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

doi:10.13925/j.cnki.gsxb.20150520

Abstract：[Objective] Two full-length sequences of S-RNase genes encoding pistil determinant factors of self-incompatibility were cloned,and bioinformatics analyses of the sequences and their decuced amino acid sequences were processed in order to facilitate molecular regulation of self-incompatibility in wild almond (Amygdalus ledebouriana Schlecht.).[Methods] The full-length sequences of S-RNase genes were cloned by RT-PCR and RACE techniques from the pistils of the wild almond.Regions of local similarity between sequences of the cloned S-RNase genes and those accessioned in GenBank were analysed by the Basic 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 deduced amino acid sequences were searched by the Conserved Domain Database (CDD).Physical and chemical parameters of the deduced amino acide sequences were computed by ProtParam.Membrane-spanning regions and their orientation of the deduced amino acid sequences were predicted by TMpred.The presence and location of signal peptide cleavage sites of the deduced amino acid sequences were predicted by SignalP 4.1 Server.The subcellular location of the deduced amino acid sequences were predicted by TargetP 1.1 Server.The secondary structure of the deduced amino acid sequences were predicted by SOPMA.Sequence alignment between the deduced amino acid sequences and ten homologous proteins of other plant species (Prunus webbii,Prunus armeniaca,Prunus avium,Prunus cerasus,Prunus dulcis,Prunus mume,Prunus persica,Prunus salicina,Antirrhinum hispanicum,Petunia integrifolia subsp,inflata) was performed by DNAMAN.Phylogenetic analysis between the deduced amino acid sequences and ten proteins of other plant species were processed by MEGA6.The main functions of the deduced amino acid sequences were predicted by ProtFun 2.2 Server.[Results] Two full-length sequences of S-RNase (PtS16-RNase and PtS17-RNase) genes from wild almond,were successfully cloned.Both the PtS16-RNase gene and the PtS17-RNase gene belonged to RNase T2 gene family.The similarity of the nucleotide sequences between the 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 ofPtS16-RNase gene and PtS17-RNase gene had a typical 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 PtS17-RNase gene was 678 bp in length,encoding a protein of 225 amino acids.Both the PtS16-RNase protein and the PtS17-RNase protein had five conserved domains (C 1-C5) and one hypervariable region (HV).Means,such as site-directed mutagenesis,could be used to modify the corresponding gene region of catalytic histidine in conserved domains 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 of hypervariable region to make S-RNase protein unable to specifically bind with pollen self-incompatibility determinant factors,therefore couldn' t initiate self-incompatibility response.Moreover,RNAi could be used to silence the target gene.It was predicted that both the PtS16-RNase protein and the PtS17-RNase protein were hydrophilic instable secretory protein.The 1-28 amino acid residues of the PtS16-RNase protein and the 1-26 amino acid residues of the PtS17-RNase protein were predicted to be signal peptide.Means,such as site-directed mutagenesis,could be used to modify corresponding gene region of signal peptide,so that S-RNase protein couldn't arrive its action site to arrest the growth of self-pollen tube.It was predicted that secondary structures of both the PtS16-RNase protein and the PtS17-RNase protein mainly presented as α-helix,extended strand and random coil.According to the prediction,evolutionary relationships of the PtS16-RNase protein and the PtS17-RNase protein were close to some other S-RNase proteins of Prunus,and homology of cross-species was higher than that of intra-species,which might suggest that S-RNase differentiation had completed before species differentiation in Rosaceae.It was predicted that main functions of the PtS16-RNase protein and the PtS17-RNase protein were hydrolase and hormone.The former was consistent with the research results that style self-incompatibility gene products are ribonucleases.The latter was consistent with the research results that S-RNase still had some other functions except ribonucleases,which could be a new research angle after proved.Ribonucleases of the T2 family played a vital role in both plant and human.A broad range of biological roles for these ribonucleases had been suggested,including scavenging of nucleic acids,degradation of self-RNA,serving as extra-or intracellular cytotoxins,and modulating host immune responses.Recently,RNaseT2 family members have been implicated in human pathologies such as cancer and parasitic diseases.Interestingly,certain functions of RNaseT2 family members are independent of their nuclease activity,suggesting that these proteins have additional functions.[Conclusion] Two full-length sequences of S-RNase gene were successfully cloned from pistils of two different Amygdalus ledebouriana Schlecht.plants.The molecular regulation of self-incompatibility in Amygdalus ledebouriana Schlecht.could be performed based on characteristics of sequences of the cloned genes and their decuced amino acid sequences.