Endo Makoto, Hakozaki Hirokazu, Kokubun Takao, Masuko Hiromi, Takahata Yoshihito, Tsuchiya Tohru, Higashitani Atsushi, Tabata Satoshi, Watanabe Masao
Faculty of Agriculture, Iwate University, Morioka, Japan.
Genes Genet Syst. 2002 Aug;77(4):277-82. doi: 10.1266/ggs.77.277.
Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.
日本百脉根作为一种潜在的豆科模式植物受到了越来越多的关注。为了研究生殖器官中的基因表达并鉴定在有性生殖中起关键作用的基因，我们从日本百脉根发育中的绒毡层细胞阶段含有花药的未成熟花芽构建了一个cDNA文库，并对从未成熟花芽cDNA文库中随机选择的919个表达序列标签（EST）进行了表征。分析的919个EST被聚类为821个非冗余EST组。数据库搜索结果显示，821个组中的436个组（53%）与公共数据库中登记的基因具有序列相似性。在这436个组中，109个组与编码功能尚未确定的假定蛋白的基因具有相似性。385个组（47%）与已知序列无显著同源性，被归类为新序列。对821个非冗余EST序列与来自日本百脉根全株的EST序列进行比较，发现从未成熟花芽中获得的474个EST序列在全株EST序列中未被发现。为了确认潜在的生殖器官特异性EST克隆的表达模式，选择了9个与全株来源的EST不匹配的克隆，并对这些克隆进行了RT-PCR分析。RT-PCR结果显示，我们发现了两个新的花药特异性克隆。一个克隆与编码人类唇腭裂相关跨膜蛋白（CLPTM1）样蛋白的基因同源，另一个克隆与公共数据库中存放的任何基因均无显著相似性。这些结果表明，这里分析的EST是在日本百脉根中寻找生殖器官特异性基因的宝贵资源。
