Cloning and phylogenetic analysis of S-RNase genes in genus Pyrus plants

Abstract:【Objective】Most pear cultivars in the world have self-incompatibility. The seed setting rate of self-pollination is far below the demand of commercial production. Studies have confirmed that pear self-incompatibility is a type of gametophyte self-incompatibility, and the pear S-RNase gene can specif- ically recognize pollen during the pollination process of stamens and pistils to prevent self-pollination. The objective of the study was to obtain the full-length sequence of the pear S-RNase gene and analyze the evolution of the pear S-RNase gene and its genetic polymorphism.【Methods】DNA was extracted from the leaves of pear cultivar Longxiang (Pyrus ussuriensns Maxim.), and specific primers were de- signed using the conservative characteristics of the apple subfamily S-RNase, and the full-length se- quence of pear S42-RNase genomic DNA was amplified by PCR. The RNA extracted from the pistil of pear cultivar Longxiang was used as the material, and the full-length cDNA sequence of the pear S42- RNase gene was obtained by using RT-PCR and RACE technology. its gene exon and HV region (Hy- per variable region) intron sequences were obtained accurately by comparing the full-length cDNA and DNA sequence of the S42-RNase. The full length sequences of other S-RNase genes cloned from Pyrusin GenBank were searched by BLAST. The phylogenetic trees of S-RNase gene coding region and HV region introns of Pyrus were constructed by multi-sequence alignment and manual adjustment with MEGA 6.06 software, and the differentiation and genetic polymorphism among the sequences were esti- mated【. Results】Bioinformatics analysis showed that the coding frame of pear S42-RNase gene are com- posed of 678 bases and encoded 226 amino acids, including a signal peptide composed of 27 amino ac- ids, five conserved regions (C1, C2, C3, RC4, C5) composed of 11, 11, 6, 8, and 7 amino acids and a HV region composed of 13 amino acids. In addition, there is a highly conserved hexapeptide region (IIWPNV) in the downstream region of HV. The full length of the genome DNA sequence is 1044 bp, and the HV region contained a 335 bp intron. The prediction of physical and chemical properties and secondary structure showed that the molecular weight of the protein encoded by the pear S42-RNase gene is 25 844.6, the pI is 8.97, the chemical formula is C1163H1785N307O333S14, and the halflife is 30h. The C1, RC4, and C5 hydrophobic regions composed of α-helices in the secondary structure were consid- ered to be involved in the stability of the enzyme structure. The C2, C3 hydrophilic regions mainly composed of β- sheets contain conserved catalytic histidine residues. It would play an important role in RNase activity. The hydrophilic region of HV between C2 and C3 was located on the surface of the pro- tein and was believed to support the selective interaction between S-RNase and pollen S gene determi- nants. Multiple sequence comparison analysis showed that the content of C+G in the coding region of pear S-RNase gene was lower than that of HV introns, the rate of conserved sites in the coding region was greater than that of introns in the HV region, and the conversion of the coding region was slightly more than that of transversion. The intron regions in the HV region were just the opposite. In addition, the intron sequence of the HV region showed a very high length polymorphism, ranging in size from 109 bp to 3130 bp. The constructed phylogenetic tree showed that the phylogenetic tree constructed by the coding region and HV introns was divided into 5 subgroups, the sequence divergence within the subgroups was smaller, the divergence between the subgroups was larger The topological structure had both similarities and differences. The average Normalized dN-dS value corresponding to the pear S- RNase codon indicated that the difference in the coding sequence of the S-RNase gene was the result of adaptive amino acid substitutions, and its evolutionary power comed from the function of the S-RNase gene to encode a specific recognition protein; The analysis also showed that in addition to the HV re- gion, there were a large number of non-synonymous amino acid substitutions in several regions, indicat- ing high sensitivity to positive selection.【Conclusion】The phylogenetic tree analysis of pear S-RNase sequences showed that the evolution of the coding region and introns in the HV region were correlated with each other under the condition of independence. In addition to the existing HV region, there were other regions in the coding region participating in the specific recognition between pear pistil and pol- len. Therefore, the pear S gene could not stay in the same HV region to determine whether it was a new gene. The index of genetic diversity showed that the sequence of the coding region of the pear S-RNase gene and the intron sequence in the HV region had different evolutionary power sources. The sequence evolution of the coding region would be caused by balanced selection, and the evolution of introns in the HV region would be under the pressure of negative selection. Based on the genetic differentiation of the coding region of the pear S-RNase gene and the intron sequence in the HV region, we could conclude that Xinjiang pear is a hybrid of Asiatic pear and Western pear.