Molecular biological analyses of receptorlike proteins responsible for sexual reproduction in Clasterium

双体Closterium peracerosum-strigosum-littorale复合体（C. pslc）是与陆地植物最接近的单细胞姐妹，在有性生殖过程方面是最具特征的藻类绿藻。两种性信息素（PR-IP和PR-IP诱导剂）参与了C. pslc有性生殖过程的研究。此外，这些信息素的可能受体分子（CpRLK1, CpRLP1）已被确定。从两个杂交系统的结果来看，PR-IP诱导剂与CpRLK1以及19-kDa亚基与CpRLPI之间存在周相互作用。为了鉴定其他信息素受体分子，利用编码信息素的cDNA作为诱饵，从有性生殖过程cDNA文库中进行了双杂交筛选。结果，以PR-IP的19kda亚基为诱饵，鉴定出有性生殖过程中表达增加的两个基因。以PR-IP诱导剂为例，多次筛选与先前鉴定的基因（Cp-01）有显著相似性的一个基因，添加PR-IP诱导剂后，Cp-01仅在mt^+细胞中表达升高。为了进一步阐明C. pslc的有性生殖，构建了粒子轰击法转化系统。制备了两个由C. pslc内源性启动子驱动的GFP构建体（CpHSP7Op和CpCABp，这两个启动子在繁殖过程中高度和组成表达），并通过粒子轰击引入细胞。这些GFP信号在轰击后48-72小时内可在细胞质中瞬时检测到。当引入带有细胞核定位选择标记基因（ble）的GFP融合构建体时，GFP信号仅在细胞核中可见。这些结果表明，该方法和策略适用于研究C. pslc有性生殖过程中未知基因产物的细胞定位。

The desmid Closterium peracerosum-strigosum-littorale complex (C. pslc), which is the closest unicellular sister to land plants, is the best-characterized of the charophycean green algae with respect to the process of sexual reproduction. Two sex pheromones (PR-IP and PR-IP Inducer) involved in the progress of sexual reproduction of the C. pslc have been well characterized. In addition, putative receptor molecules (CpRLK1, CpRLP1) for these pheromones have been identified.From the results of two hybrid systems, presences of week interactions between PR-IP Inducer and CpRLK1 and between 19-kDa subunit and CpRLPI were confirmed. To identify another receptor molecules for pheromones, two-hybrid screenings from cDNA library for sexual reproductive processes were performed using cDNAs encoding respective pheromones as baits. As a result, two genes showing an increase in expression during sexual reproduction were identified when 19-kDa subunit of PR-IP was used as a bait. In the case of PR-IP Inducer, one gene showing significant similarity with a previously identified gene (Cp-01) was screened at multiple times The expression of Cp-01 was elevated only in mt^+ cells by the addition of PR-IP Inducer.To further elucidate their sexual reproduction, the transformation system for the C. pslc by using particle bombardment method was constructed. Two GFP constructs driven by endogenous promoters of C. pslc (CpHSP7Op and CpCABp, which are highly and constitutively expressed through the reproduction processes) were prepared and introduced into the cell by particle bombardment. These GFP signals could be transiently detected in the cytoplasm during 48-72 h after bombardment. When a GFP-fusion construct with a nucleus-localized selective marker gene (ble) was introduced, the GFP signal was only visible in the nucleus. These results indicate that this method and strategy are applicable to study cellular localization of unknown gene products during the sexual reproduction in C. pslc.