Identification of ERF gene family of Taraxacum kok-saghyz Rodin and functional analysis of highly expressed genes in latex

【Objective】 This study aimed to analyze the effects of ERF transcription factor on natural rubber yield of Taraxacum kok-saghyz Rodin， elucidate the function of genes with specific expression in latex， so as to provide references for analyzing the regulation mechanism of TkERFs genes with specific high expression in rubber biosynthesis. 【Method】 Bioinformatics methods were used to identify the members of the ERF gene family in Taraxacum kok-saghyz Rodin. Transcriptome sequencing data was analyzed to identify two TkERFs genes with specific high expression in latex tissues （TkERF1 and TkERF2）. These two genes were cloned by PCR， and their conserved domains， subcellular localization， and transcriptional self-activation were analyzed. The CRISPR/Cas9 system was employed to knock out latex-specific TkERFs genes， the rubber contents in mutants were measured， and a comparative analysis was performed between the rubber content in wild types. 【Result】 This study identified the 146 members of the ERF gene family in the genome of Taraxacum kok-saghyz Rodin. Based on the results of tissue-specific characteristics， two latex-specific genes， TkERF1 and TkERF2 were identified； both genes had only one exon， and the lengths of their coding sequences （CDS） were 723 and 726 bp， encoding 241 and 252 amino acid residues， respectively. TkERF1 and TkERF2 proteins shared a low protein sequence identity of 8.63%； each possessed an AP2 domain； they belonged to the ERF subfamily， with their subcellular location in the nucleus. Transcriptional self-activation assays demonstrated that TkERF1 and TkERF2 possessed transcriptional self-activation activity， indicating they activated or inhibited the expression of downstream target genes by binding to their promoters of target genes. Tkerf1 and Tkerf2 single knockout mutants and Tkerf1/2 double knockout mutants were generated using the CRISPR/Cas9 technology. Compared with wild type， Tkerf1， Tkerf2 and Tkerf1/2 mutants exhibited extremely significant decreases in rubber content by 34.6%， 22.6%， and 31.3% （P<0.0001 or P<0.001）. 【Conclusion】 TkERF1 and TkERF2 genes with latex-specific high expression are the key nodes in the regulation network of rubber synthesis， regulating the process of natural rubber biosynthesis directly or indirectly， but this regulatory mechanisms require further investigation.