Identification Of S-genotype Of Eli And The S-alleles CDNA Cloning

The plant self-incompatibility, a universal biological phenomenon in angiosperm, is an important protective mechanism of preventing close breeding and species retrogression which was formed during the long-term evolution. As a kind of rosaceae plants, Pear is a typical fruit tree of gametophytic self-incompatibility (GSI). This characteristic results in low fruiting during the natural pollination, so it is necessary to arrange varieties for pollination or to perform artificial pollination to increase the yield. Because of the property of being good at entailing the phenotype of big fruit to the progeny, Eli pear is choosed as a parent plant for the cross-breeding of most pear trees. Researches on the S-allele genes of Eli pear should be helpful to know indirectly about the S genetype of correlative offspring, providing the scientific foundation for pear self-incompatibility. The lack of correlative reports on Eli's S-alleles impels us to research it by determining the self-incompatibility, isolating S-alleles, identifying the S genotype, and cloning the full-length cDNA of Eli pear. The main results are following as:1. Basing on the reported primary structure of pear S₁—S₉, the primers "PF" and "PR" were designed to amplify S-alleles from Eli's genomic DNA by PCR. Then two S-alleles of Eli pear were isolated and identified by Using PCR-RFLP system and TA cloning technology. The result of bioinformatic analysis suggested that one of them is a new S-allele, which was named S34 and registered at GenBank as number DB269500. Thus the Eli's S genotype is determined as S13S34.2. The 3'-end of S₁₃ and S₃₄ cDNAs were obtained by using 3'RACE technology. By assembling the 3'-end cDNA with the 5'-end from genomic DNA, the full-length cDNAs of S₁₃ and S₃₄ were obtained, then were registered at GenBank as numbers DQ414812(S₁₃) and DQ4141813 (S₃₄) respectively. The analytical result suggested that both S₁₃ and S₃₄ have 687 bp, and code 299 amino acids. The signal peptide, C1, C2, C3, RC4, C5, and HV of both S₁₃ and S₃₄ are 27, 11, 11, 6, 8, 7, and 15 a.a. respectively. Furthermore, the-re are 8 conservative 'Cys' residues forming 4 disulfide bonds, 2 conservative 'His' residues influencing the S-RNase activity, and 1 conservative 'Asn-Xaa-Ser/Thr' motif within RC4 , which plays an important role in the folding of core structure.3. The complete full-length cDNA of S₁₃ including the transcription initi-ation site.was obtained by using 5'RACE technology. The result from bioinfo-rmatic analysis predicted the secondary structure of S₁₃-RNase, and the three-dimensions structure of S₁₃-RNase was predicted in terms of the crystal structure of S₃-RNase.