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

茶树表现出一种配子体型的自交不亲和性，这种自交不亲和性由单个位点和多个等位基因（称为s基因）控制。本研究旨在鉴定茶树雌蕊组织的特异性蛋白，并阐明其自交不亲和的分子机制。从2D-PAGE后的雌蕊蛋白分析中，我们检测到两个主要的蛋白A （ca.18kDa）和B （ca.15kDa）在不同s基因型的茶品种中pH和分子量表现出多态性。2D-PAGE凝胶pvdf膜转移后，用蛋白测序仪测定蛋白A点n端氨基酸序列。根据n端氨基酸序列，合成了退化寡核苷酸引物。利用这些引物对雌蕊mRNA进行RT-PCR（3′-RACE）分析。PCR产物证实具有n端对应的核苷酸序列，并作为筛选雌蕊cDNA文库的探针。获得的全长cDNA克隆编码的发病相关蛋白PR-1与烟草PR-1具有40%的氨基酸同源性。Northern blot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B的n端也被测序，但它的n端被阻断了。因此，我们确定了裂解酶内肽酶消化蛋白质B点后从高效液相色谱分离得到的肽的一些内部氨基酸序列。通过在genbank数据库中对这些氨基酸序列的同源性搜索，发现蛋白质B是富含甘氨酸的RNA结合蛋白的成员，已知在许多植物物种中受胁迫条件（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。这些蛋白是否参与茶树的自交不亲和反应尚需进一步研究。

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.