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

茶树表现出配子体类型的自交不亲和性，受带有多个等位基因的单基因座控制(称为S基因)。本研究旨在鉴定茶树雌蕊组织所特有的蛋白质，并阐明茶树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A(约18 kDa)和B(约15 kDa)两种主要蛋白质，它们在pH和相对分子质量方面在不同S基因型的茶树品种中表现出遗传多样性。在PVDF-膜转移2D-PAGE凝胶后，用蛋白质测序仪测定蛋白A点的N-末端氨基酸序列。根据N-末端氨基酸序列，合成了简并的寡核苷酸引物。用这两条引物对雌蕊进行RT-PCR(3‘-RACE)，结果表明扩增产物中含有N端相应的核苷酸序列，并作为筛选雌蕊cDNA文库的探针。获得的全长cDNA克隆编码的致病相关蛋白(PR-1)与烟草PR-1有40%的氨基酸同源性。Northern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-末端被阻断。因此，我们测定了蛋白质B斑点经溶菌内切酶消化后，通过HPLS分级得到的一些多肽的内部氨基酸序列。在Gen Bank数据库中对这些氨基酸序列进行同源性搜索后，发现蛋白B是一种富含甘氨酸的RNA结合蛋白，在许多植物中都能在低温等逆境条件下诱导。本研究中鉴定的两种雌蕊蛋白在茶树中的生物学功能未知。这些蛋白质是否参与了茶树的自交不亲和反应，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.