Abstract:

【Objective】‘Xiangshui’lemon (Citrus limon (L.) Burm. F.) was used to study the expression of two RHF2A genes, and to screen and verify their interaction proteins by yeast two hybrid technology and BiFC, and lay a foundation for further studying the molecular mechanism of RHF2A in the process of lemon selfincompatibility. 【Method】 Two E3 ubiquitin ligase RHF2A(RING-H2 Zinc Finger2A) genes RHF2A-1 and RHF2A-2 of ‘Xiangshui’ lemon were screened from the transcriptome and ubiquitin modification group, and their full-length sequences were cloned. The sequence and protein structure of two RHF2A genes were analyzed by bioinformatics to predict the cis acting elements of their promoters. 35S-RHF2A-GFP fusion protein expression vector was constructed for subcellular localization analysis. The temporal and spatial expression patterns of two RHF2A were analyzed by real-time fluorescence quantitative PCR. The yeast two hybrid bait vector was constructed to screen the interaction proreins from the lemon yeast library. The BiFC vector was constructed to verify the interaction of the target protein in onion living cells.【Result】The RHF2A-1 and RHF2A-2 genes were obtained from ‘Xiangshui’ lemon, and the total length of ORF was 1161 and 1134 bp respectively. NCBI domain prediction found that it had a Ring/U-box domain. Promoter analysis showed that there are POLLEN1LELAT52 and GTGANTG10 related to pollen specific expression elements. Tissue expression analysis showed that RHF2A-1 gene was specifically expressed in pollen and RHF2A-2 was specifically expressed in leaves; the results of temporal and spatial expression analysis showed that the expressed of RHF2A-1 in self stigma tended to increase from the first day and reached the peak on the third day, which was more than 5 times that of hybrid stigma. Subcellular localization showed that RHF2A-1and RHF2A-2 were localized in the nucleus. The interaction protein predicted by Uniprot website shows that RHF2A can interact with KRP6, AT3G57370, UBA1, FBL17 and SK11proteins, and that RHF2A gene is involved in biological processes such as self incompatibility ubiquitination reaction pathway, gametophyte development regulation and pollen growth and development. 72 clones were screened by yeast two hybrid technology. After sequencing and blast comparison, repetitive clones were excluded. Finally, 20 candidate interaction proteins such as ABCF3 were obtained. Through one-to-one interaction verification and BiFC, it was determined that there is an interaction relationship between RHF2A-1 and ABCF3-2. 【Conclusion】 The temporal and spatial expression of RHF2A-1 gene was consistent with the germination of pollen on pistil in the process of self incompatibility; The interaction candidate proteins directly affecting pollen growth and development during the pollination were screened, which preliminarily proved that RHF2A-1 gene played an important role in the process of self incompatibility.

Key words: lemon, RHF2A, gene cloning, spatiotemporal expression, yeast two hybrid technology