory genes of TP Lotus japonicus were more abundant and active.ChIFN-ɑmediated adventitious root development constitutes a diverse molecular regulatory network through abundant regulatory pathways.4.ChIFN-ɑmediated root development regulation pathway.Transcriptome analysis indicated that ChIFN-ɑmediated adventitious root development is associated with differential expression of membrane protein genes.After treatment with exogenous CaCl₂ and LaCl₃,combined with transcriptome and qRT-PCR analysis,ChIFN-ɑwas involved in root development regulation through Ca²⁺responsive C2 domain protein kinase（Lj1g3v0026680）,which may also activate MYB,TALE,Dof Transcription factor,by Ca²⁺.This transcription factor turn promote the regulation of root formation and developmental gene transcription.5.Establish a two-hybrid system of Lotus japonicus.The cDNA library of adventitious root development was constructed by SMART technology.The library titer was 9.175×10⁸,and the amplification rate was 100%.A pGBKT7-ChIFN-ɑdecoy vector plasmid without self-activation and non-toxicity to yeast cell growth was constructed,and partial ChIFN-ɑinteraction candidate positive clones were obtained by Mating double-hybridization and defective medium screening.A yeast two-hybrid system was initially established and can be used to screen for ChIFN-ɑplant recognition/interacting proteins.