Gene expression analysis related to regulation of life cycle using Porphyra genomic information

本研究的新发现如下：1.单孢无性产孢过程中高表达基因的筛选通过cDNA微阵列和RT-PCR或北方分析，我们发现了8个与代谢相关的酶基因和一些未知基因，它们具有配子体世代特异性表达模式。这8个核苷酸序列将出现在GenBank/EMBL/DDBJ数据库中，登录号为AB 127044至AB127051.2。条斑紫菜（Porphyra yezoensis）基因表达分析及内标新技术的建立（1）以条斑紫菜配子体胚为材料，建立了条斑紫菜延伸因子-1 α（EF-1α）核酸探针原位杂交（WISH）方法。(2)我们开发了一种内标物（肌动蛋白相关蛋白4同源物），用于差异表达基因的基因表达分析。一个假定的二分核定位信号（NLS）和肌动蛋白基序内的PyARP 4氨基酸序列被发现。PyARP 4的表达水平在生活史的4个发育阶段之间没有显著变化，且低于常规肌动蛋白.化学处理诱导产生单孢子我们发现，将条斑紫菜的营养配子体菌体培养在含有10 mM尿囊素的海水培养基中，可以获得具有萌发能力的类单孢子细胞。我们还发现，这种化学处理是有效的，甚至配子体的拟线青霉，其中没有无性孢子形成通过单孢子的生活史。

New findings obtained by this study are as follows.1. Screening of highly expressed genes during asexual sporulation through monosporesWe found eight genes encoding enzymes related to metabolism and unknown genes with gametophytic generation-phase-specific expression pattern by cDNA macroarray and RT-PCR or Northern analysis. The eight nucleotide sequences will appear in the GenBank/EMBL/DDBJ databases with accession numbers AB127044 through AB127051.2. Development of a new technique for gene expression analysis and internal standard in Porphyra yezoensis(1) We developed a method for whole-mount in situ hybridization (WISH) by using elongation factor-1α (EF-1α) riboprobes and gametophytic germlings.(2) We developed an internal standard (actin-related protein 4 homologue) in gene expression analyses of differentially expressed genes. A putative bipartite nuclear localization signal (NLS) and an actin motif were found within the PyARP4 amino acid sequences. The expression level of PyARP4 did not change significantly among four developmental stages of the life cycle and was lower than that of a conventional actin.3. Induction of monospore production by chemical treatmentWe found that monospore-like cells with germinability can be obtained when vegetative gametophytic thalli of P.yezoensis were grown in enriched seawater medium that contained 10 mM allantoin. We also found that this chemical treatment is effective to even gametophytic thalli of P.pseudolinearis, which does not have asexual sporulation through monospores in the life cycle.