Molecular Cloning of cDNAs Encoding S-RNsaes-like Proteins from Self-incompatible Chinese pear 'Ya-Li' and Its Comaptible Mutant 'Kintsui li'


Naoko Norioka1, Satoko Okuhata1, Takanori Matsuura1, S.L. Li1, Hidehira Tabira2, Kosuke Inoue2, Fumio Sakiyama3 and Shigemi Norioka1

1Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
2Tottori Horticultual Experiment Station, Daiei, Tottori 689-0221, Japan
3International Buddhist University, Habikino, Osaka 583-8501, Japan

International Symposium on Asian Pears (Tottori, Japan, 2001.8.25-29)

Rosaceous plants, such as pear and apple, show gametophytic self-incimpatibility (GSI) controlled by a single locus (S-locus) with multiple alleles (S-allele), which encodes a ribonuclease expressed exclusively in pistil (S-RNase). In Japanse pear, Pyrus pyrifolia, seven S-slleles (S1~S7) are known and theirgene products in the pistil (S1~S7-RNase) have been identified. A self-incompatibility cultivar of Japanese pear, 'Osa-Nijisseiki', was found as a stylar-part mutant (sm) of self-incompatible 'Nijisseiki' (S2S4). Molecular biological analysis revealed that the self-incompatibility of 'Osa-Nijisseiki' is due to the lack of S4-RNase gene, indicationg that S-RNase is essential for GSI of Japanese pear.

Chinese pear, Pyrus usseriensis, which is a speces closely related to Japanese pear, also shows GSI but there have been very few studies for its GSI. A self-incompatible mutant of a self-incompatiblr Chinese pear 'Ya li' was found in China and called 'Kintsui li'. As well as Japanese pear, molecular biological analysis of 'Ya li' and 'Kinrsui li' will provide very importantinformations for the mechanism of GSI in Chinese pear. As the first step in this project, we have cloned cDNAs encoding S-RNase-like proteins from both cultivars and discussed the molecular basis of GSI in Chinese pear.

When pistil proteins from 'Ya Li' ware analyzed by two-dimnsional electrophoresis (2DE), S-RNase-like proteins ware not deteceted clearly in the region where Japanese pear S-RNase had migrated (called S-RNase zone}. But when tje S-RNase zone og 'Ya li' waselectroblotted onto PVDF membrane and applied to a ptotein swquencer, two S-RNase-lke sequences, YA1 and YA2, ware detected. The nucleotide sequences of cDNAs encoding YA1 and YA2 ware determined by PCR and inverse PCR. The deduced amino acid sequences of YA1 and YA2 ware 60-70% identical to those of Japanese pear S-RNases. YA1 and YA2 had two catalytic histidine residues, eight cycteine residues and an N-glycosylation site, Asn 121, which are conserved in all rosaceous S-RNase sequenced so far. The secondery structures of YA1 and YA2 predicted by PHD method were very similar to one another. Moreover, RNase activities of YA1 and YA2 were detected by RNase activity staining after isoelectric forcusing of pistil proteins of 'Ya li'. These results suggest that YA1 and YA2 are able to function as S-RNase in vivo and that GSI in Chinese pear is governed by the S-RNase as well as Japanese pear.

The same experiments were done using 'Kiutsui li'. The two sequences, KT1 and KT2, were obtained by sequence analysis of the S-RNase zone and the nucleotide sequences of cDNAs encoding KT1 and KT2 were determined by PCR and inverse PCR. The nucleotide (amino acid) sequences of KT1 and KT2 were completely identical to those of YA1 and YA2., respectively, and KT1 and KT2 also had RNase activity. These results suggest that KT1 and KT2 can act as S-RNase similarly to YA1 and YA2 and that the self-compatibility of 'Kintsui li' is due to pollen-part mutation of the S-locus of 'Ya li'. Recently cross-pollination experiments showed that pistil of 'Kintsui li' can reject pollen of 'Ya li' but pistil of 'Ya li' cannot reject pollen of 'Kintsui li'. This result supports the hypothesis that 'Kintsui li' is a pollen-part mutant of 'Ya li'.

Table 1 Amino acid sequence identities among pear S-RNases.
Chinese
pear
Japanese pear
YA2 S1 S2 S3 S4 S5 S6 S7
YA1 65.0 67.0 62.0 60.0 68.0 60.0 69.0 68.0
YA2 67.0 62.0 60.0 68.0 60.0 69.0 68.0
S1 62.0 60.0 68.0 60.0 69.0 68.0
S2 60.0 68.0 60.0 69.0 68.0
S3 68.0 60.0 69.0 68.0
S4 60.0 69.0 68.0
S5 69.0 68.0
S6 68.0


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