Expression Analysis And Functional Verification Of Auxin Response Factors (ARF) Gene Family In Citrus

Auxin response factor(ARF) plays key roles in auxin signal transduction pathway and participate in various physiological and biochemical processes. ARF proteins could specifically bind to auxin response elements(Aux REs) on promoters of auxin response genes, regulating many plant growth and development processes. In the current work, we conducted a systematic analysis of ARF gene family in citrus, and carried out functional analysis of some key genes. The main results are as follows:1. In this study, 19 non redundant ARF genes have been identified from citrus, named Ci ARF1-Ci ARF19 respectively. Most ARF proteins consist of three main domains with specific features, which are respectively N-terminal B3-type DNA binding domain(DBD), a variable middle region(MR), and C-terminal Aux/IAA domain(CTD). By implementing phylogenetic analysis, chromosome location, conserved motifs of proteins, and cis-elements in promoters of Ci ARFs, the characters of ARF gene family in citrus could be comprehensively understood.2. The expression data from q RT-PCR showed a high variability in transcript abundance of the Ci ARF genes in various tissues and organs, strongly indicating the diversified functions of the Ci ARF genes in citrus growth and development. Therefore, some Ci ARF genes demonstrated organ/tissue-specific expression patterns in sweet orange. In general, most of the Ci ARF genes exhibited low expression in reproductive organs, but a high expression level in vegetative organs except leaves in this study. By treating sweet orange callus under different concentrations of IAA and NPA, Ci ARF expression patterns were also investigated. The results indicated that eight Ci ARF genes(Ci ARF1/3/4/6/8/15/16/17) were suppressed at 6h under the IAA treatment with rising weakly at 12 h. 5 μmol/L and 10 μmol/L NPA suppressed the expression of Ci ARFs more strongerly than 1μmol/L. All these results showed that different Ci ARF genes might show different abilities to response to different concentrations of IAA and NPA treatment.3. CRISPR-Cas9 system was chosen to edit the Ci ARF8 gene. The CRISPR-Cas9 vecter was constructed and transformed into citrus callus. There were no differences between the sequence of Ci ARF8 in transformed and control materials, indicating that the CRISPR-Cas9 did not work in this study.4. The germination of overexpressed 35S::Ci ARF16 seeds was earlier than that of wild type. At the same time, the root length of transgenic seedlings was significant longer, and there were more lateral roots. All these results proved that Ci ARF16 can improve seed germination and the root growth.