Cloning and functional analysis of E3 ubiquitin ligase gene CsRNF217 in Wuzi Ougan (Citrus suavissima)

Abstract: 【Objective】Wuzi Ougan (Citrus suavissima) is a seedless mutant spontaneously derived from the fertile Ougan mandarin (Citrus suavissima) and suffers from male sterility in previous reports.The multi-omics analyses of Wuzi Ougan and Ougan indicated that the phenylpropanoid biosynthesis was the most enriched pathway and the E3 ubiquitin ligase RNF217 (CsRNF217) gene up-regulated in Wuzi Ougan was focused on. E3 ubiquitin ligases are the most important components in the ubiquitin-proteasome system (UPS) involved in the selective degradation mechanism by recognizing target pro-teins and connecting ubiquitin known as a 76-residue protein to the substrate and play an essential role in pollen development of plants. E3 ligase families have a common feature in molecular architecture de-fined by the Really Interesting New Gene (RING) domain and are divided into four groups according to a signature consisting of 40-60 residues delineated as Cys and His that act as metal ligands binding two Zinc atoms. RING type E3 ligases are the most focused and have two major forms named as RING-HC and RING-H2 type. A widespread survey has identified the repertoire of RING finger proteins in Arabi-dopsis and progresses have been made in model crops such as rice and tomato. However, the function of E3 ligase remains unknown on male sterility in Citrus. In this study, E3 ligase RNF217 gene named  as CsRNF217 was focused on according to an integrative analysis of multi-omics sequencing. Sequence characteristics, spatial and temporal expression pattern, subcellular localization and gene function of Cs-RNF217 were analyzed to improve the knowledge of RING type E3 ligase on male sterility in Wuzi Ou-gan.【Methods】In this study, CsRNF217 was cloned from anthers of Wuzi Ougan using reverse tran-scription polymerase chain reaction (RT-PCR) with gene specific primers designed in Prime Premier 5.0 according to the hit E3 (RNF217) gene in Citrus clementina. The exact nucleotide sequence was ob-tained by Sanger sequencing, and the presumed amino acid sequence and conserved domains were ana-lyzed by NCBI Conserved Domains Search. The phylogenetic tree of multi-species based on the com-plete amino acid sequences derived from selected species was established with MEGA X software. To-tal RNAs were isolated from different organs of flower and floral buds at different stages of pollen de-velopment using MiniBEST Plant RNA Extraction Kit (TaKaRa, China) according to the manufacturer's protocol. First-strand cDNA was synthesized from total RNAs using EasyScript® One-Step gDNA Re-moval and cDNA Synthesis SuperMIX (Transgen Biotech, China). The spatial and temporal expression patterns of CsRNF217 in different organs of flower and at pollen development stages were analyzed with quantitative real- time polymerase chain reaction (qRT- PCR) in Wuzi Ougan and Ougan. The whole coding sequence of CsRNF217 was subcloned into pCAMBIA 2300s vector using restriction en-zyme of BamH Ⅰ and Sal Ⅰ (TaKaRa, China) and introduced into Nicotiana tabacum using the Agro-bacterium mediated leaf-disc method to explore the role of CsRNF217 in fertility based on phenotypes of positively transgenetic tobacco. Fusion expression vector CaMV35S::CsRNF217::GFP was trans-ferred into Agrobacterium tumefaciens strain GV3101 using freeze-thaw method and introduced into Ni-cotiana benthamiana using transient expression system to identify the subcellular localization of Cs-RNF217.【Results】A homologue of E3 ligase RNF217 (CsRNF217) was isolated from the anthers of Wuzi Ougan. The whole open reading frame (ORF) of CsRNF217 was 768 bp encoding for 255 amino acids. The putative CsRNF217 protein contained a typical RING domain and an IBR region that are popular in RING-HC E3 subfamily. The phylogenetic tree based on the complete ORFs showed that all members could be mainly divided into two divergent groups of RING-HC and RING-H2 subfamily. Cs- RNF217 protein was clustered into RING-HC group and shared high identity with that of Citrus clemen- tina (97.65%) and Citrus sinensis (97.56%). Transient expression in Nicotiana benthamiana showed the green fluorescent signal of CaMV35S::CsRNF217::GFP was found in the nucleus of epidermal cells in leaves. Analyses of qRT-PCR showed that CsRNF217 had the highest expression in stamens, followed by petals, suggesting that a potential function was involved in pollen development. CsRNF217 ex-pressed at four stages of pollen development and was up-regulated at mature pollen grain stage in Wuzi Ougan compared with Ougan, respectively. Ectopically overexpression of CsRNF217 in transgenetic lines of Nicotiana tabacum (line 6, 35 and 63) showed that spread of mature pollen was limited, and the pollen viability decreased significantly, followed by a reduction of seed yields.【Conclusion】In conclu- sion, a homolog of E3 ubiquitin ligase RNF217 denoted as CsRNF217 was cloned in Wuzi Ougan. Cs-RNF217 encoded a classic RING zinc finger protein and was located in nucleus. CsRNF217 was ex-pressed much higher in stamens than in the rest of floral organs. CsRNF217 was up-regulated signifi-cantly at mature pollen stage of pollen development in Wuzi Ougan. Overexpression of CsRNF217 in Nicotiana tabacum led to a significant reduction in pollen viability and less pollen spread and yield of seeds in transgenic plants. These results suggested that CsRNF217 could play an important role in pol-len development and promise a potentially negative regulation on fertility in Wuzi Ougan.