Prediction and functional analysis of the sweet orange protein-protein interaction network.

BACKGROUND: Sweet orange (Citrus sinensis) is one of the most important fruits world-wide. Because it is a woody plant with a long growth cycle, genetic studies of sweet orange are lagging behind those of other species. RESULTS: In this analysis, we employed ortholog identification and domain combination methods to predict the protein-protein interaction (PPI) network for sweet orange. The K-nearest neighbors (KNN) classification method was used to verify and filter the network. The final predicted PPI network, CitrusNet, contained 8,195 proteins with 124,491 interactions. The quality of CitrusNet was evaluated using gene ontology (GO) and Mapman annotations, which confirmed the reliability of the network. In addition, we calculated the expression difference of interacting genes (EDI) in CitrusNet using RNA-seq data from four sweet orange tissues, and also analyzed the EDI distribution and variation in different sub-networks. CONCLUSIONS: Gene expression in CitrusNet has significant modular features. Target of rapamycin (TOR) protein served as the central node of the hormone-signaling sub-network. All evidence supported the idea that TOR can integrate various hormone signals and affect plant growth. CitrusNet provides valuable resources for the study of biological functions in sweet orange.

Stable maintenance and expression of a foreign gene in transgenic pear shoots retrieved from in vitro conservation.

Shoot-tips of transgenic pear were successfully conserved in vitro by slow-growth and cryopreservation methods. After 1 year of storage in slow growth conditions, all shoot-tips survived showing shoot re-growth. Similarly, shoot-tips showed high survival and regeneration rates after cryopreservation. The genetic stability of the transgenic GUS construct in shoots recovered from slow-growth and cryopreservation was assessed. The results from polymerase chain reaction and Southern blotting showed the stable maintenance of the GUS gene in genome, and the Single-Strand Conformation Polymorphism assay did not detect single base variation. X-Gluc staining suggested a normal expression of the GUS gene in shoots retrieved from these storage methods.

GUS gene remains stable in transgenic citrus callus recovered from cryopreservation.

The conservation of transgenic materials is very important, paticularly for their potential future use in crop development. In this study, transgenic callus cultures of 'Newhall' navel orange (Citrus sinensis Osbeck) were cryopreserved by a vitrification method. Transgenic calluses survived cryopreservation and recovered under normal culture conditions. The results of PCR (Polymerase Chain Reaction) amplification, Southern blotting and SSCP (Single-Strand Conformation Polymorphism) assay showed that the GUS gene was still maintained in the genome of callus cultures recovered from cryopreservation. X-Gluc staining further indicated GUS gene expression in callus cultures recovered from cryopreservation.

[Inheritance of organelle genomes of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and flying dragon (Poncirus trifoliata)].

Cleaved Amplified Polymorphic Sequence (CAPS) was successfully applied to analyze the organelle composition of three eight-year-old trees of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and Flying Dragon (Poncirus trifoliata). Five chloroplast and five mitochondrial universal primer pairs were used. All chloroplast primer pairs (rbcL-rbcL, rbcL-PSA I, TrnH-Trnk, TrnD-TrnT, TrnK-TrnK) and three (nad 1 exon B-nad 1 exon C, 18S rRNA-5S rRNA, nad 4 exon 1-nad 4 exon 2) of the five mitochondrial primer pairs, were efficiently amplified, but no polymorphism was detected, when the PCR products were digested by eleven restriction endonucleases, including, Hin6 I, Bus RI, Taq I, Msp I, HinfI, AluI, Dra I, EcoR I, Hind III, BamH I and Pst I respectively, three polymorphic cpDNA-CAPS markers (rbcL-rbcL/Hin 6 I, TrnD-TrnT/BusR I, TrnD-TrnT/Taq I) and one mtDNA-CAPS marker (nad 1-nad1/Msp I) were found. The results showed that cpDNA in the somatic hybrid plants came from Flying Dragon, the mesophyll parent, and mtDNA from Cleopatra mandarin, the embryogenic suspension parent uniformly. In order to prove the reliability of CAPS results, and to get more detailed information about the mtDNA inheritance, RFLP analyses was conducted. Genomic DNA of the somatic hybrids and their corresponding parents were digested by five restriction endonucleases (Dra I, EcoR I, Hind III, BamH I and Pst I), and hybridized with five mitochondrial probes (Cob, Pro 2, Pro I, atp 6, 26S rRNA) as well as one chloroplast probe, i.e. the PCR product of Flying Dragon with the primer pair of trnd 1-trnt 1. The results were in line with those of CAPS, and no novel bands were detected, which indicated that no organelle DNA recombination or rearrangement have been detected in the hybrid plants. The research showed that novel pattern of nuclear-mitochondria-chloroplast interaction could be reached via protoplast fusion.

The draft genome of sweet orange (Citrus sinensis).

Oranges are an important nutritional source for human health and have immense economic value. Here we present a comprehensive analysis of the draft genome of sweet orange (Citrus sinensis). The assembled sequence covers 87.3% of the estimated orange genome, which is relatively compact, as 20% is composed of repetitive elements. We predicted 29,445 protein-coding genes, half of which are in the heterozygous state. With additional sequencing of two more citrus species and comparative analyses of seven citrus genomes, we present evidence to suggest that sweet orange originated from a backcross hybrid between pummelo and mandarin. Focused analysis on genes involved in vitamin C metabolism showed that GalUR, encoding the rate-limiting enzyme of the galacturonate pathway, is significantly upregulated in orange fruit, and the recent expansion of this gene family may provide a genomic basis. This draft genome represents a valuable resource for understanding and improving many important citrus traits in the future.

Lycopene-epsilon-cyclase pre-mRNA is alternatively spliced in Cara Cara navel orange (Citrus sinensis Osbeck).

Lycopene-epsilon-cyclase is one of the key enzymes related to alpha-carotene metabolism in plants. A full-length cDNA of 1300 bp encoding lycopene-epsilon-cyclase (Lyce) was generated from Cara Cara navel orange, a unique navel orange containing both lycopene and beta-carotene in its pulp, with little or no alpha-carotene. The gene had a 14 bp nucleotides deletion and caused a terminal mutation. DNA sequence corresponding to the deletion region revealed that two repeats of 6 bp (AGGTGT) were flanking the region in both Cara Cara and its original variety, Washington navel oranges, but a 2 bp (AT) insertion was only found in Cara Cara which explain the alternative splicing character of the gene.