Study On The Interactions Between Determinant Factors Of Self-incompatibility In Amygdalus Ledebouriana Schlecht. By Yeast Two-hybrid System

Amygdalus ledebouriana Schlecht. is one kind of wild rare endangered plant resources. The only distributing area in China is Xinjiang. Amygdalus ledebouriana Schlecht. exhibits S-RNase-mediated gametophytic self-incompability. There has been some research on self-incompatibility gene, but most of cloned genes are not full-length, and there is no report about the interactions between determinant factors of self-incompatibility in Amygdalus ledebouriana Schlecht.. In order to lay foundation for the molecular regulation and further research of molecular mechanism of self-incompatibility in Amygdalus ledebouriana Schlecht.,SFB(encoding pollen determinant factor of slef-incompatibility) and S-RNase(encoding pistil determinant factor of slef-incompatibility) were cloned by PT-PCR and RACE techniques from Amygdalus ledebouriana Schlecht. anther and pistil respectively, bioinformatics analyses of sequences of the cloned genes and their decuced amino acid sequences were processed, the interactions between SFB protein(pollen determinant factor of self- incompatibility) and S-RNase protein(pistil determinant factor of slef-incompatibility) were indentified by yeast two-hybrid system. The main research results can be stated as follows:1. Two full-length sequences of SFB(AlsSFB16 and AlsSFB17), which encoding pollen determinant factor of self-incompatibility, were successfully cloned by RT-PCR and RACE techniques from Amygdalus ledebouriana Schlecht. anther. AlsSFB16 gene and AlsSFB17 gene both belonged to F-box gene family. The identity of nucleotide sequence between the two cloned SFB genes and SFB genes of many other plant species of Prunus was above 88%. The deduced amino acid sequences of AlsSFB16 gene and AlsSFB17 gene both had typical structure of F-box protein. Open Reading Frame(ORF) of AlsSFB16 gene was 1146 bp in length, encoding a protein of 381 amino acids; Open Reading Frame(ORF) of AlsSFB17 gene was 1131 bp in length,encoding a protein of 376 amino acids. It was predicted that the AlsSFB16 protein and AlsSFB17 protein both were slightly hydrophilic instable cytoplasmic protein, and main functions of them included biosynthesis of cofactors, energy metabolism and lyase. 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.2. Two full-length sequences of S-RNase(AlsS16-RNase and AlsS17-RNase), which encoding pistil determinant factor of self-incompatibility, were successfully cloned by RT-PCR and RACE techniques from Amygdalus ledebouriana Schlecht. pistil. AlsS16-RNase gene and AlsS17-RNase gene both belonged to RNase T2 gene family. The identity of nucleotide sequence between the two cloned S-RNase genes and S-RNase genes of many other plant species of Prunus was 83% ~ 98%.The deduced amino acid seuqences of AlsS16-RNase gene and AlsS17-RNase gene both had typical structure of S-RNase protein. Open Reading Frame(ORF) of Als16-RNase gene was690 bp in length, encoding a protein of 229 amino acids; Open Reading Frame(ORF) of AlsS17-RNase gene was 678 bp in length, encoding a protein of 225 amino acids. It was predicted that the AlsS16-RNase protein and AlsS17-RNase protein both were hydrophilic instable secretory protein, the 1-28 amino acid residues of AlsS16-RNase protein and the 1-26 amino acid residuesof AlsS17-RNase protein were signal peptide, and main functions of them included hydrolase and hormone. 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.3. AlsSFB17 gene, AlsS16-RNase gene and AlsS17-RNase gene were selected for subsequent experiment. Yeast two-hybrid system was used to indentify the interactions between protein encoded by AlsSFB17 gene and proteins encoded by AlsS16-RNase gene and AlsS17-RNase gene.Results showed that there was no interaction between AlsSFB17 protein and AlsS16-RNase protein or AlsS17-RNase protein. Combined with some previous research evidence and collaborative non-self recognition system, we don't deem that any S-RNase protein interact with a certain SLF/SFB protein, and we infer that AlsS16-RNase protein and AlsS17-RNase protein are not in the identification spectrum of Als SFB17 protein. Collaborative non-self recognition system is the most convincing model about molecular mechanism of S-RNase–based self-incompatibility.