Cloning and sequence analysis of 2 full-length S-RNase genes in Amygdalus ledebouriana Schlecht

Abstract:【Objective】Two full-length sequences of S-RNase genes encoding pistil determinant factors ofself-incompatibility were cloned, and bioinformatics analyses of the sequences and their decuced aminoacid sequences were processed in order to facilitate molecular regulation of self-incompatibility in wild al-mond(Amygdalus ledebouriana Schlecht.).【Methods】The full-length sequences of S-RNase genes werecloned by RT-PCR and RACE techniques from the pistils of the wild almond. Regions of local similaritybetween sequences of the cloned S-RNase genes and those accessioned in Gen Bank were analysed by theBasic Local Alignment Search Tool(BLAST).Open reading frames of sequences of the cloned S-RNase genes were analysed by the ORF Finder(Open Reading Frame Finder). Conserved domains of the deducedamino acid sequences were searched by the Conserved Domain Database(CDD). Physical and chemical pa-rameters of the deduced amino acide sequences were computed by Prot Param. Membrane-spanning re-gions and their orientation of the deduced amino acid sequences were predicted by TMpred. The presenceand location of signal peptide cleavage sites of the deduced amino acid sequences were predicted by Signal P 4.1 Server. The subcellular location of the deduced amino acid sequences were predicted by Target P1.1 Server. The secondary structure of the deduced amino acid sequences were predicted by SOPMA. Se-quence alignment between the deduced amino acid sequences and ten homologous proteins of other plantspecies(Prunus webbii, Prunus armeniaca, Prunus avium, Prunus cerasus, Prunus dulcis, Prunus mume,Prunus persica, Prunus salicina, Antirrhinum hispanicum, Petunia integrifolia subsp. inflata) was per-formed by DNAMAN. Phylogenetic analysis between the deduced amino acid sequences and ten proteinsof other plant species were processed by MEGA6. The main functions of the deduced amino acid sequenc-es were predicted by Prot Fun 2.2 Server.【Results】Two full-length sequences of S-RNase(Pt S16-RNase and Pt S17-RNase) genes from wild almond, were successfully cloned. Both the Pt S16-RNase gene and the Pt S17-RNase gene belonged to RNase T2 gene family. The similarity of the nucleotide sequences betweenthe two cloned S-RNase genes and those of S-RNase genes of many other plant species of Prunus was 83%-98%. The deduced amino acid sequences of Pt S16-RNase gene and Pt S17-RNase gene had a typi-cal structure of S-RNase protein. Open Reading Frame(ORF) of Pt16-RNase gene was 690 bp in length,encoding a protein of 229 amino acids; Open Reading Frame(ORF) of Pt S17-RNase gene was 678 bp inlength, encoding a protein of 225 amino acids. Both the Pt S16-RNase protein and the Pt S17-RNase pro-tein had five conserved domains(C1-C5) and one hypervariable region(HV). Means, such as site-directedmutagenesis, could be used to modify the corresponding gene region of catalytic histidine in conserved do-mains to make S-RNase protein lose its ribonuclease activity, therefore couldn't degrade self-RNA.Means, such as site-directed mutagenesis, could also be used to modify the corresponding gene region ofhypervariable region to make S-RNase protein unable to specifically bind with pollen self-incompatibilitydeterminant factors, therefore couldn't initiate self-incompatibility response. Moreover, RNAi could beused to silence the target gene. It was predicted that both the Pt S16-RNase protein and the Pt S17-RNase protein were hydrophilic instable secretory protein. The 1-28 amino acid residues of the Pt S16-RNase pro-tein and the 1-26 amino acid residues of the Pt S17-RNase protein were predicted to be signal peptide.Means, such as site-directed mutagenesis, could be used to modify corresponding gene region of signalpeptide, so that S-RNase protein couldn't arrive its action site to arrest the growth of self-pollen tube. Itwas predicted that secondary structures of both the Pt S16-RNase protein and the Pt S17-RNase proteinmainly presented as α-helix, extended strand and random coil. According to the prediction, evolutionaryrelationships of the Pt S16-RNase protein and the Pt S17-RNase protein were close to some other S-RNaseproteins of Prunus, and homology of cross-species was higher than that of intra-species, which might sug-gest that S-RNase differentiation had completed before species differentiation in Rosaceae. It was predict-ed that main functions of the Pt S16-RNase protein and the Pt S17-RNase protein were hydrolase and hor-mone. The former was consistent with the research results that style self-incompatibility gene products areribonucleases. The latter was consistent with the research results that S-RNase still had some other func-tions except ribonucleases, which could be a new research angle after proved. Ribonucleases of the T2 fam-ily played a vital role in both plant and human. A broad range of biological roles for these ribonucleaseshad been suggested, including scavenging of nucleic acids, degradation of self-RNA, serving as extra-orintracellular cytotoxins, and modulating host immune responses. Recently, RNase T2 family members havebeen implicated in human pathologies such as cancer and parasitic diseases. Interestingly, certain func-tions of RNase T2 family members are independent of their nuclease activity, suggesting that these proteinshave additional functions.【Conclusion】Two full-length sequences of S-RNase gene were successfullycloned from pistils of two different Amygdalus ledebouriana Schlecht. plants. The molecular regulation ofself-incompatibility in Amygdalus ledebouriana Schlecht. could be performed based on characteristics ofsequences of the cloned genes and their decuced amino acid sequences.