Molecular analysis of self-incompatibility gene product in tea-plants.

茶树的自交不亲和性是由一个多等位基因的单基因座（称为S基因）控制的配子体类型。本研究旨在鉴定茶树雌蕊组织特异性蛋白质，阐明自交不亲和性的分子机制。对不同S基因型茶树品种的雌蕊蛋白质进行2D-PAGE分析，发现雌蕊蛋白质A（约18 kDa）和B（约15 kDa）在pH和分子量上存在多态性。在PVDF膜转移2D-PAGE凝胶后，通过蛋白质测序仪测定蛋白A斑点的N-末端氨基酸序列。根据N-末端氨基酸序列，合成简并的寡核苷酸引物。利用这些引物对雌蕊mRNA进行RT-PCR（3 '-RACE），PCR产物经鉴定具有N端相应的核苷酸序列，并以此为探针筛选雌蕊cDNA文库。获得的全长cDNA克隆被发现编码致病相关蛋白（PR-1），显示40%的氨基酸与烟草PR-1的一致性。北方杂交分析表明，PR-1基因在雌蕊组织中特异表达，在雄蕊和叶片中未检测到。蛋白质B的N-末端也被测序，但其N-末端被封闭。因此，我们确定了从蛋白质B斑点的裂解内肽酶消化后的HPLC分级分离获得的肽的一些内部氨基酸序列。通过在Gen Bank数据库中对这些氨基酸序列进行同源性搜索，发现蛋白质B是富含甘氨酸的RNA结合蛋白的成员，已知该蛋白在许多植物物种中由胁迫条件如低温诱导。本研究中鉴定的两种雌蕊蛋白的生物学功能在茶树中尚不清楚。这些蛋白质是否参与茶树自交不亲和反应还有待进一步研究。

Tea-plants show a gametophytic type of self-incompatibility that is controlled by a single locus with multiple alleles (called S-gene). The present study is undertaken to identify some proteins specific for pistil tissue of tea-plants, and to elucidate a molecular mechanism underlysing in the self-incompatibility. From analysis of pistil proteins after 2D-PAGE,we detected two major proteins, A (ca.18kDa) and B (ca.15kDa) which showed a polymorphic pattern in pH and molecular weigh among tea cultivars different in S-genotypes. Following PVDF-mambrane transfer of the 2D-PAGE gel, N-terminal amino acid sequence of protein A spot was determined by a protein sequencer. Based on the N-terminal amino acid sequence, degenerated oligonucleotide-primers were synthesized. Using these primers, RT-PCR (3'-RACE) was performed with pistil mRNA.PCR product was confirmed to have the N-terminus corresponding nucleotide sequence, and used as a probe for screening of pistil cDNA library. A full-length cDNA clone obtained was found to encode pathogenesis related protein (PR-1) that showed 40% amino acid identity with tobacco PR-1. Northern blot analysis showed that gene expression of the PR-1 was specific for pistil tissue with no detection in stamen and leaf. N-terminus of Protein B was also sequenced, but it's N-terminus was blocked. So, we determined some internal amino acid sequences of peptides obtained from HPLS-fractionation after lysil-endopeptidase digestion of the protein B spot. From homology search of these amino acid sequences in the Gen Bank data base, protein B was found to be a member of glycine-rich RNA binding proteins that are known to be induced by stress conditions such as cold temperature in many plant species. Biological functions of the two pistil proteins indentified in this study are not known in tea-plants. Further study is needed to make clear whether these proteins are involved in the self-incompatibility reaction of tea-plants.