龙须菜四分孢子体不同发育阶段差异表达基因克隆和启动子区分析与刺激响应研究

Study on the Cloning,Promoter Analysis of Different Expressed Genes of Gracilariopsis lemaneiformis Tetrasporophyte from Different Development Stages and Stimulation Responses

龙须菜四分孢子体形成的四分孢子囊数量和四分孢子放散量可以作为育性高低的指标。龙须菜品种"981"在成熟期四分孢子放散量极低,与"鲁龙1号"有显著差异,即"981"具有低育的育性特点。从2个品种不同发育阶段数字表达谱数据中,筛选得到与育性控制相关的差异表达基因——D-乳酸脱氢酶(dld),谷氨酰胺合成酶(gln2)和热激蛋白(hsp15.4)。克隆得到dld完整的ORF与上游序列并进行测序分析,设置诱导条件检测3个基因的表达量,探究基因表达的调控因素。dld基因具有D-乳酸脱氢酶结构域、龙须菜特有的2个跨膜区、1个低复杂性区域。启动子区功能预测发现3个基因均可能受光照、茉莉酸甲酯和一些植物激素的调控。通过半定量PCR技术检测不同诱导条件下3个基因的表达变化趋势,结果显示光照与茉莉酸甲酯均可刺激3个基因的正表达。

The number of tetrasporangium formed and tetraspores released by Gracilariopsis lemaneiformis tetrasporophytes could be a judgement standard for the criteria of fertility level.The tetraspores released by cultivar 981 during its maturation is extremely low,which is significantly difference with cultivar No.1 Lulong.Thus 981 is characterized as low fertility.From the DGE data of the two strains under different development stages,significantly differentially expressed genes D-lactate dehydrogenase(dld),glutamine(gln)and hot shock protein(hsp)were screened and regarded as related to fertility control.Partial upstream and complete ORF sequences of dldwere cloned and analyzed.The expression of three genes under different induction conditions was detected to explore the gene regulation mechanism.The dldgene had one D-lactate dehydrogenase domain,two transmembrane regions which are unique to G.lemaneiformis;and one low complexity region.Promoter analysis in the upstream region showed that the expression of three genes might be regulated by light,methy jasmonate and some plant hormones.Semiquantitative RT-PCR was operated to detect gene expression under different induction conditions.Results showed that the expression level of three genes had an obviously positive correlation with light and methy jasmonate treatment.