Identification Of The Ubiquitin Pathway/26S Proteasome Genes Associated With Self-incompatibility From Citrus Reticulata Blanco Cv.‘Wuzishatangju'

Self-incompatibility(SI) is an intraspecific reproductive barrier which can prevent self-fertilization and promote out-crossing in angiosperms. Seedlessness is an excellent economical trait of Citrus, and SI is one of important factors that can result in seedless fruit. SI is a complex process involved in the ubiquitin pathway/26 S proteasome, Ca2+ signaling pathway, plant hormone signal transduction and development process. In this study, ‘Wuzishatangju' and ‘Shatangju' were used as material, to screen the ubiquitin pathway/26 S proteasome genes associated with SI by RNA-Seq and reported documents. Fourteen candidate genes were obtained, and further were screened by q-PCR. The main results were as follow:1. Mature pollen, pistils from ‘Wuzishatangju' and ‘Shatangju' and pistils of 4 h, and 1-7 days after artificial self-pollination of ‘Wuzishatangju' and cross-pollination were used for RNA-seq. Atotal of 35.67 GB base and 306,462,978 of double end reads(Q20>96%) were obtained. N50 was 1594 and the average length of unigenes was 897.99 bp. Approximately 85% were mapped to the reference transcriptome. These results suggested that RNA-Seq data is credible and canbe used for subsequent experiments.2. According to the RPKM and the annotation information,the differentially expressed unigenes were selected based on the FDR?0.001 and |log2Ratio| ?1. The number of different expression genes between WCPistils and WSPistils is 634, which 541 were up-regulated and 93 were down-regulated. There are 6,555 different expression genes in sample of YPistils compared with sample WPistils, which 2,643 were up-regulated and 3,912 were down-regulated. The different expression genes between YPollen and WPollen were 2,869, which 634 were up-regulated and 2,235 were down-regulated. According to GO annotation, these different expression genes involved in reproductive process, signaling, immune system process, response to stimulus, regulation of molecular and protein binding transcription factor activity. In the KEGG pathway, these different expression genes were involved in cysteine and methionine metabolism, plant hormone signal transduction and transcription RNA transport. Nine genes were involved in the pathway of ubiquitin-mediated proteolysis i.e. ubiquitin-activating enzyme E1(UBE1)(Unigene0031440), ubiquitin-conjugating enzyme E2(UBE2)(Unigene0049541, Unigene0034845), and ubiquitin ligase E3(UBE3)(Unigene0038751, Unigene0031435, Unigene0034401, Unigene0029897, Unigene0023597 and Unigene0035181).3. Five candidate genes i.e. SSK-21, SSK-62, SSK-98, Cullin67 and Ring were obtained according to reported literatures, Citrus sinensis database, KEGG pathway and SSH c DNA library.4. 43 were selected genes to verify the transcriptome data by q-PCR. 88.4% of these genes expression pattern were consistent with the transcriptome data, suggesting the data was reliable.5. The expression levels of these genes were detected in stigmas, styles and ovaries at 4 h, 1 d~7 d after self- and cross-pollination, as well as different tissues(leaves, petals, filaments, anthers, stigmas, styles and ovaries). Unigene0031435, Unigene0038751, Unigene0029897 and Ring were up-regulated in the styles at 3 d after cross-pollination. All had RING-finger domain suggesting Unigene0031435, Unigene0038751 and Unigene0029897 may be the key enzyme genes of the ubiquitin pathway/26 S proteasome.6. Ring had 90.0% homology with Theobroma cacao, and 84.0% consistency with MYB30-Interacting E3 ligase1 from Arabidopsis thaliana. However, there was no difference in ORF between ‘Wuzishatangju' and ‘Shatangju'. There were two bases in DNA sequence in introns. Therefore, the gene may be not the key enzyme gene in the ubiquitin pathway/26S proteasome.